ctd module 2: mock-up of quality overall summary (example)ctd module 2: mock-up of quality overall...

CTD Module 2:

Mock-up of Quality Overall Summary

(Example)

― Module 2.3 Mock-Up for Antibody Drugs ―

May 2018.

Japan Health Sciences Foundation

i

I. Introduction

The ICH M4 Guideline, the so-called CTD (Common Technical Document) Guideline, became Step 4 in

2000, and the "Guideline for preparation of documents to be attached to Application Form for manufacturing

or import of new drugs" (Notification No. 899 of the Evaluation and Licensing Division, Pharmaceutical and

Medical Safety Bureau, Ministry of Health, Labour and Welfare (MHLW), dated June 21, 2001) was issued

in Japan. (Several revisions have been made thereafter. Please refer to the Pharmaceuticals and Medical

Devices Agency (PMDA) website for the latest CTD guidelines.)

Module 2.3 Quality Overall Summary (QOS) is a key document in the review of quality domains for

marketing approval applications in Japan. When the CTD Guidelines were first issued in 2001, there had

been many opinions from the industry that they did not know how to describe Module 2.3. In view of such

opinions, specific examples (mock-up) of QOS were prepared by the Pharmaceutical Manufacturers’

Association of Tokyo (PMAT), the Osaka Pharmaceutical Manufacturer Association (OPMA), and Japan

Health Science Foundation (JHSF), and were published in 2002 (It was also published as Office

Memorandum, Evaluation and Licensing Division, MHLW, August 13, 2002).

As it is now, the number of drugs that have been approved by submitting dossier in the CTD format is

increasing, and many companies seem to have accumulated experience in preparing regulatory documents in

the CTD format.

In the original Module 2.3 Mock-Up described above, a mock-up of biological products was created by

members of the CTD-Q Implementation Review Group of JHSF for a hypothetical growth factor. As an

updated version of the Novel Biotechnological products Mock-Up, JHSF have prepared Module 2.3 Mock-

Up for Antibody Drugs.

The reasons for creating the Module 2.3 mock-up for antibody drugs are as follows:

・ Since many companies have accumulated experience in preparing regulatory documents in the CTD

format after the original mock-up was published in 2002, it was considered useful to enhance the

mock-up content based on the experiences.

・ The number of antibody drugs as new biotechnological/biological product applications has increased,

and the Guidance for Quality Assessment of Antibody Drugs (Notification No. 1214-1 of the

Evaluation and Licensing Division, Pharmaceutical and Food Safety Bureau, MHLW, dated

December 14, 2012) suggests that it is useful to provide a Module 2.3 mock-up of Antibody Drugs.

It should be noted that, like the original mock-up, the content of this mock-up was hypothetical creation

and there is a part of the submission that is not consistent overall. In addition, this mock-up summarizes

examples/references included in each CTD section and does not cover all of the necessary contents for the

approval review of individual products. Please also understand that not all illustrated items are required. The

applicants would like to use this mock-up as a reference and in an appropriate manner for each product to be

submitted.

May 2018

Japan Health Sciences Foundation

ii

The following people were responsible for creating this mock-up:

Biopharmaceuticals Working Group, Japan Health Sciences Foundation

(○: Working Group Leader).

Yuichi Akatsu Nippon Kayaku Co., Ltd.

Ryuko Igarashi MSD K.K.

Seiji Ishida Daiichi Sankyo Co., Ltd. (currently Sosei, Inc.)

Kazue Ouchi Kyowa Kirin Co., Ltd.

Takeshi Ohmura Chugai Pharma Manufacturing Co., Ltd.

○ Yoshinori Kubodera Chugai Pharmaceutical Co., Ltd.

Masashi Segawa Mochida Pharmaceutical Co., Ltd.

Sinya Takatsuka Mitsubishi Tanabe Pharma Corporation

Shogo Tanaka Chugai Pharma Manufacturing Co., Ltd.

Kouta Nagaiwa Asahi Kasei Pharma Corporation (currently Sanofi K.K.)

Hideo Nakazawa Kyowa Kirin Co., Ltd.

Yoshiko Nozawa Novartis Pharma K.K.

Yumiko Masuda Daiichi Sankyo Co., Ltd.

Tadashi Mikami Biogen Japan Ltd. (currently KM Biologics)

Koki Murakami Pfizer Japan Inc..

Akihiro Yanagita Chugai Pharmaceutical Co., Ltd.

iii

II. Mock-up for new biotechnological/biological product - Antibody drug version

In this mock-up case study, the following injection drug containing monoclonal antibody from

recombinant animal cell as the active ingredient is assumed.

・ Non-proprietary name: hutumumab (Genetical Recombinant)

・ Characteristics: Humanized monoclonal antibody, IgG1

・ Mechanism: Antibody drugs expecting cytotoxic activity via effector molecules

・ Host cell; CHO cell

・ Drug Substance: Protein concentration: Approximately 100 mg/mL

・ Drug Product: Protein concentration: 100 mg/2 mL

In this mock-up, the manufacturing process of the drug substance is not developed using an Enhanced

approach described in the ICH Q11 guideline, but is developed using a Traditional approach that has currently

accumulated company experiences.

In order to enrich the descriptive examples, the S.2.6 Manufacturing Process Development includes

examples of the development of many manufacturing methods from nonclinical drug substances to

commercial drug substances. As the S.5 Reference Standard or Reference Material, in addition to examples

in which the primary reference material and the working reference material are set, examples in which only

the primary reference material is set are also described. In addition, examples of liquid product and freeze-

dried products are described as drug products (2.3.P).

Each section contains the notes and explanation on reading this mock-up.

・ Note: General precautions for reading individual CTD sections.

・ Explanation: Supplementary explanation is provided for the contents of the example.

Japan Health Sciences Foundation (JHSA) has previously prepared an image of CTD Module 1

“Application Form” based on the original Module 2.3 Mock-Up (2002) (see Reference). In the updated

Module 2.3 Mock-Up, the image of the information included in the Application Form (Module 1.2) is shown

as cyan markers/shading. In addition, the commentaries are described as "M1.2 commentary." Please

understand that the image part of the information in the Application Form is not representative of the level

of approval because it just shows the image of the information that an applicant would write in the

Application Form at the time of application.

iv

< CTD-Q related notification > ・ Q2A: Validation of Analytical Procedures: Text and Methodology (Notification No. 755 of the

Evaluation and Licensing Division, PAB dated July 20, 1995)

・ Q2B: Validation of Analytical Procedures: Methodology (Notification No. 338 of the Evaluation and

Licensing Division, PMSB dated October 28, 1997)

・ Q3C: Impurities: Guideline for Residual Solvents (Notification No. 307 of the Evaluation and

Licensing Division, PMSB dated March 30, 1998)

・ Q3D: Guidelines for Elemental Impurities (Notification No. 09304 of the Evaluation and Licensing

Division, PFSB dated September 30, 2015)

・ Q5A: Viral Safety Assessment of Biotechnological Products Derived from Cell Lines of Human or

Animal Origin (Notification No. 329 of the Evaluation and Licensing Division, PMSB dated

February 22, 2000)

・ Q5B: Analysis of the Expression Constructs in cells used for Production of recombinant DNA

Derived Protein Products (Notification No. 3 of the Evaluation and Licensing Division, PMSB, dated

January 6, 1998)

・ Q5C: Stability Testing of Biotechnological/Biological Products (Notification No. 6 of the Evaluation

and Licensing Division, PMSB dated January 6, 1998)

・ Q5D: Derivation and Characterization of Cell Substrates Used for Production of

Biotechnological/Biological Products (Notification No. 873 of the Evaluation and Licensing

Division, PMSB dated July 14, 2000)

・ Q5E: Comparability of Biotechnological/Biological Products Subjects to Changes in Their

Manufacturing Process (Notification No. 0426001 of the Evaluation and Licensing Division, PFSB

dated April 26, 2005)

・ Q6B: Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological

Products (Notification No. 571 of the Evaluation and Licensing Division, PMSB dated May 1, 2001)

・ Q7a: Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients (Notification No.

1200 of the Pharmaceutical and Medical Safety Bureau dated November 2, 2001)

・ Q8: Pharmaceutical Development (Notification No. 0628-1 of the Evaluation and Licensing Division,

PFSB dated June 28, 2010)

・ Q11: Development and Manufacturing of Drug Substances (Chemical Entities and

Biotechnological/Biological Entities) Guidelines (Notification No. 0710-9 of the Evaluation and

Licensing Division, PFSB dated July 10, 2014)

< Reference documents >

・ HS Report No. 43: Records of briefing session for the Image of CTD-Module 1 Application Form

(Manufacturing/Specification)

・ HS Report No.68: Records of briefing session for Questionnaire on Actual Status of the

Manufacturing Process in Application Form of Biotechnological/Biological Products

1

III. Module 2.3 Mock-Up for Antibody Drugs (Examples)

TABLE OF CONTENTS

2.3. INTRODUCTION ............................................................................................................... 3

2.3.S DRUG SUBSTANCE (Hutumumab, HS Pharmaceutical) ........................................... 4

2.3.S.1 General Information (Hutumumab, HS Pharmaceutical) ............................................. 4

2.3.S.1.1 Nomenclature ..................................................................................................................... 4

2.3.S.1.2 Structure ............................................................................................................................. 5

2.3.S.1.3 General Properties ............................................................................................................ 7

2.3.S.2 Manufacture (Hutumumab, HS Pharmaceutical) .......................................................... 8

2.3.S.2.1 Manufactures ..................................................................................................................... 8

2.3.S.2.2 Description of Manufacturing Process and Process Controls .................................. 10

2.3.S.2.3 Control of Materials ......................................................................................................... 23

2.3.S.2.4 Controls of Critical Steps and Intermediates ............................................................... 35

2.3.S.2.5 Process Validation and/or Evaluation ........................................................................... 37

2.3.S.2.6 Manufacturing Process Development .......................................................................... 45

2.3.S.3 Characterisation (Hutumumab, HS Pharmaceutical) ................................................. 52

2.3.S.3.1 Elucidation of Structure and other Characteristics ..................................................... 52

2.3.S.3.2 Impurities .......................................................................................................................... 74

2.3.S.4 Control of Drug Substance (Hutumumab, HS Pharmaceutical) ............................... 75

2.3.S.4.1 Specification ..................................................................................................................... 75

2.3.S.4.2 Analytical Procedures ..................................................................................................... 77

2.3.S.4.3 Validation of Analytical Procedures .............................................................................. 86

2.3.S.4.4 Batch Analyses ................................................................................................................ 93

2.3.S.4.5 Justification of Specification........................................................................................... 97

2.3.S.5 Reference Standards or Materials (Hutumumab, HS Pharmaceutical) ................ 102

2.3.S.6 Container Closure System (Hutumumab, HS Pharmaceutical) ............................. 114

2.3.S.7 Stability (Hutumumab, HS Pharmaceutical) .............................................................. 115

2.3.S.7.1 Stability Summary and Conclusions ........................................................................... 115

2.3.S.7.2 Post-approval Stability Protocol and Stability Commitment .................................... 116

2.3.S.7.3 Stability Data .................................................................................................................. 117

2.3.P DRUG PRODUCT (Koutaiyakuhin Intravenous Infusion 100 mg) ......................... 120

2.3.P.1 Description and Composition of the Drug Product (Koutaiyakuhin Intravenous

Infusion 100 mg) ................................................................................................................................... 120

2.3.P.2 Pharmaceutical Development (Koutaiyakuhin Intravenous Infusion 100 mg) ..... 122

2.3.P.2.1 Components of the Drug Product ............................................................................... 122

2

2.3.P.2.2 Drug Product .................................................................................................................. 122

2.3.P.2.3 Manufacturing Process Development ........................................................................ 122

2.3.P.2.4 Container Closure System ........................................................................................... 122

2.3.P.2.5 Microbiological Attributes ............................................................................................. 122

2.3.P.2.6 Compatibility ................................................................................................................... 122

2.3.P.3 Manufacture (Koutaiyakuhin Injection 100 mg) ........................................................ 123

2.3.P.3.1 Manufacturer(s) ............................................................................................................. 123

2.3.P.3.2 Batch Formula................................................................................................................ 124

2.3.P.3.3 Description of Manufacturing Process and Process Controls ................................ 125

2.3.P.3.4 Controls of Critical Steps and Intermediates ............................................................. 130

2.3.P.3.5 Process Validation and/or Evaluation ......................................................................... 130

2.3.P.4 Control of Excipients (Koutaiyakuhin Intravenous Infusion 100 mg) ..................... 131

2.3.P.5 Control of Drug Product (Koutaiyakuhin Intravenous Infusion 100 mg) ................ 132

2.3.P.6 Reference Standards or Materials (Koutaiyakuhin Injection 100 mg) ................... 132

2.3.P.7 Container Closure System (Koutaiyakuhin Intravenous Infusion 100 mg) ........... 132

2.3.P.8 Stability (Koutaiyakuhin Intravenous Infusion 100 mg) ........................................... 132

2.3.A Appendices ..................................................................................................................... 133

2.3.A.1 Facilities and Facilities (Koutaiyakuhin Injection 100 mg) ....................................... 133

2.3.A.2 Adventitious Agents Safety Evaluation (Hutumumab) ............................................. 134

2.3.A.3 Excipients ....................................................................................................................... 139

3

2.3. INTRODUCTION The brand name, non-proprietary name of the drug substance, company name, dosage form, strength, and

route of administration of the drug to be applied are shown in the following table.

Brand name KOUTAI IYAKUHIN INJECTION 100mg

Non-proprietary name Hutumumab (Genetical Recombination)

Manufacturer of drug substance HS Pharmaceutical Co., Ltd.

Manufacturer of drug product HS Pharmaceutical Co., Ltd.

Dosages forms Injection

Content 100 mg/2.0 mL

Route of the administration Intravenous injection

Explanation

This mock-up does not include “Proposed Indications" which is mentioned in the 2.3 Introduction of the

ICH M4 (CTD) Guideline as an example, because it is not CMC related information.

4

2.3.S DRUG SUBSTANCE (Hutumumab, HS Pharmaceutical) 2.3.S.1 General Information (Hutumumab, HS Pharmaceutical)

2.3.S.1.1 Nomenclature Note: This is an example in the case of human antibodies.

1. International Nonproprietary Name (INN)

hutumumab

2. Japanese Accepted Names (JAN)

Nonproprietary name

Japanese names

ヒューツムマブ（遺伝子組換え）

English name

hutumumab (Genetical Recombination)

Chemical name or essence

(Japanese names)

ヒューツムマブは，ヒト HS抗原に対する遺伝子組換えヒト IgG1 モノクローナル抗体である。

ヒューツムマブは，チャイニーズハムスター卵巣細胞により産生される。ヒューツムマブは，

459 個のアミノ酸残基からなる H 鎖（γ1 鎖）2 本及び 214 個のアミノ酸残基からなる L 鎖

（κ鎖）2 本で構成される糖タンパク質（分子量：約 155,000）である。

(English name)

Hutumumab is a recombinant human monoclonal IgG1 antibody against human HS antigen.

Hutumumab is produced in Chinese hamster ovary cells. Hutumumab is a glycoprotein (molecular

weight: ca. 155,000) composed of two H-chains (γ1-chains) consisting of 459 amino acid residues

each and two L-chains (κ-chains) consisting of 214 amino acid residues each.

3. Company or laboratory code

HS-2014, rMAb xxx

4. CAS registry number

XXXXXXXX

Explanation

This mock-up exemplifies the case of approximate molecular weights such as 155,000, even though

JAN Notification (No. 0313001 of the Evaluation and Licensing Division, Pharmaceutical and Food

Safety Bureau (PFSB) dated March 13, 2009, "Administrative Procedures of Applying for Japanese

Accepted Names of Pharmaceuticals") shows an example as a range of molecular weight.

5

2.3.S.1.2 Structure

1. Chemical structure

Hutumumab is a recombinant human IgG1 monoclonal antibody composed of two H-chains (γ1

chains) consisting of 459 amino acid residues and two L-chains (κ chains) consisting of 214 amino

acid residues. It has the binding position of the N-linked sugar chain at 309 position Asn of the H-

chain. The amino acid sequences of the L-chain and H-chain of hutumumab are shown in Figure

2.3.S.1.2-1, and the major carbohydrate structures are shown in Figure 2.3.S.1.2-2.

Figure 2.3.S.1.2-1 Amino acid sequence of hutumumab

Heavy chain

EVQLVESGGG LVQPGGSLRL SCAASGFTFT SYWMSWVRQA PGKGLEWVAN

IKQEGSEKTY VDATKGRFTI TRDNAKNSLY LQMNSLRAED TAVYYCAREF

ESTMTSVNAD YYYFYMDVWG KGTTVTVSSA STKGPSVFPL APSSKSTSGG

TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV

PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPELLGGP

SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK

TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK

AKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE

NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ

KSLSLSPGK

6

Figure 2.3.S.1.2-1 Amino acid sequence of hutumumab (continued)

Light chain

ELGMTQSPSS VSASVGDRVT ITCRASHSIS TYLNWYQQKP GKAPKLLIYA

ASSLQSGVPS RFSGSGSGTD FSLTINSLQP EDFATYYCQQ TFSPSGTFGQ

GTKVELKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV

DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK LYACEVTHQG

LSSPVTKSFN RGEC

Heavy chain E1, partial pyroglutamic acid; heavy chain N309, glycosylation site; heavy chain K459, partial

processing

Light chain C214-heavy chain C232, heavy chain C238-heavy chain C238, and heavy chain C241-heavy

chain C241: disulfide bond

Figure 2.3.S.1.2-2 Major carbohydrate structure of hutumumab

(β1-4)GlcNAc(β1-2)Man(α1-6) Fuc(α1-6)

Gal0-2 Man(β1-4)GlcNAc(β1-4)GlcNAc

(β1-4)GlcNAc(β1-2)Man(α1-3)

Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; Man, mannose

2. Molecular Weight and Formula

C6536H10096N1736O2054S48:147,395.62 (protein part, four-stranded)

H-Chain C2251H3472N594O693S18:50,520.39

L-Chain C1017H1580N274O334S6:23, 181.45

Explanation The amino acid sequence described in this mock-up was used as is the amino acid sequence of humanized antibodies in JAN Notification (No. 0313001 of the Evaluation and Licensing Division, Pharmaceutical and Food Safety Bureau, dated March 13, 2009).

7

2.3.S.1.3 General Properties General characteristics of hutumumab are shown in Table 2.3.S.1.3-1.

Table 2.3.S.1.3-1 General characteristics of hutumumab

Characteristic Hutumumab

Structure See 2.3.S.1.2

Molecular-mass Approximately 150,000 Da

Absorption coefficient E (1%, 1cm) in 280 nm is 13.8.

Isoelectric point Approximately 7.4

Biological activity Hutumumab indicated CDC activity in functional evaluation using

the ● cancer cell line XX-XX expressing HS antigen.

Explanation

Since some contents in 2.3.S.1.2 and 2.3.S.1.3 in the original mock-up (2002) were duplicate, the

2.3.S.1.3 of this revised mock-up were summarized.

8

2.3.S.2 Manufacture (Hutumumab, HS Pharmaceutical)

2.3.S.2.1 Manufactures Note:

・ When the product is manufactured by two or more companies, their responsibilities should be

described.

・ External testing laboratories conducting specification tests and critical in-process control tests

should be included in this section.

HS Pharmaceutical Co., Ltd. (Culture, Purification, Storage, Testing)

2-22-2, Iwamoto town, Chiyoda ward, Tokyo, Japan

IW Analysis Center (External testing laboratory: Mycoplasma test)

1-2-3, ● town, ● prefecture, Japan

Explanation

S2.1 Manufacturer

It is recommended that the name of the manufacturer is the same written in the manufacturing

license (foreign manufacturer accreditation). The address of the head office is not required to be

described.

The storage facility for cell banks is generally considered to be the same as the manufacturing

facility for drug substance and are not described separately in this mock-up. The facility for cell

banks renewal is generally determined before the renewal in future, is not described in this mock-

up.

It is not mandatory to include the information on split storage facilities for MCBs and WCBs in this

section, because the "split storage of MCBs and WCBs in remote facilities" exemplified in the ICH

Q5D is an internal quality system.

Explanation for M1.2.

MCB and WCB renewal facilities might not be determined at the initial submission. However,

PMDA sometimes require description of MCB and WCB renewal facilities in M1.2 and each

company variously responds to these requirements according to their policy. As of such a case, if

future MCB/WCB renewal facilities are not determined, it is an example to state that the

MCB/WCB renewal facilities are "to be determined" or "no plan for renewal" in M1.2.

When a specification test is performed at external testing laboratories, the laboratories should be

described in this section.

9

Regarding in-process control test, it is not necessary to list all test items in M1.2, and the critical in-

process control tests (e.g., Mycoplasma test) should be listed in M1.2. For in-process control tests

described in M1.2, the applicant should decide whether the external testing laboratories are listed in

M1.2 or not.

10

2.3.S.2.2 Description of Manufacturing Process and Process Controls Note:

The description in this section is different among companies, because the manufacturing process

and process control of each antibody drug is different among companies.

In the manufacturing flow chart shown below, the "in-process control test" describes the in-process

control test controlled by the acceptance criteria or action limit. In addition, monitoring without

these limits are included in the text section (Sections 3 and 4). The choice of in-process control tests

described in this section (S.2.2) may depend on manufacturer.

This section is described based on the Traditional approach (not the Enhanced approach in the ICH

Q11 guideline).

11

1. Manufacturing flow chart

1) Cell culture and harvest Medium Process In-process control test

WCB thawing ↓

Medium A

Cell Culture Step 1 (250 mL flasks 1 L spinner flask 5 L spinner flask 20 L plastic bag 250 L fermenter).

↓

Medium B Cell Culture Step 2 (1000 L bioreactor).

↓

Medium C Medium D

Cell Culture Step 3 (5000 L bioreactor).

Unprocessed Bulk : Mycoplasma Bioburden Adventitious viruses Hutumumab concentration

↓ Harvest

(cell separation by filtration) Hutumumab concentration

↓ Purification process

Critical process

12

2) Purification process

Process In-process control test Culture supernatant fluid ↓ Purification 1

(Protein A column)

↓ Low pH treatment

(Virus inactivation)

↓ Purification 2

(Cation-exchange column)

↓ Purification 3

(Anion-exchange column)

↓ Concentration and Buffer

Exchange 1

↓ Purification 4

(Hydrophobic column).

↓ Virus removal filtration ↓ Concentration and Buffer

Exchange 2 Host cell proteins

↓ Conditioning ↓

Specification testing Filtration, filling and storage

Critical process

Explanation (Manufacturing Flow Chart).

・ The manufacturing process is described in the flow chart and text. The order of the flow chart and

text (which culture and purification process to write first, whether culture and purification process

are integrated or separated, etc.) should be determined by the applicant. In addition, there are many

options such as the relationship between the flow chart and the text, e.g., 1) explaining outline the

manufacturing process shown in the flow chart by text 2) using the flow chart of the manufacturing

process with almost the same content, and 3) describing the details in the flow chart and important

content with the text, and the applicant may decide. In this mock-up, the figure shown in 1) is

exemplified, but another one (see page 21-22) can be described as an example of the flow chart

shown in 2).

13

2. Batch and scale definition

See 3.2.S.2.2 for a detailed description of batch definition and batch numbering system.

Explanation (Batch and Scale)

In this mock-up, batch definitions and batch numbering systems are provided in the Module 3, and is

omitted in Module 2.3.

At the time of submission, the batch numbering system for commercial drug substance may not be

determined. In such cases, it should be stated as “to be determined”.

3. Cell culture and harvest

Note:

Parentheses for Partial Change Application/Minor Change Notification (e.g. ≪≫, 『』, “ ”)

are not described in this mock-up, because they are required only in the application form (M1.2)

and not required in the CTD (M2.3, M3)

The process parameters in the cell culture process are considered to differ among products and

manufacturers, except for those required by ICH guidelines and notifications (e.g., in vitro cell age

limit for drug substance manufacturing). Therefore, the process parameter items and their values in

this mock-up are only descriptive examples and are not recommended items or values (e.g., culture

period).

In this mock-up, the information on the composition of the cell culture media is provided in S.2.2,

but there are cases described in S.2.3.

1) Cell culture processes

The composition of the media used in cell culture is shown in Table 2.3.S.2.2-1.

① Cell Culture Step 1

After thawing one or more frozen WCB vial(s), add the cells to 250 mL flasks containing

medium A (Table 2.3.S.2.2-1), and culture at 37°C for 2 - 4 days. Add the culture fluid to 250 mL

flasks containing new medium A, and repeat this culture step at 37°C for 2 - 4 days.

The culture fluid in 250 mL flasks are added to 1 L spinner flasks containing medium A and

culture at 37°C for 2 - 4 days. The culture fluid is expanded sequentially using 1 L spinner flasks,

5 L spinner flasks, 20 L plastic bags, and 250 L fermenters (culture temperature: 37°C, culture

period: 2-4 days).

In the cell culture step 1, cell viability and cell density are monitored using the culture sample.

② Cell Culture Step 2

14

When the cell density in the 250L fermenter exceeds ●×106 cells/mL or more, add the

culture fluid to a 1000 L bioreactor containing approximately 900 L of medium B (Table

2.3.S.2.2-1) to adjust the initial cell density approximately ●×106 cells/mL and culture at 37 ±

X°C and ●% of dissolved O2 (set points) for 2 - 4 days. Add CO2 gas or sodium hydroxide

solution as needed so that the pH of the culture medium is around 7.

In the cell culture step 2, cell viability and cell density are monitored using the culture sample.

③ Cell Culture Step 3 [Critical Process]

When the cell density in the 1000L fermenter exceeds ● × 106 cells/mL or more, add the

culture fluid to a 5000L bioreactor containing approximately 4500L of medium C (Table

2.3.S.2.2-1) to adjust the initial cell density approximately ● × 106 cells/mL and culture at 37 ±

2°C and ●% of dissolved O2 (set points) for 8 - 12 days.

Add CO2 gas or sodium hydroxide solution as needed to adjust the pH of the culture fluid to

approximately 7. Add medium D (Table 2.3.S.2.2-1) as necessary after ● day of culture. When

the amount of hutumumab in the culture fluid is more than X.X mg/mL, the process shifts to the

harvest process.

The limit of in vitro cell age for drug manufacturing is ● days from MCB thawing.

In the cell culture step 3, the following in-process control tests are performed using the culture

sample (unprocessed bulk) before harvest.

(1) Mycoplasma shall be tested according to the "Mycoplasma Test (culture method, DNA

staining method)" (acceptance criteria: negative).

(2) Bioburden: Microbial Limit Test (action limit: less than 10 CFU/10 mL)

(3) Adventitious viral test: in vitro test (acceptance criteria: not detected)

(4) Hutumumab concentration: Hutumumab concentration is measured by affinity

chromatography (action limit: X.X mg/mL or more)

15

Table2.3.S.2.2- 11 Composition of media used in the cell culture process of hutumumab drug substance

Medium name Component Concentration

Medium A Basal medium AB1)

Recombinant insulin

Methotrexate

・・・

・・・

■■ g/L

■■ μg/L

■■ mmol/L

■■ g/L

■■ g/L

Medium B Basal medium AB1)

Recombinant insulin

・・・

・・・

■■ g/L

■■ μg/L

■■ g/L

■■ g/L

Medium C Basal medium CCC2)

Recombinant insulin

Porcine peptone

・・・

・・・

■■ g/L

■■ μg/L

■■ mg/L

■■ mg/L

■■ mg/L

Medium D Basal medium DDD3)

・・・

・・・

■■ g/L

■■ mg/L

■■ mg/L 1)2)3) See 2.3.S.2.3 for composition of basal medium

2) Harvest process

After completion of the cell culture step 3, harvest the whole culture fluid, remove the cells by

filtration (depth filtration), and obtain the culture supernatant through filtration of ● μm pore size

filter

In-process control test: Hutumumab concentration is measured by affinity chromatography using

culture supernatant as a sample. (Action limit: X.X mg/mL or more)

Explanation

3. Cell Culture

・ The terms of “cell culture steps 1, 2, 3” are used in the description of the cell culture, but do

not specifically require the use of these terms. For example, the applicant may use the terms

such as “seed culture, inoculum culture, production culture”, or “pre-culture and main culture”.

The same applies to the name of the equipment, for example, the applicant may use the terms

such as the culture tank, the culture equipment, the bioreactor, and the fermenter, for the

equipment to be subjected to cell culture.

16

・ The primary objective is to demonstrate the scale of the equipment, etc., and the scale

determined by the applicant should be described. The applicant should determine and describe

the scale of the cell culture vessel as nominal volume or the maximum scale available, or the

maximum space including the unusable part. In case of scale for cell culture fluid, the

applicant should determine to describe an approximate amount of standard amount of cell

culture fluid.)

・ In this mock-up, as the cell culture scale, the cell culture step 1 describes only the culture

vessel scale. The cell culture step 2 and 3 describes both the culture vessel scale and the cell

culture volume as examples.

・ Description of process parameter values is considered case-by-case, including description at

the upper and lower limits (e.g., 39°C) and target/set values (e.g., 37°C), in addition to the

range description (e.g., 37±2°C, 35°C-39°C). (There may be cases where the method of

describing the process parameter values differs between M2.3 and M1.2 of the application

form.)

・ This mock-up described both cases a set point and a range at the cultivation temperature. The

former is described as a set point for equipment, and the latter is an example of control range

of an equipment.

・ There is a flexibility of using terminology for description of manufacturing process of cell

culture process as well as other process. However, terms used in official documents such as

pharmacopoeia and ICH guidelines may be required or desirable to be used.

・ As in-process control tests for unprocessed bulk, mycoplasma, bioburden (viable count test),

and adventitious virus test were set as examples. In this mock-up, it was shown that the

adventitious virus test are tested for lot-to-lot of unprocessed bulk. However, as described in

the ICHQ5A as “It is recommended that manufacturers develop programs for the ongoing

assessment of adventitious viruses in production batches”, the scope, extent, and frequency of

testing (periodic testing) for adventitious viruses in unprocessed bulk are determined by the

applicant.

・ A combination of culture methods and DNA staining methods is exemplified as a mycoplasma

test for unprocessed bulk, but the NAT method can be used instead (see the Japanese

Pharmacopoeia Reference Information).

4. Purification process

The composition of the buffers used in the purification steps are shown in Table 2.3.S.2.2-2.

1) Purification 1 (Protein A column)

The culture supernatant is loaded on a Protein A affinity column (bed height: 25 cm, Resin: [brand

name] or equivalent) equilibrated with phosphate buffer 1 with ≤ 30 g hutumumab /L resin.

17

After washing with phosphate buffer 1, hutumumab is eluted with acetate buffer 1 (linear velocity

150 cm per hour). Obtain a fraction of OD280nm = X. X or higher.

In the Purification 1, yield and bioburden are monitored.

2) Low pH treatment (virus inactivation) [critical process]

Adjust pH of the process solution from Purification 1 with 5 mol/L acetic acid to not more than pH

3.6, and hold at least 15°C for not less than 60 minutes. Adjust pH of the solution to pH 5.0 with ●

mol/L sodium hydroxide, and filter it through a depth filter.

3) Purification 2 (cation exchange column)

The process solution obtained in the low-pH treatment step is divided into two or three fractions,

and are loaded on cation column (bed height: 25 cm, Resin: [brand name] or equivalent) equilibrated

with acetate buffer 2 at a linear velocity 150 cm/h with ≤ 30 g hutumumab/L resin. After washing with

5 times the column volume of acetate buffer 2, acetate buffer 3 serves as the initial solution and elutes

hutumumab in a linear gradient until acetate buffer 4 concentrations are 80% (linear velocity 150

cm/h). The collection starts from OD280nm = X.X or more, and the fractionation is terminated when

OD280nm = Y.Y or less. The above operation is repeated depending on the number of divisions.


4) Purification 3 (anion exchange column)

The process solution from Purification 2 is passed through an anion-exchange column (bed height:

25 cm, ([brand name] or equivalent) equilibrated with phosphate buffer 2 with ≤ 150 g hutumumab/L

resin, followed by passage through phosphate buffer 2 (linear velocity: 150 cm/h) to obtain a fraction

with OD280nm = X.X or higher.


5) Concentration and Buffer Exchange 1

The step solution of purification 3 is concentrated to 1/10 with an ultrafiltration membrane

(molecular cut-off: approximately 30,000) followed by buffer exchange with a 5-fold volume of Tris

buffer 1 of the concentrate.

In this process, the yield is monitored.

6) Purification 4 (hydrophobic column)

The process solution from Concentration and Buffer Exchange 1 is loaded on a hydrophobic

column (bed height: 30 cm, [trade name] or equivalent) equilibrated with Tris buffer 1 with ≤ 20 g

hutumumab/L resin. After washing with Tris buffer 2, hutumumab is eluted with Tris buffer 3 (linear

velocity 100 cm/h). Obtain a fraction of OD280nm = X. X or higher.


18

7) Virus removal filter filtration [critical process]

Filter the process solution of purification 4 through a virus-removal filter ([trade name] or equivalent)

(loading volume ≤ 300 L/m2 , ≤ ●MPa ) and wash with Tris buffer 4. Confirm the integrity of the virus-

removal filter. The filtrate can be kept at 5° C. for ● hours, if necessary.

In this process, the yield is monitored.

8) Concentration and Buffer Exchange 2

Concentrate the viral removal filter filtrate to 1/5 with an ultrafiltration membrane (molecular cut-

off: approximately 30,000) and exchange and concentrate with phosphate buffer 2 (the amount of which

is ● times of the concentrate), to make hutumumab concentration as approximately 100 mg/mL.

In-process control test:

1. Determine the content of host cell proteins (HCPs) in the process solution by the ELISA

method (action limit: ≤ ● ng/mg).

Yield is monitored in this process.

9) Conditioning

Add a 10% polysorbate 80 solution to the process solution of Concentration and Buffer Exchange 2

to make the concentration of polysorbate 80 as 0.5%. Make pH 6.3 with ●mol/L sodium hydroxide

solution.

10) Filtration, filling and storage

Filter (0.2 μm pore size) the process solution in the conditioning step and fill it in 1 L plastic

containers. Store at ‒20°C.

19

Table2.3.S.2.2- 22 Composition of buffers used in the purification process of Hutumumab drug substance

Name Composition

Phosphate buffer 1 20 mmol /L phosphate buffer, pH 7.5

Acetate buffer 1 100 mmol /L sodium acetate buffer, pH 3.6

Acetate buffer 2 50 mmol /L sodium acetate buffer, pH 5.0

Acetate buffer 3 10 mmol/L sodium chloride,

50 mmol /L sodium acetate buffer, pH 5.0

Acetate buffer 4 1 mol/L sodium chloride,

50 mmol /L sodium acetate buffer, pH 5.0

Phosphate buffer 2 30 mmol /L phosphate buffer, pH 6.5

Tris buffer 1 1.8 mol /L ammonium sulfate, 20 mmol /L Tris hydrochloride buffer, pH 7.5

Tris buffer 2 1.2 mol /L ammonium sulfate, 20 mmol /L Tris hydrochloride buffer, pH 7.5

Tris buffer 3 ◆◆ mmol/L sodium chloride, 20 mmol/L Tris hydrochloride buffer, pH 7.5

Tris buffer 4 ◆◆ mmol/L Tris hydrochloride buffer, pH 7.5

5. Reprocessing

If virus removal filter filtration fails to confirm the integrity of the filter, re-filtration may be

conducted up to 3 times at maximum.

Explanation

4. Purification

・ In this mock-up, the type of chromatographic resin was indicated as “[brand name] or

equivalent”. Another method of description can be a combination of functional groups and

substrates (e.g., sepharose resin bounded with protein A).

・ The type of virus removal filter was indicated as “[brand name] or equivalent”, and its pore

size was not indicated. This is because the pore size value has not been published in some

cases by the supplier or the function of filter. When the pore size information is publicly

available, only the pore size (and not describing the brand name) can be described.

・ The height of a chromatographic column may be the average bed height or the column height,

as determined by the applicant.

・ The load amount on the chromatographic columns is described as "Hutumumab is ≤ ●g/L

resin", and it means that the load amount is the weight of protein containing impurities other

than hutumumab.

・ Collection conditions in the chromatographic step were described as examples using absorbance

(OD). As other collection conditions, there may be column volume (CV) and so on as indices.

20

There may also be chromatographic steps where collection conditions are not important for

product quality.

・ Purification 3, Anion exchange chromatography: is a description of the flow-through mode (not

the binding-elution mode).

・ As description for the process parameters of low pH treatment (virus inactivation), this mock-

up shows an example of the worst case (upper or lower limit). Alternatively, the target/set point

specified in the Product Formula may be included in the submission.

・ Splitting and purifying a single process pool may not be described because of a scope of GMP.

This mock was exemplified by describing the number of divisions of process pool.

・ Whether the direction of the inequality mark of the action limit in the in-process control test is

set to the acceptable range side or the required range side of the action should be in accordance

with the policy of each manufacturer.

Explanation for M1.2

・ In-process control test

In this mock-up, not all in-process control tests described in M2.3 are shown in M1.2. The applicant

determined in-process control tests which are important from the viewpoint of quality control

among in-process control tests with acceptance criteria and action limit:

- Mycoplasma test and adventitious virus test in Cell Culture Step 3

- Bioburden in Cell Culture Step 3

Hutumumab concentration in Cell Culture Step 3 is "in-process control test with action limit" but it

is an example where the applicant determined not to be listed in M1.2, because it is an in-house

control test.

・ Hold of process solution (virus removal filtration process)

Usually, the hold conditions for in-process solution were exemplified when the applicant

determined not to be listed in M1.2 because of in-house GMP control.

21

Diagram for explanation

Figure Cell Culture and Harvest

Manufacturing process Operating conditions Medium In-process control test

WCB thawing ↓

Cell Culture Step 1 (250 mL flasks 1 L spinner flask 5 L spinner flask 20 L plastic bag 250 L Fermenter).

Temperature: 37°C Culture period: 2-4 days

Medium A

↓


Temperature: 37 ± X °C Dissolved O2: ●% Culture period: 2-4 days

Medium B

↓


Temperature: 37 ± 2 °C Dissolved O2: ●% Culture period: 8-12 days

Medium C Medium D

Unprocessed Bulk : Mycoplasma Bioburden Adventitious viruses Hutumumab concentration

↓

Harvest (cell separation by filtration)

Culture supernatant fluid : Hutumumab concentration

↓ Purification process

Critical process

22

Figure purification process

Manufacturing process Operating conditions In-process control test

Culture supernatant fluid

↓

Purification 1 (Protein A column)

Bed height; 25 cm: Maximum load:30 g/L Linear velocity (elution): 150 cm /h Fraction, according to absorbance (280 nm)

↓

Low pH treatment (Virus inactivation)

pH: ≤ 3.6 Temperature: ≥ 15°C Time: ≥ 60 minutes Neutralizing pH after inactivation: 5.0

↓

Purification 2 (Cation-exchange column)

Bed height; 25 cm: Maximum load:30 g/L Linear velocity (elution): 150 cm /h Fraction, according to absorbance (280 nm)

↓

Purification 3 (Anion-exchange column)

Bed height: 25 cm Maximum load:150 g/L Linear velocity (elution): 150 cm /h Fraction, according to absorbance (280 nm)

↓ Concentration and Buffer

Exchange 1 Molecular cut-off: about 30,000

↓

Purification 4 (Hydrophobic column).

Bed height; 30 cm: Maximum load:20 g/L Linear velocity (elution): 100 cm /h Fraction, according to absorbance (280 nm).

↓ Virus removal filter

filtration Maximum load: 300 L/m2

↓

Concentration and Buffer Exchange 2

Molecular cut-off: about 30,000 Final Hutumumab Concentration: Approximately 100 mg/mL

Host cell proteins

↓

Conditioning Polysorbate 80 concentration: 0.5% pH: 6.3

↓

Filtration, filling and storage Filter pore size: 0.2 μm Plastic containers Storage temperature : ‒20°C

Critical process

23

2.3.S.2.3 Control of Materials 1. Raw materials used in the production of drug substance

Note:

・ Information on media and buffer composition should be provided by the applicant in S.2.2 or S.2.3.

・ Master files can also be used as an information presentation method for basal medium composition.

Raw materials used in the manufacturing process of hutumumab drug substance after confirming that

they meet acceptance standards. Table 2.3.S.2.3-1 shows the raw materials used to produce the drug

substance.

Table2.3.S.2.3- 11 Raw materials used in the production of drug substance

Raw materials Process used Grade

Basal medium AB Cell culture process Supplier standard

Basal medium CCC Cell culture process Supplier standard

Basal medium DDD Cell culture process Supplier standard

Recombinant human insulin Cell culture process Supplier standard

Methotrexate Cell culture process Supplier standard

Porcine peptone Cell culture process Supplier standard

・・・・・・・・・

Sodium acetate Purification process Supplier standard

Acetic Acid Purification process Supplier standard

Sodium chloride Purification process Supplier standard

Hydroxymethyl-aminomethane Purification process Supplier standard

Ammonium sulfate Purification process Supplier standard

Purified Water Cell culture and purification processes Internal standards

Dibasic Sodium Phosphate Hydrate Purification process Japanese Pharmacopoeia

Sodium dihydrogen phosphate Purification process Japanese Pharmaceutical

Excipients

Polysorbate 80 Purification process (Conditioning

step)

Japanese Pharmacopoeia

Sodium Hydroxide Purification process (Conditioning

step)


Water for injection Purification process (Conditioning

step)


Table 2.3.S.2.3-2 to Table 2.3.S.2.3-4 shows the composition of the basal medium used in the cell culture

process.

24

Table2.3.S.2.3- 22 Composition of basal medium AB

Ingredient Concentration

L-asparagine

・・・

・・・

■■ g

・・・

・・・

(Composition contained when dissolving XX g of basal medium AB in 1 L of water).

Table2.3.S.2.3- 33 Composition of basal medium CCC


L-leucine

・・・

・・・

■■ g

・・・

・・・

(Composition contained when dissolving XX g of basal medium CCC in 1 L of water).

Table2.3.S.2.3- 44 Composition of basal medium DDD.


Glucose

・・・

・・・

■■ g

・・・

・・・

(Composition contained when dissolving XX g of basal medium DDD in 1 L of water).

Explanation

Medium Composition: Information on the composition (ingredient and concentration) of the basal

medium is a Japan-specific requirement. Information on the composition of the basal medium should

be provided in the regulatory dossier, otherwise the system of the master file is available.

Raw material: When raw materials with different hydrates (not listed in the JP but in USP/NF, EP)

are used, they can be described as "USP/EP" in this section. (e.g., sodium phosphate, dibasic,

heptahydrate)

25

2. Control of raw materials of biological origin

Note:

Although information on the host cells used in cell bank preparation are not described in this section

(section 2) but in Section 3. Information on raw materials derived from humans or animals used for

cell bank preparation except host cell are described in this section.

Humans or animal origin materials among raw material used in the manufacturing process of

hutumumab drug substance except for cell bank preparation are as follows. The following raw materials

of biological origin are considered to conform to the Standards for Biological Materials (enacted on

September 26, 2014, MHLW Notification No. 375).

Porcine peptone

Porcine peptone is derived from healthy pigs, and its manufacturing process involves heating at

●°C for more than ● min. This treatment condition is believed to be sufficient to inactivate

adventitious agents (such as viruses).

Explanation

Refer to the Standards for Biological Materials (Established on September 26, 2014, Notification No.

375, MHLW) and "Operation of the Standards for Biological Materials" (Notification No. 1002-1 of the

Evaluation and Licensing Division, PFSB, Notification No. 1002-5 of the Evaluation and Licensing

Division, Pharmaceutical and Food Safety Bureau, MHLW, dated October 2, 2014).

EMA-guidance on inactivation conditions for TSEs “Minimising the risk of transmitting animal

spongiform encephalopathy agents via human and veterinary medicinal products"(EMEA/410/01 Rev. 3,

2011) can also be referenced.

26

3. Source, history, and generation of the cell substrate

1) Expression Construct

A transgenic mouse which is deficient mouse antibody gene and is introduced human antibody

gene was immunized with human HS antigen, and the hybridoma was produced by fusing its spleen

cell with mouse myeloma cell. cDNA fragments encoding the heavy and light chains of IgG1 against

human HS antigens were generated from the mRNA of hybridomas.

Figure 2.3.S.2.3-1 shows the structure and construction flow chart of the expression vector §§§.

The cDNA fragments encoding the heavy chain of hutumumab were ligate with plasmid ●

digested by [restriction enzyme A] and [restriction enzyme B] to generate plasmid §. Separately, the

cDNA fragments encoding the light chain of hutumumab are ligate with plasmid ■ digested by

[restriction enzyme C] and [restriction enzyme D] to generate plasmid §§. Then, plasmid § and

plasmid §§ were digested with [restriction enzyme C] and [restriction enzyme D], respectively, and

DNA fragments containing encoding light chains and heavy chains were ligated to generate

expression vector §§§.

The DNA sequences of encoding the heavy and light chains of hutumumab are shown in Figure

2.3.S.2.3-2 and Figure 2.3.S.2.3-3, respectively.

Figure 2.3.S.2.3-1 Structure and construction flowchart of gene expression vector.

Explanation

DHFR (Dihydrofolate reductase): WWW is supposed to indicate the name of the animal species.

27

Figure 2.3.S.2.3-2 Hutumumab heavy chain encoding DNA sequence

1001 1100

xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx

X X X X X X X X X X X X X X

1101 1200

xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx

X X X X X X X X X X E V Q L V E S G G G L V Q P G G S L R L S C A

(hereafter abbreviated)

Figure 2.3.S.2.3-3 Hutumumab light chain encoding DNA sequence

(As in Figure 2.3.S.2.3-2)

2) Hosts cells

The host cell is CHO-XXX strain derived from Chinese hamster ovary established by §§ et al.

and obtained from ■. CHO-XXX strain lacks dihydrofolate reductase (DHFR) and has

hypoxanthine-and thymidine-requiring properties.

3) Preparation of master cell bank (MCB) (lot ZZZ)

The host cell CHO-XXX strain was introduced with the expression vector by ■ method. Next,

the genetically engineered cells were selected by culturing in medium X without ■ (containing

●% fetal bovine serum). Cultures were then performed using medium X containing methotrexate

(MTX) for gene amplification (containing ●% fetal bovine serum) and cells were selected using the

production of Hutumumab as an indicator. The cell line YYY selected here was cloned by ■

method, and the cell line ZZZ for MCB preparation was obtained and stored in suspension in a

freezing medium containing ●% fetal bovine serum and ●% dimethyl sulfoxide.

28

4. Cell banking system, characterisation, and testing

1) Preparation of Master Cell Bank (MCB) (lot ■■)

Cell line lot ZZZ for MCB preparation was cultured at 37°C using F-12 medium containing ●%

fetal bovine serum. After expansion culture, they were centrifugally concentrated, suspended in F-12

medium (medium for freezing) containing ●% fetal bovine serum and ●% dimethyl sulfoxide to

make approximately ● × 107 cells/mL cell suspension. The cell suspensions were dispensed into ●

mL plastic vials and frozen to prepare ● vials of MCB (lot ■■).

Fetal bovine serum (from the United States, Australia, and New Zealand) that met the Standards for

Biological Materials was used.

The MCB is stored in a liquid nitrogen tank.

2) Preparation of Working Cell Bank (WCB) (lot ■■)

MCBs (lot ■■) were cultured at 37°C using serum-free medium A (see Table 2.3.S.2.2-1). After

expansion culture, they were centrifugally concentrated and suspended in serum-free medium A

(medium for freezing) containing ■% dimethyl sulfoxide to make approximately ● × 107 cells/mL

cell suspension. The cell suspensions were dispensed into ● mL plastic vials and frozen to prepare

● vials of WCB (lot ■■).

The WCB is stored in a liquid nitrogen tank.

3) Characterization and Purity Tests of Cell Banks

① MCB

Table 2.3.S.2.3-5 and Table 2.3.S.2.3-6 show the results of the characterization and purity tests of

MCB (lot ■■).

Table 2.3.S.2.3-5 Characterization test results of MCB lot ■■

Test Items/analytical methods Test method Results

DNA sequence Confirm the sequence of the

Hutumumab gene with the

nucleic acid amplifications

The sequence of the expected

Hutumumab gene was

confirmed

Confirmation of

insertion/deletion of DNA

Southern blot analysis of DNA

digestion patterns with

restriction enzyme (■)

No insertion or deletion was

observed in the Hutumumab

gene.

DNA copy number DNA copy number per cell is

determined by ■ method.

Approximately ■ copies of

insertions were confirmed per

cell.

Identification (cell origin) Evaluate electrophoretic

pattern of isozymes

The electrophoretic pattern was

same with the pattern of

hamster-derived cells

29

Table 2.3.S.2.3-6 Purity test results of MCB lot ■■

Test Items/analytical

methods

Test method Results

Sterility testing Sterility test (Japanese

Pharmacopoeia)

Negative

Mycoplasma test Mycoplasma test (reference

information for Japanese

Pharmacopoeia): Culture and DNA

staining

No mycoplasma contamination

was observed.

Virus

test

Infectivity assay Examine retroviral infectivity using

sensitive cells (mink S+L-cells).

No infectivity was observed

Electron microscopy Examine for the presence of

retroviruses and retrovirus-like

particles by electron microscopy

Viruses and virus-like particles

known to be present in CHO

cells were not detected except

for type A and type C

retrovirus-like particles

Reverse

transcriptase activity

Examine reverse transcriptase

activity

No reverse transcriptase

activity was observed.

In vitro test Examine cytopathic effect and

hemagglutination after inoculation

to indicator cells (MRC-5, Vero and

CHO cells).

No viral contamination was

observed.

In vivo test Observe the condition of animals

(adult mice, suckling mice) and

embryonated chicken eggs


observed.

Antibody Production

Tests

Examine the levels of antibodies

against the viruses (LCMs, PVMs,

Reo3, bone Sendai Virus, SV5) in

the serum after inoculating and

rearing virus-free hamsters,


observed.

Bovine virus test Examine cytopathic effect and


to indicator cells (Bovine turbinate

and Vero cells).


observed.

Porcine virus test

Examined cytopathic effect and


to indicator cells (Vero cells).


observed.

30

② WCB

Table 2.3.S.2.3-7 shows the results of the characterization and purity tests of WCB (Batch ■■).

Table 2.3.S.2.3-7 Characterization and purity test results of WCB lot ■■



pattern of isozymes


matched with the pattern of



Pharmacopoeia)

Negative



Pharmacopoeia): Culture and

DNA staining


was observed.

In vitro test Examine cytopathic effect and

hemagglutination after

inoculation to indicator cells

(MRC-5, Vero and CHO cells).


observed.

In vivo test Observe the condition of

animals (adult mice, suckling

mice) and embryonated

chicken eggs


observed.

③ Characterization and purity tests of cells at the limit of in vitro cell age used for production (CALs)

Cells were cultured from WCB (lots ■■) under the manufacturing conditions described in S.2.2

and prepared CALs after the production culture process (in vitro cell age from MCB thawing: ■

days culture period). The results of the CAL characterization and purity tests are shown in Tables

2.3.S.2.3-8 and 2.3.S.2.3-9, respectively.

31

Table 2.3.S.2.3-8 Results of CAL characterization tests


DNA sequence Confirm the sequence of the

Hutumumab gene with the

nucleic acid amplifications

The sequence of the expected

Hutumumab gene was

confirmed.

Confirmation of

insertion/deletion of DNA

Southern blot analysis of DNA

digestion patterns with

restriction enzyme (■)

No insertion or deletion was

observed in the Hutumumab

gene.

DNA copy number DNA copy number per cell is

determined by ■ method.

Approximately ■ copies of

insertions were confirmed per

cell.


pattern of isozymes


same with the pattern of


32

Table 2.3.S.2.3-9 Results of CAL purity tests

Test Items/analytical

methods

Test method Results


Pharmacopoeia)

Negative



Pharmacopoeia): Culture and

DNA staining


was observed.

Virus

test

Infectivity assay Examine retroviral infectivity

using sensitive cells (mink S+L-

cells).

No infectivity was observed.

Electron

microscopy

Examine for the presence of

retroviruses and retrovirus-like

particles by electron microscopy

Viruses and virus-like particles

were not detected excluding type

A and type C retrovirus-like

particles known to be present in

CHO cells.

Reverse

transcriptase

activity

Examine reverse transcriptase

activity

No reverse transcriptase activity

was observed.

In vitro test Examined cytopathic effect and

hemagglutination after

inoculation to indicator cells

(MRC-5, Vero and CHO cells).


observed.

In vivo test Observe the condition of

animals (adult mice, suckling

mice) and embryonated chicken

eggs


observed.

Explanation

・ In vitro viral test: This section describe examples using human-derived cells (MRC-5), primate-

derived cells (Vero) and cell lines close to the producing cells (CHOs) as indicator cells. The

applicant can use other indicator cells or other assays (e.g., virus species-specific PCR) considering

the cell line origin and the type of virus that may be contaminated.

・ In vitro viral test: ICH Q5A (Notification No. 329 of the Evaluation and Licensing Division,

Pharmaceutical and Medical Safety Bureau, dated February 22, 2000) stated that "Virus test should

be performed to determine cytopathic effect and hemagglutination." However, the underlined

33

portion is considered to be a mistranslation of "hemadsorption" based on the original ICH Q5A text.

・ Retroviral infectivity tests: Homologous (ecotropic), heterotropic (xenotropic), polytropic

(polytropic) and bitropic (amphotropic) are known as host ranges for retroviruses. In this mock-up,

the infectivity test using mink S+L-cell as a test method for detecting heterotropic and bitropic

retroviruses is shown.

・ Reverse transcriptase activity: Mn++ dependent reverse transcriptase activity may be positive by

CHO cell-derived components when testing Mg++ dependent and Mn++ dependent reverse

transcriptase activity. Therefore, when the criteria for the reverse transcriptase activity test are

described, Mn++ dependent reverse transcriptase activity may be described as "Report" rather than

"not detected".

・ Antibody production test: This mock-up shows a hamster antibody production test because CHO

cells are hosts. Other antibody production tests (e.g., mouse antibody production tests) may be used

concomitantly.

・ Porcine virus test: This mock-up exemplifies the in vitro test, but it is also possible to use other

assays (e.g., PCR-based assays) or not to perform porcine virus tests in accordance with the origin

of raw materials and the results of risk assessment.

・ Identification (cell origin): Isozyme analysis has been shown to exemplify confirmatory testing of

cell types in ICH Q5D, but problems with the delivery of reagents have led to a shift toward

alternative methods of isozyme analysis (e.g., DNA-fingerprinting, barcoding). In this mock-up, the

first MCB/WCB lot and the CAL phase were tested for isozymes, but another test method was used

for future renewal. As described in the ICH Q5D, tests for the expression of target proteins are also

available for confirmatory testing of cell banks.

4) Stability of the cell bank

① Stability during culture for drug manufacturing

The DNA sequence of the gene expression construct in CAL was examined to evaluate its

stability during the culture period for manufacturing of hutumumab drug substance, and the

expected DNA sequence was confirmed as shown in Table 2.3.S.2.3-8. Therefore, the stability of

cells was confirmed during the culture period for manufacturing of hutumumab drug substance.

② Stability during storage

Vials of MCB and WCB are thawed at the time of use or once per ● year to monitor cell

viability.

5) Renewal of cell bank.

The renewal method of MCB shall be in accordance with the preparation procedure of the MCB lot

■■ using the cell line batch ● for the production of MCB, or existing MCB (see Section 5.1). The

renewal method of WCB conforms to the preparation method of WCB lot ■■. The prepared MCB and

34

WCB can be used when the test meets the acceptance criteria shown in Table 2.3.S.2.3-10 and Table

2.3.S.2.3-11, respectively.

Table 2.3.S.2.3-10 Characterization test and purity test at the time of MCB renewal

Test Items/analytical methods Acceptance criteria

Identification (cell origin) Confirmed to be derived from hamster cells

Sterility testing Negative

Mycoplasma test Not detected

Virus

test

Infectivity assay No observation of viral contamination.

Electron microscopy Viruses and virus-like particles were not

detected excluding retrovirus-like particles

known to be present in CHO cells

Reverse transcriptase activity No observation of viral contamination.

In vitro test (indicator cells: MRC-5, Vero

and CHO-cells)

No observation of viral contamination.

In vivo test (adult mice, suckling mice and

embryonated chicken eggs)


Antibody production test (HAP) No observation of viral contamination.

Table 2.3.S.2.3-11 Characterization and Purity Tests at renewal of WCB

Test Items/analytical methods Acceptance criteria

Confirmation of hamster cells Confirmed to be derived from hamster cells

Sterility testing Negative

Mycoplasma test Not detected

Virus test In vitro test (indicator cells:

CHO cells)


In vivo test (adult mice) No observation of viral contamination.

Explanation 5) Renewal of cell bank.

The numbers of indicator cells and species used for the viral test at the time of WCB renewal was

in accordance with the ICH Q5A.

35

2.3.S.2.4 Controls of Critical Steps and Intermediates Note:

The policy of setting Critical Process may depend on manufacturer/applicant. In this mock-up, it is

shown the example that the applicant considered Critical Process are:

1. Process steps that greatly affect the quality attributes of the desired product

2. Process steps for inactivating/removing viruses

This section is based on the Traditional approach and not the Enhanced approach described in the

ICH Q11 guideline.

1. Critical Process

1) Cell Culture Step 3

Since it is a cell culture step to produce hutumumab, it was set as a critical process.

2) Low pH treatment (virus inactivation)

It was set up as a critical process because it is a step set up intended to inactivate a potential virus.

3) Virus removal filter filtration

It was set up as a critical process because it is a set-up step intended to remove potential viruses.

2. In-process control test

In-process control tests shown in Table 2.3.S.2.4-1 were established as part of product quality control.

Table 2.3.S.2.4-1 In-Process Control Tests for Manufacturing Process of Hutumumab Drug Substance

Target process/target

sample

Test items Acceptance criterion

Cell Culture Step 3

(unprocessed bulk)

Mycoplasma test (culture method,

DNA staining method)

Acceptance criteria: Negative

Bioburden (microbial numeration

tests)

Action limit: < 10 CFU/10 mL

Adventitious viral test (in vitro test) Acceptance criteria: Not

detected

Hutumumab concentration (affinity

chromatography)

Action Limit: ≥ X.X mg/mL

Harvest

Culture supernatant fluid

Hutumumab concentration (affinity

chromatography)

Action Limit: ≥ X.X mg/mL

Concentration and Buffer

Exchange 2

Host cell-derived proteins (ELISA

method)

Action Limit: ≤ ■ ng/mg

3. Critical intermediate

Not applicable.

36

Explanation

・ Critical Process: The term Critical Step was used in M2.3.S.2.4 of the ICH M4 Guideline. Then, the

Critical Process was defined in Notification No. 0210001 of February 10, 2005 (Notification No.

0210001 of the Evaluation and Licensing Division, Pharmaceutical and Food Safety Bureau) as a

process that has an impact on quality and includes process conditions, tests, and other relevant

parameters that need to be operated within pre-determined control values to ensure that the drug

substance conforms to the specifications. The definition of this Critical Process includes Japanese-

specific concepts not found in Europe and the United States in the CTDs, and there are differences

in the definition and the description methods on the regulatory dossier between the Critical Step in

foreign countries and the Critical Process in Japan.

・ Setting of Critical Process: In the setting of Critical Processes, processes for which significant

removal of impurities and viruses has been confirmed can be selected by the applicant.

・ Critical intermediates: intermediates that are isolated and stored for a long time during the

manufacturing process.

37

2.3.S.2.5 Process Validation and/or Evaluation Note.

This section is based on the Traditional approach and not the Enhanced approach described in the

ICH Q11 guideline.

1. Consistency of the manufacturing process

1) Cell culture processes

The consistency of the cell culture process of hutumumab drug substance was evaluated at

commercial scale (production cell culture scale: 5000 L). Cell Culture Step 1 and Cell Culture Step

2 are processes intended for expansion cultivation and are not considered to have a direct effect on

product quality, so the test results are not presented in this section (see 3.2.S.2.5 for test results of

Culture Step 1 and Culture Step 2). Table 2.3.S.2.5-1 shows the results of in-process control tests

for Culture Step 3 that may affect product quality.

The results obtained and the results of the S.4.4 batch analysis confirmed the good consistency of

the cell culture process of hutumumab.

Table 2.3.S.2.5-1 Test results for cell culture step 3 (scale: 5000 L).

Corresponding

Drug substance

lot number

Mycoplasma

(Acceptance

Criteria :Negative

Bioburden

(Action Limit:

< 10 CFU/10 mL.

Adventitious viruses

(Acceptance Criteria:

not detected)

Hutumumab

concentration

(Action Limit:

≥ X.X mg/mL).

XXX Negative 2 CFU/10 mL Not detected 0.4 mg/mL

YYY Negative 0 CFU/10 mL Not detected 0.4 mg/mL

ZZZ Negative 0 CFU/10 mL Not detected 0.3 mg/mL

2) Harvest

Table 2.3.S.2.5-2 shows the results of in-process control tests for harvest steps. From the obtained

results, it was confirmed that the harvest step has good consistency.

Table 2.3.S.2.5-2 Harvest Step Test Results

Correspond to drug substance lot

number

Hutumumab concentration

(Action limit: ≥ X.X mg/mL)

XXX 0.4 mg/mL

YYY 0.3 mg/mL

ZZZ 0.3 mg/mL

3) Purification process

Process evaluation was performed on the purification process of hutumumab drug substance at

38

commercial scale. The yield and bioburden test results for each purification step are shown in Table

2.3.S.2.5-3 and Table 2.3.S.2.5-4, respectively. The results of in-process control tests for the

concentration and buffer exchange steps are shown in Table 2.3.S.2.5-5 with the results of the host

cell-derived protein (HCP) and hutumumab concentrations.

From the obtained results, the purification process of hutumumab confirmed good consistency.

39

Table 2.3.S.2.5-3 Yield results for each step of the purification process.

Corresponding

drug substance

lot number

Yield (%)

Protein A

column

Viral

inactivation

process

Cation-

exchange

column

Anion-

exchange

column

Concentration

and Buffer

Exchange 1

Hydrophobic

column

Virus removal

filter filtration

Concentration

and Buffer

Exchange 2

XXX ■■ ■■ ■■ ■■ ■■ ■■ ■■ ■■

YYY ■■ ■■ ■■ ■■ ■■ ■■ ■■ ■■

ZZZ ■■ ■■ ■■ ■■ ■■ ■■ ■■ ■■

Table 2.3.S.2.5-4 Bioburden test results for each step of the purification process.

Corresponding drug substance

lot number

Bioburden (CFU/mL)

Protein A column Cation-exchange column Anion-exchange column Hydrophobic column

XXX ■ ■ ■ ■

YYY ■ ■ ■ ■

ZZZ ■ ■ ■ ■

Table 2.3.S.2.5-5 Results of two-step in-process control test (HCP) for concentration and buffer replacement

Corresponding drug substance

lot number

HCP(ng/mg)

(Action Limit: ≤ XX ng/mg)

XXX ■■

YYY ■■

ZZZ ■■

40

2. Removal of impurities

1) Product-related impurities

Each in-process solution was analyzed by size exclusion chromatography, and the levels of the

resulting high and low molecular weight regions are shown in Table 2.3.S.2.5-6. The level of the

high molecular weight region was reproducibly reduced in the ■ step and ■■ step, with ●~●% in

the drug substance. The levels in the low molecular weight region were reproducibly reduced in the

■ step and the ■■ step, and in the ●~●% in the drug substance. Based on the above, it was

confirmed that these product-related impurities can be reproducibly reduced by properly controlling

the manufacturing process of hutumumab.

Table 2.3.S.2.5-6 Levels of High and Low Molecular Weight Regions by Size Exclusion Chromatographic Analysis in Each In-process Solution

In-process solution

Drug Substance Lot XXX Drug Substance Lot YYY Drug Substance Lot ZZZ

High-

molecular-

weight

region (%)

Low

molecular

weight

region

High-

molecular-

weight

region (%)

Low

molecular

weight

region

High-

molecular-

weight

region (%)

Low

molecular

weight

region

Protein A column ■■ ■■ ■■ ■■ ■■ ■■

Cation-exchange

column ■■ ■■ ■■ ■■ ■■ ■■

Anion-exchange

column ■■ ■■ ■■ ■■ ■■ ■■

Concentration and

Buffer exchange 2 ■■ ■■ ■■ ■■ ■■ ■■

Drug Substance ■■ ■■ ■■ ■■ ■■ ■■

41

2) Process-Related Impurities

1. Host cell-derived DNA

Table 2.3.S.2.5-7 shows the results of measuring the amount of DNA in each in-process

solution by the ● method. The DNA amount in the culture supernatant solution was ● ×10x

to ● ×10x pg/mg, but it was reproducibly reduced in the ■ step, ■■ step and ■■■ step, and

was below the quantitation limit in the drug substance. From the above, it was confirmed that

the manufacturing process of hutumumab can be reproducibly reduced DNA by properly

controlling the process.

Table 2.3.S.2.5-7 Residual amount of host cell-derived DNA in each in-process solution

In-process solution

Host cell-derived DNA (pg/mg)

Drug Substance

Lot XXX

Drug Substance

Lot YYY

Drug Substance

Lot ZZZ

Culture supernatant fluid ■■ ■■ ■■

Protein A column ■■ ■■ ■■

Anion-exchange column ■■ ■■ ■■

Concentration and Buffer Exchange 2 < ■■ < ■■ < ■■

Drug Substance < ■■ < ■■ < ■■

2. Host cell proteins

Table 2.3.S.2.5-8 shows the results of measuring the amounts of host cell proteins in each

in-process solution by the ELISA method. The amount of host cell protein in the culture

supernatant solution was ● × 10x to ● ×10x ng/mg, and it was reproducibly reduced in the ■

step, ■■ step, and ■■■ step, and in the drug substance, it was ● to ● ng/mg. Thus, it was

confirmed that the manufacturing process of hutumumab can be reproducibly reduced host cell

protein by properly controlling the process.

Table 2.3.S.2.5-8 Residual amount of host cell-derived proteins in each process solution

In-process solution

Host cell proteins (ng/mg)

Drug Substance

Lot XXX

Drug Substance

Lot YYY

Drug Substance

Lot ZZZ



Cation-exchange column ■■ ■■ ■■


Hydrophobic column ■■ ■■ ■■

Concentration and Buffer Exchange 2 ■■ ■■ ■■

Drug Substance ■■ ■■ ■■

42

3. Protein A

Protein A is an impurity that leaches from the protein A column resin of the purification

step. Table 2.3.S.2.5-9 shows the results of measuring protein A levels in the process solution

by the ELISA method. Protein A, which was §~§§ ng/mg in the protein A chromatography

fraction, was reproducibly reduced in the § step and in the §§ step, which were below the

quantification limit in the drug substance. From the above, it was confirmed that the

production process of hutumumab can be reproducibly reduced protein A by properly

controlling the process.

Table 2.3.S.2.5-9 Residual amount of protein A in each process solution

In-process solution

Protein A (ng/mg)

Drug Substance

Lot XXX

Drug Substance

Lot YYY

Drug Substance

Lot ZZZ


Cation-exchange column ■■ ■■ ■■


Concentration and Buffer exchange 2 < ■■ < ■■ < ■■


4. Recombinant insulin

Recombinant insulin is a component of the medium used in the cell culture process. The

amounts of insulin in the different process solutions were measured by ELISA. The results are

shown in Table 2.3.S.2.5-10. In the culture supernatant solution, insulin was § ng/mg, and it

was reproducibly reduced in the § and §§ steps, which were below the limit of quantification in

the drug substance. From the above, it was confirmed that insulin can be reproducibly reduced

recombinant insulin by properly controlling the manufacturing process of hutumumab.

Table 2.3.S.2.5-10 Residual amount of transgenic insulin in each process solution

In-process solution

Recombinant insulin (ng/mg)

Drug Substance

Lot XXX

Drug Substance Lot

YYY

Drug Substance

Lot ZZZ


Protein A column < ■■ < ■■ < ■■


⑤ Methotrexate

Methotrexate is a component of the medium used in the cell culture process. The amount of

43

methotrexate in each process solution was measured by GC-MS. The results are shown in

Table 2.3.S.2.5-11. In the culture supernatant solution, methotrexate was § pg/mg, and it was

reproducibly reduced in the § step, which was below the quantification limit in the drug

substance. Thus, it was confirmed that methotrexate can be reproducibly reduced by properly

controlling the manufacturing process of hutumumab.

Table 2.3.S.2.5-11 Residual amount of methotrexate in each process solution

In-process solution

Methotrexate (pg/mg)

Drug Substance

Lot XXX

Drug Substance Lot

YYY

Drug Substance

Lot ZZZ


Protein A column < ■■ < ■■ < ■■


Explanation

・ In this mock-up, descriptive examples for the removal of product-related impurities and process-

related impurities in the purification process are shown. On the other hand, actual measurement

data may not always be required by showing risk assessment results (e.g., to explain that the

residual amount of impurities in the drug substance is at a safe level even if not removed during the

purification process).

・ Because of the scientific limitations of fully classifying product-related substances, this mock-up

provides an example that does not specify the product-related substance.

44

3. Reprocessing

In the virus removal filter filtration step of the purification process, the filter is re-filtered when the

filter integrity test fails, etc. Therefore, the number of re-filtration steps of virus removal filter filtration

was studied in a verified scale-down model. As the result, no change was observed within the range of

the test items tested up to three times of re-filtration (Table 2.3.S.2.5-16).

Table 2.3.S.2.5-16 Refiltration results of virus removal filters.

Number of

times of re-

filtration

Yield

Size exclusion chromatography Biological activity

CDC-activity assay

(Unit/mg)

Main peak

(%)

High-molecular-

weight region

(%)

Low-molecular-

weight region

(%)

0 ■■ xx.x x.x x.x xx


2 ... ... ... ... ...


Footnote 1) Number 0 is the result of the fraction before virus removal filter filtration

45

2.3.S.2.6 Manufacturing Process Development Note.

This section is based on the Traditional approach and not the Enhanced approach described in the ICH

Q11 guideline.

1. Manufacturing Process Development

Table 2.3.S.2.6-1 outlines the process development history of hutumumab.

Process A was established as a manufacturing method for drug substance for toxicity and clinical phase I

studies. Subsequently, Process B was established as a manufacturing method for drug substance for clinical

Phase II trials. Process C1 was established as a manufacturing method of the drug substance for clinical

phase III trial, and Process C2 was established as a manufacturing method of the commercial drug

substance by scaling up from Process C1.

Major changes from Process A to Process B are as follows:

The manufacturing facility was changed from facility A to facility B.

The cell bank used for manufacturing was changed from MCB to WCB.

The harvest method was changed from centrifugation to filter filtration for productivity

improvement.

Major changes from Process B to Process C1 are as follows:

The scale of cell culture step 3 was changed from 1000L to 2000L, together with the scales of

cell culture step 1, cell culture step 2, and purification step.

The medium used in cell culture step 3 was changed from Medium Z to Medium C.

The resin of anion exchange column chromatography was changed for productivity

improvement.

The storage temperature of the drug substance was changed from ‒70°C to ‒20°C.

Hydrophobic column chromatography step was added for the improvement of impurity

removal capability. In addition, the concentration step and buffer exchange step were added

before hydrophobic column step.

Major changes from Process C1 to Process C2 are as follows:

The manufacturing facility was changed from facility B to facility C.

The scale of cell culture step 3 was changed from 2000 L to 5000 L, together with the scales of

cell culture step 1, cell culture step 2, and Purification Process.

Drug substance composition was unchanged across all manufacturing methods from Process A to Process

C2.

46

Table 2.3.S.2.6-1 Overview of the Development History of the Manufacturing Methods for Hutumumab Manufacturing process Process A Process B Process C1 Process C2

Use Toxicity/ Phase I clinical studies Phase II clinical studies Phase III clinical studies Commercial production

Production facility Site A Site B Site B Site C Cell bank used for

manufacturing MCB (lot ■■). WCB (lot ■■)

Cell Culture Step 1 Cell culture using medium A

Cell Culture Step 2 Cell culture in a 300 L bioreactor using medium B Cell culture in a 500 L bioreactor using medium B

Cell culture in a 1000 L bioreactor using medium B

Cell Culture Step 3 Cell culture in a 1000 L bioreactor using medium Z Production culture in a 2000 L bioreactor using medium C

Production culture in a 5000 L bioreactor using medium C

Harvest Cell separation by centrifugation and filtration

Cell separation by filter filtration

Purification 1 Protein A column chromatography Low pH treatment Virus inactivation by low pH treatment.

Purification 2 Cation exchange column chromatography Purification 3 Anion exchange column

chromatography (resin A) Anion exchange column chromatography (resin B)

Concentrated buffer exchange (Not performed) Implementation Same as on the left Purification 4 (Not performed) Hydrophobic column chromatography

Virus removal filter filtration Filtration through virus removal filters Concentration and buffer

exchange Implementation Same as on the left

Preparation Implementation Same as on the left Filtration and packing Final filtration, packing and storage at ‒70ºC. Final filtration, packing and storage at ‒20ºC.

47

2. Comparability Assessment of Drug Substances

1) Comparability of drug substance batches in manufacturing Process A and drug substance batches in

Process B

Comparability was assessed by the tests shown in Table 2.3.S.2.6-2 for the drug substance lot ■

produced in Process A and thedrug substance lot ■■ produced in Process B. As the result, the

process changes had no significant effect on the product quality, and the drug substance of Process A

and Process B were judged to be comparable.

Table 2.3.S.2.6-2 Evaluation of Equivalence/Equivalence of Drug Substances for Process A and B

Test items Drug substance

batch ■

(Process A)

Drug substance

batch ■■

(Process B)

Description ● ■

Peptide map ● ■

IEF ● ■

N-terminal amino acid analysis by Edman's method ELG ELG

pH 6.3 6.3

Glycosylation Profile: Afucosyl Glycans (%) ● ■

Ion exchange chromatography: Main peak (%) ● ■

Size exclusion chromatography: Main peak (%) ● ■

Capillary electrophoresis (non-reducing conditions): Main

peak (%) ● ■

Host cell-derived DNA (pg/mg) ● ■

Host cell proteins (ng/mg) ● ■

Protein A (ng/mg) ● ■

Biological activity (CDC-activity assay) (Unit/mg) ● ■

Content (mg/mL) 102 97

48

2) Comparability of drug substance batch of Process B and drug substance batch of Process C1

Comparability was assessed by the tests shown in Table 2.3.S.2.6-3 for the drug substance lot ■

produced in Process B and for the drug substance lot ■■ produced in Process C1. As the result, the

process changes did not have any significant impact on product quality, and the drug substance of

process B and process C1 were judged to be comparable.

Table 2.3.S.2.6-3 Comparability Evaluation of Drug Substances of Process B and Process C1


batch ■

(Process B)

Drug substance

batch ■■

(Process C1)

Description ● ■

Peptide map ● ■

IEF ● ■

pH 6.3 6.3

Glycosylation Profile: Afucosyl Glycans (%) ● ■

Ion exchange chromatography: Main peak (%) ● ■

Size exclusion chromatography: Main peak (%) ● ■

Capillary electrophoresis (non-reducing conditions): Main

peak (%) ● ■

Host cell-derived DNA (pg/mg) ● ■

Host cell proteins (ng/mg) ● ■

Protein A (ng/mg) ● ■

Biological activity (CDC-activity assay) (Unit/mg) ● ■

Content (mg/mL) 99 102

3) Comparability of Batches of Drug Substance for Process C1 and Batches of Drug Substance for

Process C2

Comparability was assessed by the tests shown in Table 2.3.S.2.6-4 for the drug substance lots

produced in Process C1 and for the drug substance lots produced in Process C2. As the result, the

process changes did not have any significant impact on product quality, and the drug substance of

process C1 and process C2 were judged to be comparable.

49

Table 2.3.S.2.6-4 Equivalence/Equivalence Evaluation of Drug Substances of Process C1 and Process C2


batch

■

(Process C1)

Drug substance

batch

■■

(Process C1)

Drug substance

batch

■■■

(Process C2)

Drug substance

batch

■■■■

(Process C2)

Drug substance

batch

■■■■■

(Process C2)

Description ● ● ● ● ●

Peptide map ● ● ● ● ●

Isoelectric focusing capillary electrophoresis ● ● ● ● ●

pH 6.3 6.2 6.2 6.3 6.3

Glycosylation Profile: Afucosyl Glycans (%) ● ● ● ● ●

Ion exchange chromatography: Main peak (%) ● ● ● ● ●

Size exclusion chromatography: Main peak (%) ● ● ● ● ●

Capillary electrophoresis (non-reducing conditions): Main peak

(%)

● ● ● ● ●

Host cell-derived DNA (pg/mg) ● ● ● ● ●

Host cell proteins (ng/mg) ● ● ● ● ●

Protein A (ng/mg) ● ● ● ● ●

Biological activity (CDC-activity assay) (Unit/mg) ● ● ● ● ●

Content (mg/mL) 99 101 102 100 101

50

3. Development history of the control strategies

Table 2.3.S.2.6-5 summarizes the development history of the specifications for the investigational hutumumab drug substance.

Table 2.3.S.2.6-5 Development History of Drug Substance Specifications

Test items Drug Substance Specifications for the

Process A

Drug Substance Specifications for the

Process B


Process C1


Process C2 Description Colorless to pale

yellow, clear or slightly white turbid

solution

Colorless to pale yellow, clear or

slightly white turbid solution





Identification Peptide map Report Similar peaks are observed at the same retention time as the reference material

Similar peaks are observed at the same retention time as the reference material

Similar peaks are observed at the same retention time as the reference material.

Isoelectric focusing Report ― ― ― CDC activity Observe a dose-

response curve dependent on Hutumumab concentration

― ― ―

pH 5.5~7.0 5.5~7.0 5.8~6.8 5.8~6.8 Glycosylation Profile: G0 (%) Report Report G0: ≤ ●% G0: ≤ ●% Purity test Ion-exchange chromatography

Main peak (%) Acidic region (%) Basic region (%)

≥ ●% Report Report


≥ ●% ≤ ●% ≤ ●%

≥ ●% ≤ ●% ≤ ●%

Size exclusion chromatography Main peak (%) High-molecular-weight region (%) Low-molecular-weight region (%)


≥ ●% ≤ ●% Report

≥ ●% ≤ ●% ≤ ●%

≥ ●% ≤ ●% ≤ ●%

51

Table 2.3.S.2.6-5 Development History of Drug Substance Specifications (continued)

Test items Drug Substance

Specifications for the

Process A

Drug Substance


Process B

Drug Substance


Process C1

Drug Substance


Process C2

Capillary electrophoresis (non-reducing

conditions)

Main peak (%).

Report

≥ ●%

≥ ●%

≥ ●% Host cell proteins (ng/mg) Report ≤● ≤● -*

Host cell-derived DNA (pg/mg) Report ≤● ≤● -

Protein A (ng/mg) Report ≤● ≤● -

Endotoxin <▲ <▼ <■ <●

Microbial limit test ≤● ≤● ≤■ ≤▲

Biological activity (CDC-activity assay) (Unit/mg) ■~■ Unit/mg ▲ ~▲ Unit/mg ●~● Unit/mg ●~● Unit/mg

Content (mg/mL) 85~115mg/mL 90~110mg/mL 90~110mg/mL 90~110mg/mL

* Host cell-derived proteins were established as in-process control studies (see 2.3.S.2.2)

Explanation

Table 2.3.S.2.6-5: ICH Q11 Guideline, "Changes in relevant control system associated with the development of the manufacturing process should be

briefly described in Section 3.2.S.2.6," and this section describes the changes in specifications during the development phase but can also be included in the

S.4.4 Batch Analyses.

52

2.3.S.3 Characterisation (Hutumumab, HS Pharmaceutical) 2.3.S.3.1 Elucidation of Structure and other Characteristics

Note:

・ This section is only an example of the description, does not describe all of the necessary contents

for the approval review of individual products, and all of the contents exemplified are not required.

Appropriate content should be provided in this section for each individual product on the basis of

the applicant's policy.

・ For characterization of antibody drugs, please refer to the ICH Q6B Guidelines (Notification No.

571 of the Evaluation and Licensing Division, PSFB, dated May 1, 2001) and the Guidance for

Quality Evaluation of Antibody Drugs (Notification No. 1214-1 of the Evaluation and Licensing

Division, PFSB, dated December 14, 2012).

Characterization studies were performed using the Hutumumab Primary Reference Material Lot PRS-1 (see

2.3.S.5).

1. Primary structure

1) Peptide mapping

After reductive alkylation by dithiothreitol and iodoacetamide, enzymatic digestion was

performed by lysyl endopeptidase (Lys-C) and peptidyl-ASP metalloendopeptidase (Asp-N), and

the digests were analyzed by reverse phase column-connected LC/MS.

Figure 2.3.S.3.1-1 and Figure 2.3.S.3.1-2 show the chromatogram of the digest, the assignment of

each peak, and the peptide fragment expected to be obtained by Lys-C enzymatic digestion of

hutumumab (bold is the peptide fragment identified by MS). Table 2.3.S.3.1-1 and Table 2.3.S.3.1-2

show the structure, theoretical molecular weight, and actual values of each fragment. Figures

2.3.S.3.1-3 and 2.3.S.2.3.S.3.1-4 and Tables 2.3.S.3.1-3 and 2.3.S.3.1-4 show the results from Asp-

N enzyme digestion. From the obtained results, it was confirmed that it matched the amino acid

sequence expected from the gene sequence.

53

Figure 2.3.S.3.1-1 Peptide mapping chromatogram of Lys-C digested Hutumumab.

0

20

40

60

80

100

0 5 10 15 20 25 30 35 40 45 50 55 60 65

ｍA

Uat

XX

Xn

m

Time (min)

LH9 LH2

LH19

LH11

LH26

LH5

LH20 LH23

LL4 LL5

LL10

LL8

LH1

LL9 LH12

LH8

LH18

LH7

LH21 LH12

LH3

LH14

LL7

LH15

LH17 LL6 LH1

LL3

LL11

LH24

LH4

LH22 LL1

LH6

LL2

LH10

LH25

LH16

54

Figure 2.3.S.3.1-2 Amino acid sequences after Lys-C peptide maps.

Light chain

E L G M T Q S P S S V S A S V G D R V T I T C R A S H S I S T Y L N W Y Q Q K P G K A P K L L I Y A

A S S L Q S G V P S R F S G S G S G T D F S L T I N S L Q P E D F A T Y Y C Q Q T F S P S G T F G Q

G T K V E L K R T V A A P S V F I F P P S D E Q L K S G T A S V V C L L N N F Y P R E A K V Q W K V

D N A L Q S G N S Q E S V T E Q D S K D S T Y S L S S T L T L S K A D Y E K H K L Y A C E V T H Q G

L S S P V T K S F N R G E C

Heavy chain

E V Q L V E S G G G L V Q P G G S L R L S C A A S G F T F T S Y W M S W V R Q A P G K G L E W V A N

I K Q E G S E K T Y V D A T K G R F T I T R D N A K N S L Y L Q M N S L R A E D T A V Y Y C A R E F

E S T M T S V N A D Y Y Y F Y M D V W G K G T T V T V S S A S T K G P S V F P L A P S S K S T S G G

T A A L G C L V K D Y F P E P V T V S W N S G A L T S G V H T F P A V L Q S S G L Y S L S S V V T V

P S S S L G T Q T Y I C N V N H K P S N T K V D K R V E P K S C D K T H T C P P C P A P E L L G G P

S V F L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K

T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K A L P A P I E K T I S K

A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E

N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q

K S L S L S P G K

LL1

LL2

LL3 LL4 LL5 LL6

LL7 LL8 LL9

LL11

LL10

LH1

LH3

LH7 LH8 LH9

LH10

LH2

LH4 LH5 LH6

LH12 LH11 LH14 LH13

LH15 LH16

LH17 LH18 LH19

LH20 LH22 LH21

LH23 LH25 LH24

LH26

55

Table 2.3.S.3.1-1 LC/MS analysis results (light chain) of Lys-C digested peptide maps.

Peaking Peptide fragment Theoretical Actual LL1 E1-K39 4287.1 4287.1 LL2 L45-K103 6100.9 6100.9 LL3 V104-K107 487.3 487.3 LL4 R108-K126 2101.1 2101.1 LL5 S127-K145 2068.0 2068.0 LL6 D146-K149 599.3 599.3 LL7 S150-K168 2135.0 2135.0 LL8 V169-K173 1501.8 1501.8 LL9 A174-K178 624.3 624.3 LL10 V191-K207 1831.9 1831.9 LL11 V208-C214 811.3 811.3

Table 2.3.S.3.1-2 LC/MS analysis results (heavy chain) of Lys-C digested peptide maps.

Peaking Peptide fragment Theoretical Actual LH1 E1-K43 4543.2 4543.2 LH2 G44-K52 1028.6 1028.6 LH3 Q53-K58 676.3 LH4 T59-K65 796.4 LH5 G66-K76 1277.7 1277.7 LH6 N77-K121 5407.4 5407.4 LH7 G122-K133 1137.6 1137.6 LH8 G134-K145 1185.6 1185.6 LH9 S146-K158 1263.7 1263.7

LH10 D159-K217 6128.0 6128.0 LH11 P218-K222 545.3 545.3 LH12 R226-K230 627.4 627.4 LH12 S231-K234 451.2 451.2 LH14 T235-K258 2504.3 2504.3 LH15 D261-K286 2897.4 2897.4 LH16 F287-K300 1676.8 1676.8 LH17 P303-K328 3230.7 3230.7 LH18 A339-K346 837.5 837.5 LH19 T347-K350 447.3 447.3 LH20 G353-K362 2342.2 2342.2 LH21 N363-K372 1103.6 1103.6 LH22 G373—K404 2543.1 2543.1 LH23 T405-K421 1171.9 1171.9 LH24 L422-K426 574.3 574.3 LH25 S427-K451 2986.4 2986.4 LH26 S452-K459 787.4 787.4

56

Figure 2.3.S.3.1-3 Peptide mapping chromatogram of Asp-N digested Hutumumab.

(Figure : omission)

Figure 2.3.S.3.1-4 Amino Acid Sequence after Asp-N Digested Peptide Map.

(Figure : omission)

Table 2.3.S.3.1-3 LC/MS analysis results (heavy chain) of Asp-N digested peptide maps.

(Table : omission)

Table 2.3.S.3.1-4 LC/MS analysis results (heavy chain) of Asp-N digested peptide maps.

(Table : omission)

Explanation

In this mock-up, the amino acid sequence of each peptide fragment obtained in the peptide map was

identified by comparing the molecular weight of each peptide fragment inferred from the gene sequence

with the results of LC/MS analysis. In some cases, the amino acid sequence of each peptide fragment is

identified by MS/MS analysis.

2) N-and C-terminal amino acids

1. N-terminal amino acid

Lys-C digested peptide map-tandem mass spectrometry (MS/MS analysis) under reducing

conditions confirmed that approximately 90% of the N-terminal glutamines of the heavy and

light chains were pyroglutamylated.

2. C-terminal amino acid

57

Lys-C digested peptide map-MS/MS analysis under reducing conditions confirmed that the

Lys at the C-terminal of the heavy chain was about 90% deleted. And, the deletion body of

Gly-Lys was partially recognized.

2. Higher-order structure

1) Disulfide bonds and sulfhydryl groups

① Disulfide bond

The results of Lys-C digested peptide map-MS/MS analyses under reducing and non-reducing

conditions confirmed the presence of two and three intra-and intra-heavy disulfide bonds (light

chain: Cys23-Cys88; Cys134-Cys194; heavy chain: Cys22-Cys96; Cys156-Cys212; Cys273-

Cys333), and two and one inter-and heavy-light chain disulfide bonds (heavy chain Cys238-heavy

chain Cys238, heavy chain Cys241-heavy chain Cys241, light chain Cys214-heavy chain Cys232),

respectively.

Figure 2.3.S.3.1-5 Lys-C enzyme digested peptide map (reducing conditions).

(Figure : omission)

Figure 2.3.S.3.1-6 Lys-C enzyme digested peptide maps (non-reducing conditions).

(Figure : omission)

Table 2.3.S.3.1-5 Lys-C enzyme digested peptide map (reducing conditions) analysis results.

(Tables: omission)

(The entries may include, for example, peak IDs for expected Cys residues, positions of peptide

fragments and Cys, theoretical molecular weights, actual values or peak IDs for disulfide bonds, S-S

bond positions, theoretical molecular weights, actual values, etc.)

Table 2.3.S.3.1-6 Lys-C enzyme digested peptide map (non-reducing conditions) analysis results.

(Table : omission)

58

② Sulfhydryl group

The content of 0.3 to 0.5 mol free sulfhydryl groups per 1 mol was confirmed by colorimetric

determination using 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reaction).

2) Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy revealed a maximum at 1630 cm-1, confirming the

structures of β-sheet-dominant characteristic of IgGs.

Figure 2.3.S.3.1-7 Fourier transform infrared spectra.

3) Far-UV and near-UV circular dichroism spectra.

Far-UV circular dichroism spectral analyses revealed a negative maximum in 217 nm and a

positive maximum in 198 nm, confirming the structures of β-sheet predominantly characteristic of

IgGs.

Near-UV circular dichroism spectrometry revealed that the maximum wavelength of 295 nm by

tryptophan, the maximum wavelength of 285 nm by tryptophan and tyrosine, the fine peak between

255 nm and 280 nm by phenylalanine and tyrosine, and the totally negative signal by disulfide bond

were observed, confirming that the spectrum was unique to IgGs.

0

20

40

60

80

100

吸光

度（％

）

波長（cm‐1）

1700 1690 1680 1670 1660 1650 1640 1630 1620 1610 1600

Abs

orba

nce

(%)

Wave length (cm-1)

59

Figure 2.3.S.3.1-8 Far-UV circular dichroism spectra.

Figure 2.3.S.3.1-9 Near-UV circular dichroism spectra

Explanation

In this mock-up, multiple methods (Fourier transform infrared absorption spectra, far-UV and

near-UV circular dichroism spectra) are exemplified as analytical methods for higher-order

structures, but the analytical methods are appropriately selected and described by the applicants.

‐5000

0

5000

10000

190 200 210 220 230 240

[θ] (deg・

cm2/dmol)）

波長（nm）

‐60

‐40

‐20

0

20

40

60

250 260 270 280 290 300 310 320 330 340 350

[θ] (deg・

cm2/dmol)）

波長（nm）

Wave length (nm)

Wave length (nm)

60

3. Carbohydrate structure

1) N-linked oligosaccharides

Hutumumab has a consensus sequence (Asn309-Ser-Thr) at one position in the CH2 domains of

the heavy chains.

Lys-C digested peptide map analysis under reducing conditions and SDS-capillary

electrophoresis (CE-SDS) confirmed that 99% of the heavy chain had undergone N-linked glycan

modification at the 309th Asn residue.

Released sugar chains with peptidic N-glycosidase F (PNGase F) treatment were derivatized with

2-aminobenzamide (2-AB), and the derivatives were analyzed by normal-phase HPLC/MS. The

major carbohydrate structures were G0F, G1F, and G2F complex-type double-stranded fucosylated

glycans with 0 to 2 added galactose at the ends shown below, and the percentages of each

carbohydrate structure to the total carbohydrate structures were approximately ■%, ■%, and ■%,

respectively. Fucose-free afucosyl sugar chains (G0, G1, and G2 sugar chains) and high mannose-

type sugar chains (M5 to M7) were identified, and the percentages of each carbohydrate structure to

the total sugar chain structures were less than ■% and less than ■%, respectively (of which ■% is

M5). (Figure 2.3.S.3.1-10, Figure 2.3.S.3.1-11)

Figure 2.3.S.3.1-10 HPLC chromatogram of N-linked Glycan

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20 25 30

吸光

度

保持時間（分）

G0

G0F

M5

G1

G1F

M6 G2F

G2 M8 M9 M7

Abs

orba

nce

Retention time (min)

61

Figure 2.3.S.3.1-11 Sugar chain profile of hutumumab

Type Structure

G0F

G1F

G2F

G0

G1

G2

High mannose

(M5)

High mannose

(M6)

High mannose

(M7)

Fuc(α1-6)


Gal1


Man(β1-4)GlcNAc(β1-4)GlcNAc

Gal(β1-4)GlcNAc(β1-2)Man(α1-3)



Fuc(α1-6)

GlcNAc(β1-2)Man(α1-3)






Fuc(α1-6)

(β1-4)GlcNAc(β1-2)Man(α1-3) Gal1






Man(α1-3)

Man(α1-6) Man(α1-6)


Man(α1-3)

Man(α1-3)

Man(α1-6)

Man(α1-2)Man(α1-3)

Man(α1-6)


Man1 (α1-2)Man(α1-3)

(α1-2)Man(α1-6)

Man(α1-2)Man(α1-3)

Man(α1-6)


62

2) O-linked oligosaccharides

The absence of O-linked glycan modifications was confirmed by Lys-C digested peptide map

analyses and HPAEC /PAD under reducing conditions.

3) Sialic acid

Free sugar chains from PNGase F treatment were derivatized with 2-AB, and the derivatives

were analyzed by normal-phase HPLC/MS. The results detected sugar chains with N-

acetylneuraminic acid. Also, by monosaccharide compositional analyses, the content of N-

acetylneuraminic acid was about ■mol per hutumumab 1 mol.

N-glycolylneuraminic acid was not detected.

4. Physicochemical Properties

1) Molecular-mass

Electron spray ionization-mass spectrometry (ESI-TOF/MS) revealed peaks of 150119 Da, peaks

of 149962 Da and 149806 Da, and peaks of 147350 Da in PNGase F treated animals, consistent

with the theoretical molecular weights expected from amino acid sequences and sugar chain

structures (Figure 2.3.S.3.1-12 to Figure 2.3.S.3.1-15).

PNGase F treated after reductive alkylation revealed peaks of 50473 Da and 23169 Da, consistent

with the expected theoretical molecular weights of the heavy and light chains.

Figure 2.3.S.3.1-12 Mass spectrum of Hutumumab (before removal of sugar chains)

0

2000

4000

6000

8000

10000

149000 149500 150000 150500 151000

相対

強度

Mass（Da）

149806

149962

150119

Rel

ativ

e In

tens

ity

63

Figure 2.3.S.3.1-13 Mass spectrum of Hutumumab (after removal of sugar chains)

Figure 2.3.S.3.1-14 Mass spectrum of Hutumumab (after reductive alkylation, heavy chain)

0

2000

4000

6000

8000

10000

146500 147000 147500 148000 148500

相対

強度

Mass（Da）

147350

50473

Rel

ativ

e In

tens

ity

Rel

ativ

e In

tens

ity

64

Figure 2.3.S.3.1-15 Mass spectrum of Hutumumab (after reductive alkylation, light chain)

Table 2.3.S.3.1-7 Molecular Mass analysis result

Condition Molecular species Theoretical

molecular weight

(Da)

Actual

measurement (Da)

- G0F added 149809 149806

- G1F added 149966 149962

- G2F added 150122 150119

PNGase F treatment Sugar chain

removed

147353 147350

Reductive

alkylation

PNGase F treatment

Sugar chain

removed (heavy

chain)

50477 50473

Sugar chain

removed ( light

chain)

23172 23169

2) Electrophoresis

1. SDS-capillary gel electrophoresis

Under non-reducing conditions, a major peak of monomer with an area percentage of ■% and

0

2000

4000

6000

8000

10000

20000 20500 21000 21500 22000 22500 23000 23500 24000 24500 25000

相対

強度

Mass（Da）

23169

Rel

ativ

e In

tens

ity

65

two minor peaks of high molecular weight with ■% and low molecular weight with < ■% were

observed (Figure 2.3.S.3.1-16).

Under reducing conditions, two main peaks of heavy and light chains and a peak of unbound

heavy chains of sugar chains (area percentage ■%) were observed (Figure 2.3.S.3.1-17).

Figure 2.3.S.3.1-16 SDS-capillary electrophoresis electropherogram (non-reducing conditions).

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20

吸光

度


High-molecular-

weight region

Low molecular

weight region

Abs

orba

nce

Retention Time (min)

66

Figure 2.3.S.3.1-17 SDS-capillary electrophoresis electropherogram (reducing conditions).

2. Capillary isoelectric focusing

The main peak and two basic peaks were observed, and the percentages of the area were

approximately ■%, ■%, and ■%, respectively (Figure 2.3.S.3.1-18). The isoelectric points were

7.40, 7.51 and 7.54, respectively. The bioactivities of basic peak 1 and basic peak 2 were ■% and

■% of hutumumab, respectively.

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20

吸光

度


Heavy chain without carbohydrate

Light chain

Heavy chain


Abs

orba

nce

67

Figure 2.3.S.3.1-18 Capillary isoelectric focusing electropherogram

3) Liquid chromatography

1. Size exclusion chromatography

The main peak of monomer with area percentage ■% and the high molecular weight peak with

area percentage ■% and the low molecular weight peak with area percentage ■% were observed

(Figure 2.3.S.3.1-19). The component included in the high molecular weight peak was mainly

dimer. Components included in the low molecular weight peak were presumed to be truncated

heavy and light chains (see 3.2.S.3.1).

The biological activity of the high molecular weight fraction was ■% of hutumumab (see

3.2.S.3.1).

0

0.2

0.4

0.6

0.8

1

7 7.2 7.4 7.6 7.8 8

吸光

度

PI

Basic peak 1

Basic peak 2

pI marker pI marker

Principal peak A

bsor

banc

e

68

Figure 2.3.S.3.1-19 Size exclusion chromatography chromatogram

2. Cation-exchange chromatography

Main peak with area percentage ■% and acidic region with area percentage ■% and basic region

with area percentage ■% were observed (Figure 2.3.S.3.1-20).

Analysis of the acidic region confirmed that it contained deamidated bodies, fragments of ■%

(see 3.2.S.3.1). It was also confirmed that the basic region contains Lys and Gly-Lys deletions at the

C-terminus of the heavy chain (see 3.2.S.3.1). The bioactivity of the deamidated and deleted

fractions was 70% and 80% of hutumumab, respectively (see 3.2.S.3.1).

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20

吸光

度


8 13 18

High-molecular-

weight region

Low molecular

weight region

Enlarged view Principal peak

Abs

orba

nce


69

Figure 2.3.S.3.1-20 Cation exchange chromatography chromatogram

4) Others

① Absorption coefficient

The extinction coefficient (E1%, 1 cm (280 nm)) was 13.8.

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20

吸光

度


Basic region Acidic region

Principal peak


Abs

orba

nce

70

5. Biological activities

1) Binding property

① Binding to HS antigens

Binding to HS-antigens was assessed by enzyme-linked immunosorbent assay (ELISA).

Hutumumab showed binding to HS antigen in a dose-dependent manner with a 50% binding

concentration of XX μg/mL (Figure 2.3.S.3.1-21).

As a result of HS-antigen binding kinetic analysis using the surface-plasmon resonance (SPR)

method (Fig. 2.3.S.3.1-22), the equilibrium dissociation constant (KD) was XX nmol per liter.

Figure 2.3.S.3.1-21 Binding to HS antigen_ Dose relationship diagram

0

0.2

0.4

0.6

0.8

1

1 10 100 1000

吸光

度

抗体濃度（μg/mL)

Abs

orba

nce

Antibody concentration (μg/mL)

71

Figure 2.3.S.3.1-22 Binding sensorgrams for the interaction of hutumumab with HS antigen

‐10

0

10

20

30

40

50

60

70

80

90

100

-100 0 100 200 300 400 500

強度

時間（秒）

500nmol/L

100nmol/L

50nmol/L

10nmol/L

1nmol/L

Inte

nsity

Time (sec)

72

2) Functional property

① Complement-dependent cytotoxicity (CDC)

CDC activity was measured fluorometrically using resazurin. In the presence of complement,

Hutumumab and the ■ cancer cell line XX-XX expressing HS antigen were cultured. Hutumumab

caused cytotoxicity from XX μg/mL to ■cancer cell line XX-XX, reached a maximum at XX

μg/mL, and CDC activity was detected (Figure 2.3.S.3.1-23).

Figure 2.3.S.3.1-23 CDC Activity: Dose Relationship Diagram

0

20

40

60

80

100

0.01 0.1 1 10 100

細胞

死割

合（％

）


Rat

io o

f dea

d ce

lls (%

)


73

② Antibody-dependent cytotoxicity (ADCC)

ADCC activity was measured by the 51Cr release method. ■■ cells expressing hutumumab and

HS-antigen were cultured in the presence of human peripheral blood mononuclear cells (PBMC).

Hutumumab did not cause concentration-dependent cytotoxicity of ■■ cells at concentrations up to

XX μg/mL, and no ADCC activity was detected (Fig. 2.3.S.3.1-24).

Figure S.3.1-24 ADCC Activity: Dose Relationship Diagram

Explanation

• Tests for functional characterization are established based on the properties of antibody drugs

and antigens, expected efficacy, etc. In this mock-up, the ADCC activity and CDC activity were

selected, because the antibody drug which expects the cytotoxic activity through the effector

molecules was assumed in the mock-up.

• Methods for measuring ADCC activity and CDC activity in this mock-up are an example, and it

is advisable to describe a method established by the applicant. Since the biological properties

depend on the characteristics of the antibody and vary from antibody to antibody, the analytical

results of each test item are also exemplified.

0

5

10

15

20

0.01 0.1 1 10 100

細胞

障害

活性

（％

）


Cyto

toxi

city

act

ivity

(%)


74

2.3.S.3.2 Impurities Note:

・ For complex large-molecules such as antibody products, clearly distinguishing between product-

related substances and product -related impurities may have scientific limitations. This mock-up

shows examples described as cases where both product-related substances and product-related

impurities cannot be clearly classified (e.g., peak areas on ion-exchange chromatography, etc.).

Refer to 2.3.S.4.4 for test results of related substances (ion exchange chromatography) and impurities

(high and low molecular weight regions of size exclusion chromatography) derived from hutumumab in the

drug substance.

Refer to 2.3.S.4.4 for results of tests on endotoxin and bioburden (microbial limit) for process-related

impurities in the drug substance. Refer to 2.3.S.2.5 for test results of host cell-derived proteins, host cell-

derived DNA, protein A, recombinant insulin, and methotrexate in the drug substance.

Explanation

・ Residual solvent: Among solvents used in the manufacturing process of the drug substance for this

mock-up, only acetic acid (class 3 solvent) used in the purification process is corresponds to the ICH

Q3C guideline (Notification No. 307 of the Evaluation and Licensing Division, Pharmaceutical and

Medical Safety Bureau dated March 30, 1998). Considering the drug substance manufacturing

process of this mock-up, it is estimated that the acetic acid residue in the drug substance is

sufficiently low, so this mock-up does not show an example of the description of the residual

solvent.

・ Elemental Impurities: ICH Q3D Guidelines (Notification No. 0930-4 of the Evaluation and

Licensing Division, Pharmaceutical and Food Safety Bureau, September 30, 2015) explain that "

specific controls on elemental impurities up to the biotech drug substance are generally not needed".

Also, there is no modification step to intentionally add metal elements in the manufacturing process

of this mock-up. Therefore, S.3.2 of this mock-up does not provide an example of elemental

impurity description.

75

2.3.S.4 Control of Drug Substance (Hutumumab, HS Pharmaceutical) Note: Test items, analytical procedures, acceptance criteria, digits of test results, significant figures, etc.

are only exemplified, and are written in reference to Japanese Pharmacopoeia, etc.

2.3.S.4.1 Specification Table 2.3.S.4.1-1 summarizes the specifications for the drug substance of hutumumab.

Table 2.3.S.4.1-1 Specifications for Hutumumab Drug Substance

Test items Acceptance criteria

Description Colorless to pale yellow, clear or slightly white

turbid solution

Identification Peptide map Similar peaks are observed at the same retention

time as the reference material.

pH 5.8~6.8

Sugar chain profile Afucosyl sugar chains (G0): ≤ ■%

Purity test Ion-exchange chromatography Main peak: ≥ ■%

Acidic region: ≤ ■%

Basic region: ≤ ■%

Size exclusion chromatography Main peak: ≥ ■%

High molecular weight region: ≤ ■%

Low-molecular-weight region: ≤ ■%

Capillary electrophoresis

(non-reducing conditions)

Main peak: ≥ ■%

Endotoxin < ● EU/mg

Microbial limit test Total aerobic microbial count: ≤ ■ CFU/10 mL

Fungal count: ≤ ■ CFU/10 mL

Biological activity (CDC activity assay) ●~● Unit/mg

Content of protein 90~110 mg/mL

Explanation

Specifications

Test items, analytical methods, and acceptance criteria are just example.

Description

"White turbidity" was used as a translation of the English-language “opalescent” in clarity.

Peptide map

The notation of the acceptance criteria is an example and may have a variety of notations. In

addition, there may be a case in which a test is performed on a solution obtained by mixing the

76

sample solution and the standard solution, or a case in which the acceptance criteria is set for only a

typical part of the peaks obtained in the peptide map.

Name of the peak used in the purity test

Peak names used in various types of chromatography and capillary electrophoresis are an example

and not by way of limitation.

77

2.3.S.4.2 Analytical Procedures Note:

This section was an image according to "Specifications and Analytical Procedures" described in

Module 1, Application form that were before publishing the Notification “Handling of changes in

approval matters related to drug quality (Notification No. 0309-1 of the Evaluation and Licensing

Division, Pharmaceutical and Narcotic Bureau, Notification No. 0309-1 of the Evaluation and

Licensing Division, Pharmaceutical and Narcotic Safety Bureau, dated March 9, 2018)

System suitability: System suitability should be set with consideration of the test method as

appropriate.

The specifications and analytical procedures for hutumumab drug substance are as follows:

1. Content specification

The product contains 90 - 110 mg/mL of protein.

2. Description

The product is a clear or slightly white, turbid, colorless to pale yellow liquid.

3. Identification test; Peptide map

Weigh a portion of the product and hutumumab reference material, equivalent to ■ mg of protein,

freeze-dry, add ■ μL of XXX buffer solution and ■ μL of ■ mmol/L dithiothreitol solution, and

allow to stand at ■ºC for ■ minutes. Next, add ■ μL of ■ mmol/L iodoacetoamide solution and

allow to stand in the dark for ■ minutes, and then add ■ μL of ■ mmol /L dithiothreitol solution. To

this solution, add ■ μL of ■ mol/L tris hydrochloric acid buffer solution and ■ μL of lysyl

endopeptidase solution, and allow to stand at 37ºC for ■ hours. Add ■ μL of trifluoroacetic acid, and

use these solutions as the sample solution and standard solution, respectively. Perform the test with

100μL each of the sample solution and standard solution as directed under Liquid Chromatography

according to the following conditions, and compare the chromatograms: similar peaks appear at the same

retention times.

Operating conditions:

Detector: An ultraviolet absorption photometer (wavelength: 210nm).

Column: A stainless steel column 4.6 mm in inside diameter and 20 cm in length, packed with

octadecylsilanized silica gel for liquid chromatography (5 μm in particle diameter).

Column temperature: A constant temperature of about 25ºC

Mobile phase A: water and trifluoroacetic acid (1000:1).

Mobile phase B: A mixture of acetonitrile and trifluoroacetic acid (1000:1)

Flow of mobile phase: Control the gradient by mixing the mobile phase A and B as directed in the

78

following table.

Time after injection

(Min)

Mobile phase A

(vol%)

Mobile phase B

(vol%)

0~■ ■ →■ ■ →■

（Omission）（Omission）（Omission）

Flow rate: 1 mL per minute

System suitability:

System performance: When the procedure is run with 100 μL of the standard solution under the

above operating conditions, the resolution between the peak with the retention time of about XX

minutes and the peak with the retention time of about YY minutes is not less than 1.5.

Figure 2.3.S.4.2-1 Example chromatogram from Identification test (Peptide Map)

Standard Solution: Working reference material lot RS-1

4. pH

5.8~6.8

5. Sugar chain profile

Take an appropriate amount of the product, replace the solvent with buffer solution for enzymatic

treatment, and concentrate the solvent until the total amount is ■ μL. Add ■ μL of the peptide-N-

0

20

40

60

80

100

0 5 10 15 20 25 30 35 40 45 50 55 60 65

ｍA

Uat

XX

Xn

m

時間（min）Time (Min)

79

glycosidase solution to this solution, and allow to stand at 37ºC for ●-● hours. Add ■ μL of diluted

trifluoroacetic acid (1 in XXX), and evaporate to dryness. Dissolve ■ mg of 2-aminobenzamide in (■:

■) with ■ mL of a mixture of dimethylsulfoxide and acetic acid (100), and dissolve ■ mg of sodium

cyanoborohydride in this solution. Add ■ μL of this solution to the residue after evaporation to dryness,

allow to stand at 65ºC for ●-● hours, cool, add dropwise the total amount of the solution to a reagent

removal filter equilibrated with a mixture of water, water and acetic acid (100) (■:■), and acetonitrile,

and wash the solution with a mixture of acetonitrile, and acetonitrile and water (■:■). Add dropwise ■

mL of water to the filter, recover the eluting 2-aminobenzamide-labeled sugar chain solution, evaporate

the solvent to dryness under reduced pressure, add ■ mL of a mixture of the mobile phase A and the

mobile phase B (■:■), and use this solution as the sample solution. Perform the test with 50 μL of the

sample solution as directed under Liquid Chromatography according to the following conditions.

Determine each peak area of the sample solution by the automatic integration method, and calculate the

amount of afucosyl sugar chains by the area percentage method: not more than ■%.

Operating conditions

Detector: fluorometer (excitation wavelength: 330 nm, fluorescence wavelength: 420 nm).


amidated silica gel for liquid chromatography (5 μm in particle diameter).


Mobile phase A: Add ■ μmol/L ammonia solution to ■ mol/L formic acid, and adjust the pH of

the solution to ■.

Mobile phase B: acetonitrile

Flow of the mobile phase by changing the mixing ratio of the mobile phase A and the mobile phase

B as follows to control the concentration gradient.


Minutes

Mobile phase A

(vol%)

Mobile phase B

(vol%)

0~■ ■ →■ ■ →■


Flow rate: 0.5 mL per minute

Area Measurement Range: For ■ minutes after injection of the sample solution

System suitability test

Performance of the system: Dissolve ■ mg of the standard solution of sugar chains in ■ mL of a

mixture of acetonitrile and water (■:■), and use this solution as the system suitability test solution.

When the procedure is run with 50μL of the system suitability test solution under the above operating

conditions, XXXX and afucosyl sugar chains are eluted in this order with the resolution between these

peaks being not less than 1.5.

80

6. Purity test (1) Ion exchange chromatography

Take an appropriate amount of the product, add the mobile phase A to make a solution of ■ mg

protein/mL, and use this solution as the sample solution. Perform the test with 50 μL of the sample

solution as directed under Liquid Chromatography according to the following conditions. Determine

each peak area of the sample solution by the automatic integration method, and calculate the amounts of

them by the area percentage method: the main peak is not less than ■%, the acidic region is not more

than ■%, and the basic region is not more than ■%.

Operating conditions:


Columns: A polyether ether ketone tube an inside diameter 4.0 mm and a length of 20 cm is packed

with 10 μm cation-exchange resins for liquid-chromatography.


Mobile phase A: Dissolve ■ g of disodium hydrogen phosphate anhydride, ■ g of sodium

dihydrogen phosphate monohydrate and ■ g of sodium chloride in water to make ■ mL, adjust the

pH to 6.5 with phosphoric acid, and add water to make ■ mL.

Mobile phase B: Dissolve ■ g of disodium hydrogen phosphate anhydride, ■ g of sodium

dihydrogen phosphate monohydrate and ■ g of sodium chloride in water to make ■ mL, adjust the

pH to 6.5 with phosphoric acid, and add water to make ■ mL.

Flow of mobile phase: Control the gradient by mixing the mobile phase A and B as directed in the

following table.


(Min)

Mobile phase A

(vol%)

Mobile phase B

(vol%)

0~■ ■ →■ ■ →■



Area: About 2 times as long as the retention time of the main peak beginning after the solvent peak.

System suitability:

Test for required detectability: Take an appropriate amount of hutumumab reference material, add

the mobile phase A to make a solution containing ■ mg of protein per mL, and use this solution as

the solution for system suitability test. When the procedure is run with 50 μL of the solution for

system suitability test under the above operating conditions, the SN ratio of the main peak is not less

than ■.

System performance: When the procedure is run with 50 μL of the solution for system suitability test

under the above operating conditions, 0 K peak and 1 K peak are eluted in this order with the

resolution between these peaks being not less than ■.

81

Figure 2.3.S.4.2-2 Chromatogram of ion exchange chromatography of Representative batch ■

7. Purity test; (2) Size exclusion chromatography

Take an appropriate amount of the product, add the mobile phase to make a solution containing ■ mg

of proteins per milliliter, and use this solution as the sample solution. Perform the test with 50 μL of the

sample solution as directed under Liquid Chromatography according to the following conditions.

Determine each peak area of the sample solution by the automatic integration method, and calculate the

amounts of them by the area percentage method: the main peak is not less than ■%, the high molecular

weight region is not more than ■%, and the low molecular weight region is not more than ■%.




hydrophilic silica gel for liquid chromatography (5 μm in particle diameter).

Column A temperature: constant temperature around 25ºC

Mobile phase: Dissolve ■ g of anhydrous disodium hydrogen phosphate, ■ g of sodium

dihydrogen phosphate monohydrate and ■ g of sodium chloride in water to make ■ mL, and adjust

the pH to 6.5 with phosphoric acid, if necessary.


Area: About 2 times as long as the retention time of the main peak beginning after the solvent peak.

System suitability

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20

吸光

度


Acidic region Basic region

Main peak

Abs

orba

nce

Retention Time (Min)

82

Test for required detectability: Take an appropriate amount of hutumumab reference material, and

add the mobile phase A to make a solution containing ■ mg of protein per mL. When the procedure is

run with 50 μL of this solution under the above operating conditions, the SN ratio of the hutumumab

peak is not less than ■.

System performance: When the procedure is run with 50 μL of the molecular weight marker solution

under the above operating conditions, ■ and ▲ are eluted in this order with the resolution between

these peaks being not less than 1.5.

Figure 2.3.S.4.2-3 Chromatogram of size exclusion chromatography of Representative lots ■

8. Purity test; (3) Capillary electrophoresis (non-reducing conditions)

Take an appropriate amount of the product, and add water to make a solution containing ■ milligram

of proteins per 1 mL. To ■ μL of this solution add ■ μL of non-reducing sample buffer solution and

■ μL of a solution of mmol/L iodoacetoamide, warm at 70ºC for ■ minutes, and use this solution as

the sample solution. Perform the test with the sample solution as directed under Capillary

Electrophoresis according to the following conditions. Determine each peak area of the sample solution

by the automatic integration method, and calculate the amount of the peak by the area percentage method

using the peak area divided by the migration time of each peak: the principal peak is not less than ■%.



Capillary, a capillary tube of fused silica with an inner diameter of 50 μm and an effective length of 20

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20

吸光

度


8 13 18

High-molecular-weight region Low molecular weight region

Enlarged view

Main peak


Abs

orba

nce

83

cm

Capillary temperature: A constant temperature around 25°C

Electrolyte solution: SDS capillary electrophoresis buffer

Sample introduction method: Electrical introduction method

Electrophoresis; ■ Bolt

Running time: 15 minutes

System suitability

Test for required detectability: Take an appropriate amount of hutumumab reference material, and

add water to make a solution containing ■ mg of protein per mL. Take ■ μL of this solution, and

add ■ μL of water. To ■ μL of this solution add ■ μL of non-reducing sample buffer solution and

■ μL of a solution of ■ mmol/L iodoacetoamide, and warm at 70ºC for ■ minutes. When the

procedure is run with this solution under the above operating conditions, the SN ratio of hutumumab is

not less than 10.

System performance: Take an appropriate amount of hutumumab reference material, and add water

to make a solution containing ■ mg of protein per mL. To ■ μL of this solution add ■ μL of non-

reducing sample buffer solution and ■ μL of a solution of ■ mmol/L iodoacetoamide, and warm at

70ºC for ■ minutes. Confirm that the elution profiles of this solution are equivalent to those of the

standard electropherogram (see Figure 2.3.S.4.2-4) when the procedure is run with this solution under

the above operating conditions.

Figure 2.3.S.4.2-4 Electropherogram of Representative batch ■

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20

吸光

度


High molecular

weight region

Low molecular

weight region


Abs

orba

nce

84

9. Endotoxin

< ● EU/mg

10. Microbial limit

The total aerobic microbial count per 10 mL of the product is not more than ●● CFU and the total

fungal count is not more than ● CFU.

11. Biological activity

Take an appropriate amount of the product and hutumumab reference material, add the bioactivity test

medium to make solutions so that each contains ■ mg of proteins in 1 mL, and use these solutions as

the sample stock solution and standard stock solution, respectively. With the sample stock solution and

the standard stock solution, add the bioactivity test medium to make 2-fold dilution series of the sample

stock solution and the standard stock solution as ■, ■, ■, ■, ■, ■, ■, and ■ μg/mL and use

these series as the sample dilution series and the standard dilution series, respectively. Thaw the ■ cells,

wash with phosphate-buffered saline, and suspend in a suitable amount of the bioactivity test medium to

determine the cell number and viability. Add the test medium to make 1 mL contain ■ × 106 cells, and

use this solution as ■ cell suspension. Add ■ μL each of the sample solution series and the standard

solution series to 3 wells in column ■ to ■ of the microplates. Add ■ μL of the test medium in wells

of column ■ of the microplates for the blank series. Add ■ μL of the ■ cell suspension to each

well. After allowing to stand for ■ minutes at ■ºC, add ■ μL of the complement solution to each

well, and allow to stand for ■ minutes at ■ºC. Add ■ μL of Alamar Blue solution to each well, and

shake for ■ to ■ minutes at ■ºC. Perform the test with the solution in each well as directed under

Fluorescence Photometry, and determine the fluorescence intensity at the excitation wavelength of 530

nm and the fluorescence wavelength of 590 nm. Using the analysis software, generate a calibration curve

from the fluorescence intensity of the standard dilution series by 4-parameter logistic regression,

determine the relative potency of product, and determine the bioactivity of the product by the following

formula: ■ to ■ Unit/mg.

Bioactivity of the product (Unit per mg) = Relative potency × Bioactivity of hutumumab reference

material (Unit per mg)

System suitability

（Omission）

12. Assay

Take an appropriate amount of the product, add the assay buffer solution so that the absorbance at

280nm is between ● and ■, and use this solution as the sample solution. Determine the absorbance AT

85

of the sample solution at 280 nm as directed under Ultraviolet-visible Spectrophotometry, using the

buffer solution for assay as the blank.

Protein content (mg/mL) = AT/(1.38 × l) × dilution factor

1.38: Extinction coefficients (absorbance of hutumumab 1 mg in 1 mL)

l: Cell path length (cm)

13. Reagents・test solutions

（Omission）

Explanation

・ Overalls

Examples are provided and might include inconsistencies or unrealistic statements.

・ Sugar chain profile, purity test (ion exchange chromatography, size exclusion chromatography)

In view of the trend of the general test method Liquid Chromatography in PDG, the system

repeatability is not shown in the tests by the area percentage method.

・ Purity test (ion exchange chromatography, size exclusion chromatography)

As “Test for required detectability” of the system suitability, it is also possible to confirm that the

subpeak obtained from the standard solution prepared at the same concentration as the sample

solution is within a predetermined range.

・ Purity test (capillary electrophoresis (non-reducing conditions))

The system repeatability is not shown in the test as same as liquid chromatography with the area

percentage method.

・ Biological activity

For calculating method of analytical results, a statistical method should be established based on the

applicant's policy.

The method for judging the validity (acceptance condition) of the test was designated as "system

suitability", but the term is an example. The contents of the acceptance condition for the system

suitability are not illustrated because they vary depending on the test method, the analysis method

of the measurement results, and the like.

・ Reagents and test solutions

In this mock-up, the section of “reagents and test solutions” was described in the bottom of section

S.4.2. In other describing way, “reagents and test solutions” can be described at each test site.

Product names of marketed reagents, etc. can be described as “[product name] or equivalent” if

they may be changed in the future.

86

2.3.S.4.3 Validation of Analytical Procedures Note:

The following grey markers are examples where the applicant wishes to emphasize that the test was

conducted in accordance with the ICH Q2 guidelines.

Analytical method validation was performed on the test items listed in Table 2.3.S.4.3-1 based on the

"Text on Analytical Method Validation (Implementation items)" (Notification No. 755 of the Evaluation

and Licensing Division, Pharmaceutical and Medical Safety Bureau, dated July 20, 1995) and the "Text on

Analytical Method Validation (Implementation methods)" (Notification No. 338 of the Evaluation and

Limitation Division, Pharmaceutical and Medical Safety Bureau, dated October 28, 1997). The description

and pH were not validated, because they are in accordance with the Japanese Pharmacopoeia General Rules

and General Tests. Endotoxin was tested for reaction interfering factors. Microbial limits were evaluated

according to the suitability of the assay in the presence of the product.

Analytical validation was performed as described above to confirm the appropriateness of the test method.

Table 2.3.S.4.3-1 Validation of Test Items

Analytical method

parameters

Test items

Specificity

Linearity

Accuracy

Repeatability

Intermediate

precision

Detection lim

it

Quantitation

limit

Range

Identification test (peptide map) ○ - - - - - - -

Sugar chain profile ○ ○ ○ ○ ○ - ○ ○

Purity test: IEX ○ ○ ○ ○ ○ ○ ○ ○

Purity test: SEC ○ ○ ○ ○ ○ ○ ○ ○

Purity test: CE-SDS/non-

reduction

○ ○ ○ ○ ○ ○ ○ ○

Biological activity (measurement

of CDC activity)

○ ○ ○ ○ ○ - - ○

Content of protein - ○ ○ ○ ○ - - ○

○: Validated items

-: Items not validated

Analytical method validation results for each test item are as follows:

87

Table 2.3.S.4.3-2 Validation Results of the Identification Test (Peptide Map)

Validation

characteristics

Implementation details Results

Specificity Blank, ▲▲mab, and

hutumumab drug substance

were measured.

Hutumumab showed a characteristic profile.

It showed a different profile from ▲▲mab.

Table 2.3.S.4.3-3 Validation Results of Glycosylation Profiles

Validation

characteristics


Specificity Hutumumab Drug Substance and

blank solution were measured.

The peak derived from the sugar chain was

observed in the hutumumab drug substance. In

the blank solution, no peak was observed at

the elution position of each sugar chain.

Linearity Solutions of 5 levels ■ to 120%

were measured.

G0F:

Correlation coefficient: 0.9■■,

y-intercept: ● (95% CI ● to ●), residual

sum of squares: ●

G0: ...

Others total: …

Accuracy Three levels solutions of 80 -

120% were measured in triplicate

G0: ●% (95 % CI ● ~ ●%)

Other total: …

Repeatability Hutumumab drug substance was

measured in six replicates

G0:

Standard deviation: ●(90 %CI ● ~ ●)

Relative standard deviation: ●%

Others total: ...

Intermediate

precision

Hutumumab drug substance was

measured under 6 conditions with

■,■, and ■ as factors of

variability.

G0:

Standard deviation: ●(90 %CI ● ~ ●)


Others total: ...

Quantitation limit The % level solution was

measured in triplicate

●%

Range Hutumumab drug substance concentration of

80-120%

88

Table 2.3.S.4.3-4 Validation Results of Purity Test by Ion Exchange Chromatography

Validation

characteristics


Specificity Hutumumab drug substance and


In the blank solution, no peak was observed in

the elution position of the peak derived from

hutumumab.

Linearity 5 levels solution between ■

and 120% were measured.

Correlation coefficient :0.9■■

Y-intercept: ● (95 %CI ● ~ ●)

Residual sum of squares ●

Accuracy Three levels solution of 50-

150% were measured in

triplicate.

●% (95 %CI ● ~ ●%)


measured in six replicates.

Main peak

Standard deviation: ● (90 %CI ● ~ ●%)


Acidic region: …

Basic region: …

Intermediate

precision


measured under 6 conditions

with ■,■, and ■ as factors of

variability.

Main peak

Standard deviation: ● (90 %CI ● ~ ●)


Acidic region: …

Basic region: …

Detection limit The ■% level solution was


●%

Quantitation limit The ■% level solution was


●%

Range ● ~ ●%

89

Table 2.3.S.4.3-5 Validation Results of Purity Test by Size Exclusion Chromatography

Validation

characteristics





the elution position of the peak derived from

hutumumab.

Linearity 5 levels solution between ■ and

120% were measured.



Residual sum of squares : ●

Accuracy Three levels solution between 50

and 150% were measured in

triplicate.

●% (95 %CI ● ~ ●%)



Main peak



High-molecular-weight region: …

Low molecular weight region: …

Intermediate

precision



■, ■, and ■ as factors of

variability.

Main peak



High-molecular-weight region: …

Low molecular weight region: …

Detection limit Three repeated measurements at

the ■%level

●%

Quantitation limit Three repeated measurements at

the ■%level

●%

Range ● ~ ●%

90

Table 2.3.S.4.3-6 Validation Results of Purity Test by Capillary Electrophoresis (Non-reducing conditions)

Validation

characteristics


Specificity Hutumumab drug substance

and blank solution were

measured.


the migration time of the peak derived from

hutumumab.

Linearity 5 levels solution between ■

and 120% were measured.




Accuracy Three level solutions between

50 - 150% were measured in

triplicate.

●% (95 %CI ● ~ ●%)

Repeatability Hutumumab drug substance

was measured in six replicates.

Main peak



Intermediate

precision

Hutumumab drug substance

was measured under 6

conditions with ■, ■, and ■

as factors of variability.

Main peak



Detection limit Three repeated measurements

at the ■%level

●%

Quantitation limit Three repeated measurements

at the ■%level

●%

Range ● ~ ●%

91

Table 2.3.S.4.3-9 Validation Results of Biological Activity (CDC Activity Assay)

Validation

characteristics




Hutumumab drug substance showed cytotoxic

activity. Blank solution showed no cytotoxic

activity.

Linearity Five level solutions of ■ ~ ■

Unit/mL (equivalent to 70% to

130% of normal analyses) were

measured.




Accuracy Three level solutions of ■ ~ ■

Unit/mL (equivalent to 70-130%

of normal analyses) were

measured.

● % (95 %CI ● ~ ●%)

Repeatability Hutumumab drug substance were



Relative standard deviation :●%

Intermediate

precision




variability.


Relative standard deviation : ●%

Range ● ~ ● Unit/mg

92

Table 2.3.S.4.3-10 Validation Results of Contents of protein

Validation

characteristics


Linearity Five level solutions of ■ ~ ■

mg/mL (equivalent to 80% to

120% of normal analysis) were

measured.




Accuracy Five level solutions of ■ ~ ■

mg/mL (equivalent to 80% to

120% of normal analysis) were

measured.

●% (95 %CI ● ~ ●%)

Repeatability Hutumumab drug substance were



Relative standard deviation :●%

Intermediate

precision




variability.


Relative standard deviation : ●%

Range Hutumumab drug substance: 80 - 120 mg/mL

Explanation

・ Table 2.3.S.4.3-1: It is not mandatory to compile the assay performance parameters for all studies as

in this table.

・ Table 2.3.S.4.3-2 (Peptide Map), specificity: "Hutumumab has a characteristic profile Hutumumab

showed a characteristic profile. It showed a different profile from ▲▲mab." is an image of cases

where other antibody product (▲▲mab) have been produced and tested at the same facility. Because

of the high structural similarity of antibody between antibody products, specificity may be required to

allow their discrimination from other antibodies.

93

2.3.S.4.4 Batch Analyses

Table 2.3.S.4.4-1 Batch analysis of the drug substance Lot No. DS-P001 DS-0001 Manufacturing scale 1000 L 1000 L Manufacturing process Process A Process A Manufacturing date 20YY/MM/DD 20YY/MM/DD Manufacturing site Site A Site A Use Stability study, XXXX Standards (ST-1); Toxicology

studies; Phase I trials; XXXX Test items Results Results Description Pale yellow, slightly white turbid

solution Clear and colorless solution

Identification Peptide map Peaks of hutumumab-derived peptides were observed

Peaks of hutumumab-derived peptides were observed

Isoelectric focusing Three bands were observed near pI ●, ▲ and ■

Three bands were observed near pI ●, ▲ and ■

CDC activity Dose-response curves dependent on hutumumab concentration were observed

Dose-response curves dependent on hutumumab concentration were observed

pH 6.2 6.4 Sugar chain profile G0: ●% G0: ●% Purity Ion-exchange

Chromatography Main peak: ●% Acidic region: ●% Basic region: ●%

Main peak: ●% Acidic region: ●% Basic region: ●%

Size exclusion Chromatography

Main peak: ●% High-molecular-weight region : ●% Low molecular weight region: ●%

Main peak: ●% High-molecular-weight region : ●% Low molecular weight region: ●%

Capillary electrophoresis (Non-reducing condition)

Main peak: ●% Main peak: ●%

Host cell proteins ● ng/mg ● ng/mg Host cell-derived DNA ● pg/mg ● pg/mg Protein A ● ng/mg ● ng/mg

Endotoxin < ● EU/mg < ● EU/mg Microbial limit TAMC: ● CFU/10 mL

TYMC: ● CFU/10 mL TAMC: ● CFU/10 mL TYMC: ● CFU/10 mL

Biological activity (CDC activity assay) ● Unit/mg ● Unit/mg Content of protein 99 mg/mL 104mg/mL

94

Table 2.3.S.4.4-1 Batch analysis of drug substance (continued) Lot No. DS-0002 DS-0010 DS-0011 Manufacturing scale 1000 L 1000 L 1000 L Manufacturing process Process B Process B Process B Manufacturing date 20YY/MM/DD 20YY/MM/DD 20YY/MM/DD Manufacturing site Site B Site B Site B Use Reference material (ST-

2)、XXXX Clinical phase II study; Primary reference material (PRS-1); XXX

Working reference material (RS-1), XXXX

Test items Results Results Results Description Clear pale yellow liquid Clear and colorless


Identification

Peptide map Showed a band pattern similar to that of the reference material lot ST-1

Showed a band pattern similar to that of the reference material lot ST-2

Showed a similar elution pattern to that of the primary reference material lot PRS-1

Isoelectric focusing Showed a band pattern similar to that of the reference material lot ST-1

- -

CDC activity Dose-response curves dependent on hutumumab concentration were observed

- -

pH 6.3 6.4 6.5 Sugar chain profile G0: ●% G0: ●% G0: ●% Purity

Ion-exchange Chromatography





Main peak: ●% High-molecular-weight region : ●% Low-molecular-weight region: ●%



Capillary electrophoresis (non-reducing conditions)

Main peak: ●% Main peak: ●% Main peak: ●%

Host cell proteins ● ng/mg ● ng/mg ● ng/mg Host cell-derived DNA ● pg/mg ● pg/mg ● pg/mg Protein A ● ng/mg ● ng/mg ● ng/mg

Endotoxin < ● EU/mg < ● EU/mg < ● EU/mg Microbial limit TAMC: ● CFU/10 mL


TAMC: ● CFU/10 mL TYMC: ● CFU/10 mL

Biological activity (CDC activity assay) ● Unit/mg ● Unit/mg ● Unit/mg Content of protein 99mg/mL 104mg/mL 98mg/mL

95

Table 2.3.S.4.4-1 Batch analysis of drug substance (continued) Lot No. DS-0013 DS-0014 Manufacturing scale production 2000 L 2000 L Manufacturing process Process C1 Process C1 Manufacturing date 20YY/MM/DD 20YY/MM/DD Manufacturing site Site B Site B Use Stability study

XXXX Phase III Stability study XXXX

Test items Results Results Description Colorless, slightly white-turbid


Identification Peptide map With a similar elution pattern to that of the routine reference material lot RS-1

With a similar elution pattern to that of the routine reference material lot RS-1

pH 6.3 6.2 Sugar chain profile G0: ●% G0: ●% Purity Ion-exchange

Chromatography Main peak: ●% Acidic region: ●% Basic region: ●%



Main peak: ●% High-molecular-weight region: ●% Low-molecular-weight region: ●%

Main peak: ●% High-molecular-weight region: ●% Low-molecular-weight region: ●%



Host cell proteins ● ng/mg ● ng/mg Host cell-derived DNA ● pg/mg ● pg/mg Protein A ● ng/mg ● ng/mg

Endotoxin < ● EU/mg < ● EU/mg Microbial limit TAMC: ● CFU/10 mL


Biological activity (CDC activity assay) ● Unit/mg ● Unit/mg Content of protein 99 mg/mL 101 mg/mL

96

Table 2.3.S.4.4-1 Batch analysis of drug substance (continued) Lot No. DS-0017 DS-0018 DS-0019 Manufacturing scale 5000 L 5000 L 5000 L Manufacturing process Process C2 Process C2 Process C2 Manufacturing date 20YY/MM/DD 20YY/MM/DD 20YY/MM/DD Manufacturing site Site C Site C Site C Use Stability study

XXXX Stability study XXXX

Stability study XXXX

Test items Results Results Results Description Clear and colorless

solution Clear pale yellow liquid Clear and colorless

solution Identification

Peptide map With a similar elution pattern to that of the routine reference material lot RS-1



pH 6.2 6.4 6.3 Sugar chain profile G0: ●% G0: ●% G0: ●% Purity

Ion-exchange Chromatography









Main peak: ●% Main peak: ●% Main peak: ●%

Host cell proteins ● ng/mg ● ng/mg ● ng/mg Host cell-derived DNA ● pg/mg ● pg/mg ● pg/mg Protein A ● ng/mg ● ng/mg ● ng/mg

Endotoxin < ● EU/mg < ● EU/mg < ● EU/mg Microbial limit TAMC: ● CFU/10 mL


TAMC: ● CFU/10 mL TYMC: ● CFU/10 mL

Biological activity (CDC activity assay) ● Unit/mg ● Unit/mg ● Unit/mg Content of protein 102 mg/mL 100 mg/mL 101 mg/mL

TAMC, total aerobic microbial count; TYMC, total fungal count

97

2.3.S.4.5 Justification of Specification

1. Justification of Specification

In establishing the specifications, the results of a total of 5 batches, including 3 batches produced by the

commercial Process C2 and 2 batches produced by Process C1 (see 2.3.S.4.4), the results of analytical

method validation (see 2.3.S.4.3), and the results of stability studies (see 2.3.S.7), were considered.

1) Description

Considering the results of the batch analysis and stability studies, the specifications for the

description of hutumumab drug substance were set as clear or slightly white turbid, colorless to pale

yellow.

2) Identification test (peptide map)

The results of the 5 batches of hutumumab drug substance (see 2.3.S.4.4) were all "similar peaks at

the same retention time as the working reference material." Therefore, the acceptance criterion was set

as "the same peak is observed at the same retention time as that of the working reference material".

3) pH

Five lots of hutumumab drug substance tested ranged from 6.2 to 6.4 (see 2.3.S.4.4). Based on the

above, the acceptance criterion of pH was set as 5.8 to 6.8.

4) Sugar chain profile

The main mechanism of action of hutumumab is to bind to HS antigen-expressing cells and inhibit

the growth of HS antigen-expressing cells by complement-dependent cytotoxicity (Xxxxx et al.

2005) 。Effector function is part of the mechanism of action of hutumumab, and a link between the

percentage of afucosyl sugar chains and biological activity has been reported (Yyyyy et al. 2006).

Therefore, in the sugar chain profile analysis of hutumumab, the specification was set for G0 minus F,

which is the highest proportion among the afucosyl sugar chains.

The results of the test for the afucosyl carbohydrate "G0 minus F" in the sugar chain profile analysis

of the five lots of hutumumab drug substance were ■% to ■% (see 2.3.S.4.4). The acceptance

criterion of afucosyl sugar chains (G0 minus F) was set as ■% or less, taking into account the mean +

3× standard deviation = ■% and the variability of the analytical method (see 2.3.S.4.3).

5) Purity test

(1) Ion-exchange chromatography

The analytical results for 5 batches of hutumumab drug substance (2.3.S.4.4) are shown in the

table below. Considering the following results and the stability test results (2.3.S.7), the acceptance

criteria for the main peak, acidic region, and basic region were set as ≥ ■%, ≤ ■%, and ≤ ■%,

98

respectively.

Batch analysis results Ion exchange chromatography test results of five lots of hutumumab drug

substance

Main peak Acidic region Basic region

Minimum ■ ■ ■

Maximum ■ ■ ■

Mean ■ ■ ■

Standard deviation ■ ■ ■

(2) Size exclusion chromatography


table below. The following results and stability test results (2.3.S.7) were considered, and the

acceptance criteria for the main peak, high molecular weight region, and low molecular weight

region were set as ≥ ■%, ≤ ■%, and ≤ ■%, respectively.

Batch analysis results Size Exclusion Chromatography Test Results of Five Lots of Hutumumab

Drug Substance

Main peak High-molecular-

weight region (%)

Low molecular weight

region

Minimum ■ ■ ■

Maximum ■ ■ ■

Mean ■ ■ ■

Standard deviation ■ ■ ■

(3) Capillary electrophoresis (non-reducing conditions)


table below. The following results and stability test results (2.3.S.7) were considered, and the

acceptance criterion of the main peak was set as ≥ ■%.

Batch analysis results Capillary electrophoresis test results of five lots of hutumumab

drug substance

Main peak

Minimum ■

Maximum ■

Mean ■

Standard deviation ■

99

6) Endotoxin

Based on the formula of the K/M value shown in the Japanese Pharmacopoeia Reference

Information "Setting the endotoxin standard value", the endotoxin acceptance criterion of hutumumab

is calculated as follows.

5.0 (EU/kg) =X.X (EU/mg)

YYY (mg)

The acceptance criterion for endotoxin for hutumumab drug substance were set as < ■ EU per 1 mg

of hutumumab protein, taking into account the safety factor (■) that accounted for the variability in the

test method.

7) Microbial limit

Five lots of hutumumab drug substance tested ranged from 0 to 5 CFU/10 mL (see 2.3.S.4.4).

Acceptance criterion for microbial limits for hutumumab drug substance were set as total aerobic

microbial count and fungal count, ≤ ■ CFU/ 10 mL, respectively.

8) Biological activity (CDC activity assay)

From the analytical method validation result (2.3.S.4.3) of this biological activity test (CDC

activity method), the intermediate precision was ■ ~ ■%. Batch analyses of hutumumab drug

substances ranged from ■ to ■ Unit/mg (see 2.3.S.4.4).

Considering the above results and the stability study results, the acceptance criterion for the

bioactivity of hutumumab were set to be ■ to ■ Unit/mg.

9) Contents of protein

The test results for the content of 5 lots of hutumumab drug substance ranged from 99 to 102 mg

/mL. The test results were used to calculate the variation (mean ± 3× SD) in the manufacturing

experiences, which was 96.6 to 103.4 mg/mL. Based on the above manufacturing results of the drug

substance of hutumumab, the variability of the test method, and the results of the stability test, the

acceptance criterion of the content was set as "90 to 110 mg/mL"

2. Items not used as specifications and test methods

1) Host cell proteins

The host cell protein is set as an in-process control test at concentration and buffer exchange 2

step at which hutumumab concentration is highest during the manufacturing process , and which

can measure the host cell protein most sensitively. Therefore, it was not set as a specification test

for the drug substance.

100

2) Host cell derived DNA

Host cell derived DNA content was consistently low (approximately ■ pg/mg or below) or

below the quantitation limit across all drug substance batches from process A to process C2. In

addition, it was confirmed that the host cell derived DNA was consistently removed in the

purification process (see 2.3.S.2.5).

Therefore, it was determined that no specification for host cell-derived DNA was required for

the drug substance.

3) Protein A

Protein A content was consistently below the quantitation limit (< ■ ng/mg) throughout the

drug substance batches from process A to process C2. In addition, it was confirmed that protein A

was consistently removed in the purification process (see 2.3.S.2.5). Therefore, it was judged that

the specification setting of protein A is not necessary for the drug substance.

Explanation

・ Description

As the justification for establishing the specification, an example of using manufacturing

experiences (batch analysis) is shown.

・ pH

An example of using manufacturing experiences (batch analysis) as justification for the

specification is shown. Formulation development, etc. may be included as a justification for

establishing specification.

・ Sugar chain profile

As example of using the manufacturing experiences (batch analysis) and its statistical analysis

value, and the variability of the analytical method as the justification for the specification is shown.

- Reference: In general, detailed information on references are described in Module 3 (in this

case, 3.2.S.4.5). Electronic linking from Module 2.3 to Module 3 is not mandatory.

- Setting acceptance criteria based on statistical values: In the absence of a sufficient number of

manufacturing experiences (batch analysis results), it might be difficult to use statistical methods to

set acceptance criteria. Statistical methods include multiple methods (e.g., mean ±■SD,

confidence interval, etc.), and the choice of which is based on the applicant's policy.

- Statistical values can be listed in tables or in text only. The example described is the latter.

・ Ion-exchange chromatography

An example using manufacturing experiences (batch analysis) and its statistical analysis value,

variability of analytical method, and stability test results as the justification for setting the

specifications is shown.

・ Size exclusion chromatography

101

An example of using manufacturing experiences (batch analysis) and its statistical analysis value

and stability test results as the justification for setting the specifications is shown.

・ Capillary-electrophoresis



・ Endotoxin

This is an example using the reference information of the Japanese Pharmacopoeia and

manufacturing results (batch analysis) at commercial facility as the justification for setting the

specification.

・ Microbial limit

An example of using manufacturing experiences (batch analysis) at commercial facility as a

justification for establishing the specification.

・ Biological activity

As a justification for establishing the specification, variability of analytical methods (particularly,

because the variability of the cell-based assay is greater than that of the physicochemical tests), and

the results of the stability test are used as examples.

・ Contents of protein



102

2.3.S.5 Reference Standards or Materials (Hutumumab, HS Pharmaceutical) Note: Since it is possible to set only the primary reference material and not to set the working reference

material, the case where both are set and the case where only the primary reference material is set are

shown.

Examples for setting the primary reference material and the working reference material,

respectively

1. Primary reference material

Primary reference materials are used in the renewal of primary and working reference materials and

specification tests for hutumumab drug substance and drug product.

1) Current primary reference material

Current primary reference materials were prepared by diluting hutumumab drug substance with ■

mol/L of ●● buffer (pH ■). The results of characterization and release test are showed in Table

2.3.S.5-1.

103

Table 2.3.S.5-1 Analytical Results of Primary Reference Materials

Lot No. PRS-1

Lot No. of drug substance DS-0010

Drug substance manufacturing process Process B

Date of manufacturing of drug substance 20YY/MM/DD

Date of preparation of reference material 20YY/MM/DD

Test items Results

Description Clear and colorless liquid

Identification Peptide mapping Showed a similar peak profile to that

of the former primary reference

material (Lot No. ST-2)

pH 6.5

Purity test Ion-exchange chromatography Main peak: ●%

Acidic region: ●%

Basic region: ●%

Size exclusion chromatography Main peak: ●%

High-molecular-weight region : ●%

Low-molecular-weight region: ●%

Capillary electrophoresis (non-reduced

condition)

Main peak: ●%

Biological activity (CDC activity assay) ● Unit/mg

Content of protein 20.4 mg/mL

Characterization Primary structure Primary sequence

confirmation by peptide

mapping with LC/MS.

See section 2.3.S.3.1 Elucidation of

Structure and other Characteristics.

Heterogeneity of

charge variants

Ion-exchange

chromatography



Oligosaccharide

analysis

Liquid chromatography See section 2.3.S.3.1 Elucidation of


2) Renewal of Primary Reference Materials

Prepare the primary reference material by diluting hutumumab drug substance with ■ mol/L of ●●

buffer (pH ■). Perform the tests according to Table 2.3.S.5-2 and confirm that the results meet the

acceptance criteria. Also, confirm that it is the same as the primary reference material before renewal

by characterization (■■■ and ▲▲▲). Test methods of description, identification, pH, purity tests,

biological activity and content of protein are followed for "the specifications and test methods for

104

hutumumab drug substance", and current primary reference material is used as the reference material

for this tests.

Table 2.3.S.5-2 Specifications for Primary Reference Materials



Identification Peptide mapping Show a similar peak profile to former

primary reference material

pH 5.8~6.8

Purity test Ion-exchange chromatography Main peak: ≥ ●%

Acidic region: ≤ ●%

Basic region: ≤ ●%

Size exclusion chromatography Main peak: ≥ ●%

High molecular weight region: ≤ ●%

Low-molecular-weight region: ≤ ●%


condition)

Main peak: ≥ ●%

Biological activity (CDC activity assay) ● ~ ● Unit/mg

Content of protein 18.0~22.0mg/mL

Explanation

This mock-up provides an example of a case description of renewal for the primary reference material. If

the primary reference material is not renewed, state it.

3) Stability

Store at ‒60°C or below with a retest period of 2 years.

Explanation

Since the retest period or shelf life of the primary reference material (same applies to working reference

materials) is controlled under the GMP, it is not considered necessary to mention it in M2.3, but this

mock-up provides an example of the case description.

105

2. Working reference material

Working reference materials are used in specification tests for hutumumab drug substance and drug

product.

1) Current working reference material

Current working reference materials were prepared by diluting hutumumab drug substance with ■

mol/L of ●● buffer (pH ■).

The results of the release test are shown in Table 2.3.S.5-3.

Table 2.3.S.5-3 Analytical Results of Working Reference Materials

Lot No. RS-1





Test items Results


Identification Peptide mapping Showed a similar peak profile to that of the

primary reference material (Lot No. PRS-

1)

pH 6.5

Purity test Ion-exchange chromatography Main peak: ●%

Acidic region: ●%

Basic region: ●%


High molecular weight region : ●%

Low molecular weight region: ●%


condition)

Main peak: ●%


Content of protein 21.0 mg/mL

2) Renewal of Working Reference Materials

Prepare the working reference materials by diluting hutumumab drug substance with ■ mol/L of

●● buffer (pH ■). Perform the tests according to Table 2.3.S.5-4 and confirm that the results meet

the acceptance criteria. Test methods of description, identification, pH, purity tests, biological activity

and content of protein are followed for "the specifications and test methods for hutumumab drug

substance", and current primary reference material is used as the reference material for this tests.

106

Table 2.3.S.5-4 Specifications for Working Reference Material



Identification Peptide mapping Show a similar peak profile to the primary

reference material

pH 5.8~6.8




Size exclusion chromatography Main peak : ≥ ●%

High-molecular-weight region : ≤ ●%



conditions)

Main peak: ≥ ●%

Biological activity (CDC activity assay) ●~● Unit/mg

Content of protein 18.0~22.0 mg/mL

3) Stability

Store at ‒60°C or below with a shelf life of 2 years.

107

3. History of reference materials at the time of development

Table 2.3.S.5-5 shows the release test results of reference materials used in the development phase.

Table 2.3.S.5-5 Release test results of reference materials at the time of development

Lot No. of reference materials ST-1 ST-2

Lot No. of drug substances DS-0001 DS-0002

Drug substance manufacturing process Process A Process B

Date of manufacturing of drug substance 20YY/MM/DD 20YY/MM/DD

Date of preparation of reference material 20YY/MM/DD 20YY/MM/DD

Use XXX YYY

Test items Results Results

Description Clear and colorless liquid Clear and pale yellow liquid

Identification Peptide map (Not tested) Peaks of hutumumab-

derived peptides were

observed

Isoelectric focusing Three bands were observed

around pI ●, ▲ and ■.

(Not tested)

CDC activity Dose-response curve depend

on hutumumab

concentration was observed.

(Not tested)

pH 6.3 6.3

Purity Ion-exchange

chromatography

Main peak: ●%

Acidic region: ●%

Basic region: ●%

Main peak: ●%

Acidic region: ●%

Basic region: ●%

Size exclusion

chromatography

Main peak: ●%

High-molecular-weight

region : ●%

Low-molecular-weight

region: ●%

Main peak: ●%


region : ●%


region: ●%

Capillary electrophoresis

(non-reduced condition)


Biological activity (CDC activity assay) Set the value as

● Unit/mg

● Unit/mg

Content of protein 20.4 mg/mL 18.8 mg/mL

108

Table 2.3.S.5-5 Analysis results of reference material at the time of development (continued)

LotNo. ofreferencematerials ST-1 ST-2 C

hara

cter

izat

ion

Primary structure:

Primary sequence confirmation by

peptide mapping with LC/MS.

It was confirmed that the protein

was hutumumab.

Conforms to ST-1

Charge heterogeneity :

Ion-exchange chromatography

The percentages of isoforms

with 0, 1 and 2 lysines added to

the C-terminal of the heavy

chain (K0, K1 and K2) were,

●%,●% and ●%,

respectively.

Conforms to ST-1

Oligosaccharide analysis:

Liquid chromatography

Fucosyl biantennary glycans

with 0 to 2 added galactose at

the terminal (G0F, G1F, and

G2F) were ●% of the total

glycans, and afucosyl glycans

(G0) were ●%.

Conforms to ST-1

109

The examples of set only the primary reference material (not set the working reference

material), the same composition as that of the drug substance, and not set the stability of

reference material, are described.

1. Hutumumab reference material

Hutumumab reference materials are used in renewal of reference materials and specification tests for

hutumumab drug substance and the drug product.

1) Current reference material

Current hutumumab reference materials are dispensed hutumumab drug substance. Results of

characterization and release test are presented in Table 2.3.S.5-1.

Table 2.3.S.5-1 Analytical Results of Hutumumab Reference Material

Lot No. PRS-1





Test items Results


Identification Peptide map See section 2.3.S.3.1 Elucidation of


pH 6.4

Purity Ion-exchange chromatography Main peak: ●%

Acidic region: ●%

Basic region: ●%


High-molecular-weight region: ●%

Low-molecular-weight region: ●%


condition)

Main peak: ●%


Content of protein 104 mg/mL

Characterization Primary structure: Primary sequence

confirmation by peptide mapping with LC/MS.



Charge heterogeneity: Ion-exchange

chromatography



Carbohydrate analysis: See section 2.3.S.3.1 Elucidation of

110

Liquid chromatography Structure and other Characteristics.

2) Renewal of Hutumumab Reference Materials

Prepare the hutumumab reference materials by dispensing hutumumab drug substance. Perform the

tests according to Table 2.3.S.5-2 and confirm that the results meet the acceptance criteria. Also,

confirm that it is the same as the primary reference material before renewal by characterization (■■

■ and ▲▲▲). Test methods of description, identification, pH, purity tests, biological activity and

content of protein are followed for "the specifications and test methods for hutumumab drug

substance", and current primary reference material is used as the reference material for this tests.

Hutumumab reference materials were stored at -60°C or below.

Table 2.3.S.5-2 Specification of Hutumumab Reference Material


Description Colorless or pale yellow, clear liquid.

Identification Peptide map Show a similar peak profile to former reference

material

pH 5.8~6.8




Size exclusion chromatography Main peak : ≥ ●%

High molecular weight region : ≤ ●%

Low molecular weight region: ≤ ●%

Capillary electrophoresis (non-

reduced conditions)

Main peak: ≥ ●%

Biological activity (CDC activity assay) ● ~ ● Unit/mg

Content of protein 90~110 mg/mL

3) Reference material at the time of development

Table 2.3.S.5-3 shows the results of the analysis of the reference material used in the development.

Table 2.3.S.5-3 Analytical results of reference material at the time of development

Reference material lot number ST-1 ST-2

Drug substance lot number DS-0001 DS-0002

Drug substance manufacturing process Process A Process B

111

Date of manufacturing of drug substance 20YY/MM/DD 20YY/MM/DD

Date of preparation of reference material 20YY/MM/DD 20YY/MM/DD

Use XXX YYY

Test items Results Results

Description Clear and colorless liquid Clear and pale yellow liquid

Identification Peptide mapping (Not tested) Peaks of hutumumab-derived

peptides were observed

Isoelectric focusing Three bands were observed

around pI ●, ▲ and ■.

(Not tested)

CDC activity Dose-response curve depend

on hutumumab concentration

was observed

(Not tested)

pH 6.3 6.3

Purity Ion-exchange

chromatography

Main peak: ●%

Acidic region: ●%

Basic region: ●%

Main peak: ●%

Acidic region: ●%

Basic region: ●%

Size exclusion

chromatography

Main peak: ●%


region: ●%


region: ●%

Main peak: ●%


region: ●%


region: ●%

Capillary

electrophoresis (non-

reduced condition)


Biological activity (CDC activity assay) Set the value as ● Unit/mg. ● Unit/mg

Content of protein 104mg/mL 98mg/mL

112

Table 2.3.S.5-3 Analytical results of reference material at the time of development (Continued)

Reference material lot number ST-1 ST-2

Characterization Primary structure:

N-terminal amino

acid analysis by

peptide mapping

with LC/MS/MS.

Primary sequence at the N-

terminal of the heavy chain;

EVQLVES:

Conforms to ST-1

Charge

inhomogeneity :

Ion-exchange

chromatography

The percentages of isoforms

with 0, 1 and 2 lysines added

to the C-terminal of the heavy

chain (K0, K1 and K2) were,

●%,●% and ●%,

respectively.

Conforms to ST-1

Glycosylation analysis:

Liquid

chromatography

Fucosyl biantennary glycans

with 0 to 2 added galactose at

the terminal (G0F, G1F, and

G2F) were ●% of the total

glycans, and afucosyl glycans

(G0) were ●%.

Conforms to ST-1

Explanation

・ Primary and working reference materials

As it is not mandatory to set the working reference material in discrimination from the primary

reference material, examples of both case with and without setting working reference materials are

shown.

・ Preparation of reference materials

The examples for reference materials of both case, diluting and not diluting are shown.

・ Renewal of reference materials

An example of additional characterization in addition to release test when renewing the primary

reference material was shown.

If biological activity is set in units per protein-weight representation, such as Units per milligram,

then bioactivity shifts (drifts) result from the renewal of reference materials are considered minor. On

the other hand, when the biological activity is defined as the relative potency (percentage) with

respect to the reference material, drift of the activity value due to the renewal of the reference material

113

should be kept in mind.

・ Stability

Described examples of cases where the retest period or shelf life is set are shown in this mock-

up. A variety of other methods are available, such as writing stability data for reference materials

and extending the shelf life based on the results of stability studies.

Unlike drug substances and drug products, the retest period rather than the shelf life can be set

for reference materials.

・ Results of analysis of reference material at the time of development

"Release Test Results" indicates the results at the time of the initial analysis rather than the results

of the retest test.

114

2.3.S.6 Container Closure System (Hutumumab, HS Pharmaceutical)

Containers and closure systems used in the storage of hutumumab drug substance are as follows:

・ Container: 1 L plastic container (Material: Polyethylene)

・ Plastic caps (material: polypropylene)

Explanation

In this M2.3 mock-up, the following information are omitted to be described in case that those information

are described in M3 because of in-house control.

・ Specifications and Test Methods for Containers and Closure Systems

・ Methods to use (Pre-use sterilization/sterilization) as described in S.2.2 or A.1

・ Information on suitability of the container

115

2.3.S.7 Stability (Hutumumab, HS Pharmaceutical)

2.3.S.7.1 Stability Summary and Conclusions

Stability studies were conducted on Hutumumab Drug Substance under long-term storage conditions,

accelerated conditions, and stress conditions. The containers used for the following stability studies are made

of the same material as those used in the manufacturing of the Hutumumab Drug Substance:

1. Long-term stability study (-20°C)

Three batches of Hutumumab Drug Substance (Lot No. XXX, YYY, and ZZZ) manufactured at pilot

scale were studied for their stabilities under long-term storage condition at-20°C for 12 months in 100

mL plastic containers. All testing results did not change compared with baseline at the initial testing

point and met the acceptance criteria.

2. Accelerated stability study (5°C)

Three batches of Hutumumab Drug Substance (Lot No. XXX, YYY, and ZZZ) were studied for their

stabilities under accelerated condition at 5 °C for X months in 100 mL plastic containers. All test results

showed no change compared with baseline at the initial testing point.

3. Stress stability study (25°C)

A batch of Hutumumab Drug Substance (Lot No. XXX) was studied for its stability under at 25°C

for Y months in 15 mL plastic containers. Size exclusion chromatography showed a 1% reduction in the

peak area percentage of the monomer peak. Ion exchange chromatography showed a 20% reduction in

the peak area percentage of the main peak. No changes were observed in other testing items compared

with baseline at the initial testing point.

4. Conclusion

Based on the above testing results, the storage temperature and shelf life of Hutumumab Drug

Substance were set at-20°C and 12 months.

Explanation

・ ICH Q1A guideline describes that the stress testing for drug substance is to be conducted under light

exposure and oxidative conditions, but ICH Q5C guideline for biotechnological/biological products

does not require that. Since drug substances for biopharmaceuticals are generally solution and are

cryopreserved, stability testing under light exposure conditions is not considered mandatory and

would be conducted by the applicant for in-house data if needed.

116

2.3.S.7.2 Post-approval Stability Protocol and Stability Commitment

Three batches of Hutumumab Drug Substance (Lot No. XXX, YYY, and ZZZ) manufactured at the pilot

scale are ongoing, and the study protocol is shown in Table 2.3.S.7.2-1.

In addition, a long-term storage study of three batches of Hutumumab Drug Substance manufactured at

the commercial production scale will be conducted according to the ICH Q5C guideline, and the study

protocol is shown in Table 2.3.S.7.2-1.

Table 2.3.S.7.2-1 Stability study protocol (-20°C).

Test items Storage period (months)

0 3 6 9 12 18 24* 36*

Description + + + + + + + +

pH + + + + + +

Ion-exchange chromatography + + + + + + + +

Size exclusion chromatography + + + + + + + +

Capillary-electrophoresis + + + + + + + +

Protein content + + + + + + + +

Biological activity + + + + + + + +

* Applicant will decide whether to do or not do internally.

Explanation

In accordance to the matrixing method based on the prior knowledge of the development stage, pH at three

and nine months is planned not to measure.

117

2.3.S.7.3 Stability Data

Table 2.3.S.7.3-1 Long-term stability study (-20°C) of Hutumumab Drug Substance lot XXX.

Table 2.3.S.7.3-2 Long-term stability study (-20°C) of Hutumumab Drug Substance lot YYY.

Table 2.3.S.7.3-3 Long-term stability study (-20°C) of Hutumumab Drug Substance lot ZZZ.

Table 2.3.S.7.3-4 Accelerated stability study (5°C) of Hutumumab Drug Substance lot XXX.

Table 2.3.S.7.3-5 Accelerated stability study (5°C) of Hutumumab Drug Substance lot YYY.

Table 2.3.S.7.3-6 Accelerated stability study (5°C) of Hutumumab Drug Substance lot ZZZ.

Table 2.3.S.7.3-7 Stress stability study (25 ) of Hutumumab Drug Substance lot XXX

118

Table 2.3.S.7.3-1 Long-term stability study (-20 ± x °C) of Hutumumab Drug Substance lot XXX.

Item Specification Storage period (months)

0 3 6 9 12

Description Colorless to pale yellow, clear or


■■ ■■ ■■ ■■ ■■

pH 5.8~6.8 ■■ ■■ ■■ ■■ ■■

Ion-exchange

chromatography

Main peak: ≥ ●%

Acidic region: ≤ ●Y%


■■ ■■ ■■ ■■ ■■

Size exclusion

chromatography

Main peak: ≥ ●%

High molecular weight region: ≤ ●%


■■ ■■ ■■ ■■ ■■

Capillary-

electrophoresis

Main peak: ≥ ●% ■■ ■■ ■■ ■■ ■■

Protein content 90~110 mg/mL ■■ ■■ ■■ ■■ ■■

Biological activity ● ~ ● Unit/mg ■■ ■■ ■■ ■■ ■■

(Omission).

119

Table 2.3.S.7.3-4 Accelerated stability study (5 ± 3 °C) of Hutumumab Drug Substance lot XXX.

Item Storage period (months)

0 X X X

Description ■■ ■■ ■■ ■■

pH ■■ ■■ ■■ ■■

Ion-exchange chromatography ■■ ■■ ■■ ■■

Size exclusion chromatography ■■ ■■ ■■ ■■

Capillary-electrophoresis ■■ ■■ ■■ ■■

Protein content ■■ ■■ ■■ ■■

Biological activity ■■ ■■ ■■ ■■

(Omission).

Table 2.3.S.7.3-7 Stress stability study (25±2°C) of Hutumumab Drug Substance lot XXX.

Item Storage period (weeks)

0 X X X

Description ■■ ■■ ■■ ■■

pH ■■ ■■ ■■ ■■

Ion-exchange chromatography ■■ ■■ ■■ ■■

Size exclusion chromatography ■■ ■■ ■■ ■■

Capillary-electrophoresis ■■ ■■ ■■ ■■

Protein content ■■ ■■ ■■ ■■

Biological activity ■■ ■■ ■■ ■■

120

2.3.P DRUG PRODUCT (Koutaiyakuhin Intravenous Infusion 100 mg)

2.3.P.1 Description and Composition of the Drug Product (Koutaiyakuhin Intravenous Infusion 100 mg)

Note:

There may be a variety of cases for the description way of this section, such as in overfilled/non-overfilled

cases.

① Case of liquid formulation

The drug product is filled in a glass vial (5 mL) with a solution of pH 6.3 containing Hutumumab as the

active ingredient. The composition of the drug product is shown in Table 2.3.P.1-1.

Table 2.3.P.1-1 Composition of the Drug Product

Components Specification Purpose of use One vial

Amount per unit

Hutumumab (Genetical

Recombination)

Manufacturer's

specification

(Refer to 2.3.S.4)

Active ingredients 100 mg

Dibasic Sodium Phosphate

Hydrate

Japanese

Pharmacopoeia

Buffering agent X.X mg

Sodium dihydrogen phosphate Japanese

Pharmaceutical

Excipients

Buffering agent X.X mg

Sodium Hydroxide Japanese

Pharmacopoeia

pH adjuster Suitable amount

Polysorbate 80 Japanese

Pharmacopoeia

Surfactant X.X mg

■■■ Japanese

Pharmacopoeia

Stabilizer XX mg

Water for injection Japanese

Pharmacopoeia

Solvent Total 2.0 mL

121

② Case of lyophilized formulation

The drug product is filled in a glass vial (5 mL) with a solution of pH 6.3 containing Hutumumab as the

active ingredient.

The drug product is a lyophilized powder in which a solution containing Hutumumab as the active

ingredient is fed into glass vials (10 mL). The formulation of the drug product are shown in Table 2.3.P.1-1.

Table 2.3.P.1-1 Composition of the Drug Product

Components Specification Purpose of

use

Amount per vial

Labeled amount

Hutumumab (Genetical

Recombination)

Manufacturer's

specification

(see 2.3.S.4)

Active

ingredients

100 mg

Dibasic Sodium Phosphate

Hydrate

Japanese

Pharmacopoeia

Buffering

agent

X.X mg

Sodium dihydrogen phosphate Japanese

Pharmaceutical

Excipients

Buffering

agent

X.X mg

Sodium Hydroxide Japanese

Pharmacopoeia

pH adjuster XX mg

Polysorbate 80 Japanese

Pharmacopoeia

Surfactant X.X mg

■■■ Japanese

Pharmacopoeia

Stabilizer XX mg

Sucrose Japanese

Pharmacopoeia

Excipient 50 mg

122

2.3.P.2 Pharmaceutical Development (Koutaiyakuhin Intravenous Infusion 100 mg) 2.3.P.2.1 Components of the Drug Product

(Omission)

2.3.P.2.2 Drug Product (Omission)

2.3.P.2.3 Manufacturing Process Development (Omission)

2.3.P.2.4 Container Closure System (Omission)

2.3.P.2.5 Microbiological Attributes (Omission)

2.3.P.2.6 Compatibility (Omission)

123

2.3.P.3 Manufacture (Koutaiyakuhin Injection 100 mg)

2.3.P.3.1 Manufacturer(s) Note:

・ This case assumes sharing of manufacturing processes in several companies. In such case, each

company’s name and address, and the responsibility should be described. When one company conduct

manufacture, test and release, the description of the shared responsibility in this section can be

simplified.

・ The following example shows the case of liquid formulation in two cases of a liquid/lyophilized

formulation described in Section P.3.2-P.3.3.

Responsibility: Preparation for Bulk of Drug Product, Sterile filtration, Vial filling, Stoppering and

Crimping, Storage

Name of manufacturer: Setagaya Plant, HS Pharmaceutical Co., Ltd.

Manufacturer's address: X-XX-X, Kamiyoga, Setagaya-ku, Tokyo, Japan

Responsibility: Packaging and Labeling

Name of Manufacturer: ●● Pharmaceutical Co., Ltd.

Manufacturer's Address: X-XX-X, ●●, ■■-ku, Kawasaki City, Kanagawa Prefecture, Japan

Responsibility: Quality test

Name of the Laboratory: ■■ Pharmaceutical Co., Ltd. Testing Laboratory

Address of the laboratory : X-XX-X, ●●, Shibuya-ku, Tokyo, Japan

124

2.3.P.3.2 Batch Formula The batch size of the drug product is planned to be ● - ● L. The formulation of the drug product is

shown in Table 2.3.S.P.3.2-1.


Table 2.3.S.P.3.2-1 Batch Formula

Components Amount for ● L Amount for ■ L

Hutumumab (Genetical Recombination) XX g XX g

Dibasic Sodium Phosphate Hydrate XX g XX g

Sodium dihydrogen phosphate XX g XX g

Sodium Hydroxide XX g XX g

Polysorbate 80 XX g XX g

■ ■ ■ XX g XX g

Water for injection To make ● L To make ■ L


Table 2.3.S.P.3.2-1 Batch Formula

Components Amount for ● L Amount for ■ L

Hutumumab (Genetical Recombination) XX g XX g

Dibasic Sodium Phosphate Hydrate XX g XX g

Sodium dihydrogen phosphate XX g XX g

Sodium Hydroxide XX g XX g

Polysorbate 80 XX g XX g

■ ■ ■ XX g XX g

Sucrose XX g XX g

Water for injection To make ● L To make ■ L

Explanation in M1.2.

・ Applicant should describe “Range” or “Representative value” for Standard batch size.

125

2.3.P.3.3 Description of Manufacturing Process and Process Controls 1. Manufacturing Process Flow

The drug product manufacturing process is depicted in Figure 2.3.P.3.3-1.


Figure 2.3.P.3.3-1 Flow diagram of the manufacturing process of the drug product

Operation Materials In-process tests

Process of Compounding

Drug Product

Compounding Hutumumab drug

substance

■■■

Phosphate Buffer X Process Control 1

pH, bioburden

Filtration

Aseptic filtration process Process Control 2

Filter integrity test

Filling process

Filling Glass vial Process Control 3

Filling volume

Plugging Rubber stopper

Clamping process Aluminum cap

Packaging and Labeling

Process

Labels, Paper Boxes

Critical process

126


Figure 2.3.P.3.3-1 Flow diagram of the manufacturing process of the drug product

Operation Materials In process tests

Process of Compounding Drug Product

Compounding Hutumumab drug substance

Sucrose

■■■

Phosphate Buffer X Process Control 1

pH, bioburden

Filtration

Aseptic filtration process Process Control 2

Filter integrity test

Filling process

Filling Glass vial Process Control 3

Filling volume

Stopper placement Rubber stopper

Freeze-drying

Loading and freezing

Primary drying

Secondary drying

Clamping process Aluminum cap

Packaging and Labeling Process Labels, Paper Boxes

Critical step

127

2. Manufacturing process

(Below is the production method when the standard batch size for ● L.)


1) Process of compounding drug product

Prepare a solution dissolved ■■■ with phosphate buffer X (see 2.3.S.2.3). After thawing the drug

substance, add it to the ● L tank. Add ■■■ solution previously prepared to this solution, and adjust

the final concentration to be 50 mg/mL Hutumumab, and the final concentration to be ● mg/mL

■■■, then stir until the solution is homogenized. Determine the pH and bioburden (microbial count)

of this solution as in-process control, and confirm that it is within the range of pH ● to ●, and ≤

● CFU/● mL, respectively.

The solution is filtered through a hydrophilic filter with a pore size of 0.2 μm and transfer to a

steam-sterilized tank.

2) Sterile filtration process (Critical process)

Filtrate aseptically the solution in previous process with a [material] filter with a pore size of 0.2

μm. Perform integrity test for filter as in-process control and confirm compliance.

3) Filling process (Critical process)

Fill compounded drug product solution into the cleaned and dried sterilized colorless glass vials

(volume ● mL, JP compliant) using a fill pump in a clean room (in-process control: Filling volume

●±● mL). The filled vials are stoppered with washed and steam-sterilized rubber stoppers

(Materials: ***, JP compliant).

4) Clamping process

Clamp a stoppered vial with an aluminum cap using a Clamping machine.

5) Packaging process

Label and package in paper boxes.

128


1) Process of compounding drug product

Prepare a solution dissolved sucrose and ■■■ with phosphate buffer X (see 2.3.S.2.3). After

thawing the drug substance , add it to the ● L tank . Add ■■■ solution previously prepared to this

solution, and adjust the final concentration to be 50 mg/mL Hutumumab, and the final concentration

to be ● mg/mL ■■■ and 25 mg/mL sucrose, then stir until the solution is homogenized. Determine

the pH and bioburden (microbial count) of this solution as in-process control, and confirm that it is

within the range of pH ● to ●, and ≤ ● CFU/● mL, respectively.

The solution is filtered through a hydrophilic filter with a pore size of 0.2 μm and transfer to a

steam-sterilized tank.

2) Sterile filtration process (Critical Process)

Filtrate aseptically the solution in previous process with a [material] filter with a pore size of 0.2

μm. Perform integrity test for filter as in-process control and confirm compliance.

3) Filling process (Critical Process)

Fill compounded drug product solution into the cleaned and dried sterilized colorless glass vials

(volume ● mL, JP compliant) using a fill pump in a clean room (in-process control: Filling volume

●±● mL). The filled vials are partially stoppered with washed and steam-sterilized rubber stoppers

(Materials: ***, JP compliant).

4) Lyophilization process (Critical Process)

(1) Loading and freezing

After setting the shelf temperature of the freeze-dryer to ●°C, put the partially stoppered

vials, and freeze at the shelf temperature ●°C for ● hours.

(2) Primary drying

Raise the shelf temperature from ●°C to ●°C at ● Pa (● mbar) for ● hours, and then

dry primarily at ●°C for ● hours.

(3) Secondary drying

After completion of the primary drying, warm up to ●°C for ● hours. Then, dry

secondarily at ● Pa (● mbar) for ● hours.

(4) Stoppering completely and lift out

Recompress to ● Pa using nitrogen, stopper them completely. After that, recompress to

atmospheric pressure with compressed air at humidity not more than ●%.

5) Clamping process

Clamp a stoppered vial with an aluminum cap using a Clamping machine.

129

6) Packaging process

Label and package in paper boxes.

130

2.3.P.3.4 Controls of Critical Steps and Intermediates (Omission)

2.3.P.3.5 Process Validation and/or Evaluation (Omission)

131

2.3.P.4 Control of Excipients (Koutaiyakuhin Intravenous Infusion 100 mg) Note:

This case shows that all excipients are compliant with “Japanese Pharmacopoeia” or “Japanese

Pharmaceutical Excipients”. Therefore, the summary of 3.2.P.4.2 to 3.2.P.4.4 is not described in this

section (2.3.P.4).

When excipients are not compliant with the Japanese Pharmacopoeias, the specifications and test

methods, validation results of analytical methods, and justification of the specifications and test methods

should be described.

The specifications of the excipients used in this drug product are shown in Table 2.3.P.4-1. No novel

excipients are used in the manufacture Drug Product.

Excipients of human or animal origin are not used.

Table 2.3.P.4-1 Specification for excipient Ingredient Specification

Dibasic Sodium Phosphate Hydrate Japanese Pharmacopoeia

Sodium dihydrogen phosphate Japanese Pharmaceutical Excipients

Sodium Hydroxide Japanese Pharmacopoeia

Sucrose (in the case of lyophilized

formulation)


Hydrochloride Japanese Pharmacopoeia

Polysorbate 80 Japanese Pharmacopoeia

■ ■ ■ Japanese Pharmacopoeia

Water for injection Japanese Pharmacopoeia

132

2.3.P.5 Control of Drug Product (Koutaiyakuhin Intravenous Infusion 100 mg) (Omission)

2.3.P.6 Reference Standards or Materials (Koutaiyakuhin Injection 100 mg) (Omission)

2.3.P.7 Container Closure System (Koutaiyakuhin Intravenous Infusion 100 mg) (Omission）

2.3.P.8 Stability (Koutaiyakuhin Intravenous Infusion 100 mg) (Omission)

133

2.3.A Appendices 2.3.A.1 Facilities and Facilities (Koutaiyakuhin Injection 100 mg)

1. Production facility

Manufacturing facilities for the drug substance

Iwamoto Plant, HS Pharmaceutical Co., Ltd.

2. Manufacturing equipment

Major manufacturing equipment for the drug substance are shown in Table 2.3.A.1-1. Refer to 3.2.A.1

for more information.

Table 2.3.A.1-1 Major equipment used to produce the drug substance

Process Equipment

Cell Culture Step 1 250 L Fermenter

Cell Culture Step 2 1000 L Bioreactor

Cell Culture Step 3 5000 L Bioreactor

Purification 1 (Protein A column) Column and Chromatographic apparatus

Purification 2 (cation exchange column) Column and Chromatographic apparatus

Purification 3 (anion exchange column) Column and Chromatographic apparatus

Purification 4 (hydrophobic column) Column and Chromatographic apparatus

Concentration and Buffer Replacement 1 Dialysis/Ultrafiltration device

Concentration and Buffer Substitution 2 Dialysis/Ultrafiltration device

Explanation

・ In this mock-up, only information on manufacturing facilities and equipment of the drug substance which

are important for biopharmaceuticals was provided, and information on manufacturing facilities and

equipment of the drug product was not provided.

・ As major equipment, fixed equipment which can affect the product quality are listed in this mock-up.

134

2.3.A.2 Adventitious Agents Safety Evaluation (Hutumumab)

1. Non-viral adventitious agents

1) Safety evaluation of cell substrates

Table 2.3.A.2-1 shows the results of evaluation of non-viral adventitious agents in MCB and WCB.

Table 2.3.A.2-1 Results of evaluation of non-viral adventitious agents of cell substrates

Test substance Test items Acceptance criteria Results

MCB lot ■■ Sterility Negative Negative

Mycoplasma Not detected Not detected

WCB lot ■■ Sterility Negative Negative

Mycoplasma Not detected Not detected

2) Control practices for non-viral adventitious agents in the unprocessed bulk, drug substance, and drug

product

In all batches of the unprocessed bulk, test for bioburden and mycoplasma will be performed as in-

process control tests (see 2.3.S.2.2).

In all batches of the drug substance, test for bioburden and endotoxin will be performed as

specification tests (see 2.3.S.4).

In all batches of the drug product, sterility and endotoxin tests will be performed as specification

tests. (see 2.3.P.5).

3) Control of other non-viral adventitious agents in raw materials

Cell culture medium used in the manufacturing process shall be used after filter sterilization with a

filter with a pore size less than 0.2 μm.

For information on raw materials of biological origin, see 2.3.S.2.3.

135

2. Viral adventitious agents

1) Safety evaluation of cell substrates

The results of evaluation of viral adventitious agents in MCB, WCB and CAL are shown in Table

2.3.A.2-2.

Table 2.3.A.2-2 Results of evaluation of viral adventitious agents of cell substrates Test substance

Test items Acceptance criteria Results

MCB Lot ■■

Retrovirus and endogenous virus testing

Infectivity assay Not detected Not detected Electron microscopy No virus particles other

than type A or type C retrovirus-like particles known to be present in CHO cells

No virus particles other than type A and type C retrovirus like particles known to be present in CHO cells were observed.

Reverse transcriptase activity

Not detected Not detected

Tests for non-endogenous or adventitious viruses

In vitro test a) Not detected Not detected In vivo test b) Not detected Not detected Bovine virus test c) Not detected Not detected Porcine virus test d) Not detected Not detected Antibody production assay; HAP:


WCB Lot ■■


In vitro test a) Not detected Not detected In vivo test b) Not detected Not detected

CAL Retrovirus and endogenous virus testing

Infectivity assay Not detected Not detected Electron microscopy No virus particles other

than type A or type C retrovirus-like particles known to be present in CHO cells

No virus particles other than type A and type C retrovirus like particles known to be present in CHO cells were observed.

Reverse transcriptase activity



In vitro test a) Not detected Not detected In vivo test b) Not detected Not detected

a) Cells used in the in vitro study: MRC-5, Vero, CHO

b) Animals used in the in vivo study: suckling mice, adult mice, and embryonated chicken eggs

c) Cells used in the bovine viral test: Bovine turbinate and Vero

d) Cells used in the porcine virus test; Vero

2) Safety assessment of unprocessed bulk

Adventitious virus testing will be performed as an in-process control test in all batches of

unprocessed bulk (see 2.3.S.2.2).

Table 2.3.A.2-3 shows the results of an adventitious viral test (in vitro test) performed on three

batches of unprocessed bulk. Also, the number of retrovirus-like particles in the three unprocessed

bulk batches shown in the table below was evaluated by transmission electron microscopy with the

results of ■ × 106, ▼ × 106, and ● × 106 particles per mL.

136

Table 2.3.A.2-3 Results of adventitious virus testing of unprocessed bulk

Unprocessed Bulk Test items Acceptance criteria Results

Lot ●● Adventitious viruses

(in vitro test) a).


Lot ■■ Not detected Not detected

Lot ▲▲ Not detected Not detected

a) Cells used in the In vitro study: MRC-5, Vero, CHO

3) Safety evaluation of other raw materials

The porcine peptone used in the cell culture process is derived from a healthy pig and is believed to

inactivate the virus in the heat treatment conditions of the production method.

4) Viral clearance studies

① Selection of model viruses

In the viral clearance study of the purification process of Hutumumab, MuLV, MMV, and Reo3

were used as model viruses. Characteristics of these viruses are shown in Table 2.3.A.2-4.

Table 2.3.A.2-4 Model viruses used for viral clearance studies

Virus Family Genome Envelope Size (nm).

Murine leukemia virus (MuLV) Retroviruses RNA Yes 80-110

Mouse minute virus (MMV) Parvovirus DNA No 18-24

Reovirus type 3 (Reo3) Reovirus RNA No 60-80

② Validity of the scale-down model.

The validity of the scale-down model for use in viral clearance studies was assessed and

considered valid. See 3.2.A.2 for information on validity for scale-down models.

③ Results of viral clearance studies

Inactivation and removal of MuLV, MMV, and Reo3 were confirmed in the purification process of

Hutumumab. The test results are shown in Table 2.3.A.2-5. MuLV had total clearances of ≥18.1

during the purification process. Table 2.3.A.2-6 shows the results of time-dependent evaluation of

viral inactivation in the low pH treatment process.

137

Table 2.3.A.2-5 Viral clearance of Hutumumab purification process

Process Clearance capacity (Log reduction value)

MuLV MMV Reo3

Protein A

chromatography 4.5a) 4.2b) 1.5a) 1.6b) 3.0a) 2.5b)

Low pH treatment c) 4.4 4.5 ― ― ― ―

Anion exchange

column chromatography 3.5 a) 3.8b) 3.0a) 2.9b) 3.0a) 3.5b)

Virus removal filter

filtration >6.1 >6.0 4.0 4.2 >6.1 >5.9

Total clearance value d) >18.1 8.4 >11.4

―: Not tested

a) Test with a new resin

b) Tests on reused resins

c) See Figure 2.3.A.2-6 for data on hold time 60 min

d) Of the two tests at each step, the small LRV values were summed

Figure 2.3.A.2-6 Virus inactivation during the low pH treatment step (pH 3.6, 15°C).

Hold time (min)

Clearance capacity (Log reduction value)

MuLV

Study 1 Study 2

0 ― ―

10 3.5 3.7

30 4.0 4.2

60 4.4 4.5

120 4.5 4.6

4. Retroviral risk assessment

The risk assessment for retroviruses to be administered to patients was calculated as follows:

The maximum amount of Hutumumab administered to the patient is expected to be ■ mg,

which corresponds to ▲ mL as the culture supernatant solution. Also, the maximum number of

retrovirus-like particles in the unprocessed bulk, measured by electron microscopy, of ● × 106

particles/mL, was used to calculate.

The number of retrovirus-like particles contained in the unprocessed bulk corresponding to one

dose of Hutumumab is assumed to be (▲ mL) × (● × 106 particles/mL) = ▼▼. Using a total

clearance value of ≥ 18.1 for the MuLV in the purification process, the drug product contains less

than 10-X.X particles per dose.

138

Explanation

・ Table 2.3.A.2-1, Safety evaluation of non-viral adventitious agents for cell substrates: In this mock-up,

since bioburden and mycoplasma is tested in unprocessed bulk, CAL was not tested for bioburden and

mycoplasma.

・ 1., 3) Information on raw materials of biological origin should be included in 2.3.S.2.3 and/or 2.3.A.2

and the corresponding Module 3. The applicant should determine the balance between the amounts of

information provided in S.2.3 and A.2.

・ Table 2.3.A.2-2: As described in ICH Q5A guideline, viral testing (in vitro, in vivo) in WCBs is not

mandatory if viral testing (in vitro, in vivo) is performed in CALs. In this mock-up, viral testing (in

vitro, in vivo) was performed on WCBs, which is an example where developers voluntarily tested

beyond the requirements of the ICH Q5A guideline.

・ 4) Viral clearance studies, Table 2.3.A.2-5:

Reasons for not evaluating MMV and Reo3 at the low-pH treatment step: The evaluation for the

two viruses was omitted because it was empirically known that the low-pH treatment could not

be expected to inactivate the viruses.

This is an example only chromatography steps for which clearance could be expected were

characterized.

As two repeated tests of the chromatographic step, this mock-up exemplified the results of each

of the new resin and the reused resin. Other combinations for the two repeated tests are possible.

・ 4) Viral clearance studies, Table 2.3.A.2-6:

The test results of the virus inactivation step are shown in a tabular form. In other way, the

results can be graphically represented as inactivation curves.

139

2.3.A.3 Excipients The drug product does not use novel excipients.

140

CTD- Module 2: Mock-up of Quality Overall Summary

(Example described) ― Module 2.3 for antibody drugs Mock-up (example) ―

Publication date: May 7, 2018.

Published by Japan Health Sciences Foundation

2-11-1 Herb Kanda Building, Iwamoto-cho, Chiyoda-ku,

Tokyo, 101-0032, Japan

Phone 03(5823)0361

FAX 03(5823)0363

（Tomio Inokuchi, Secretariat of Japan Health Sciences Foundation）

Unauthorized reproducing and copying the document is prohibited without

permission from the publisher.

© 2018 Japan Health Sciences Foundation

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