Evolution of Specifications and Analytical

Methods During Synthetic Peptide Drug

Development

Michael Verlander, D.Phil., Proactive Quality Compliance

Biologics Monographs 1 – Peptides & Insulins Expert Committee Member

Webinar, March 14, 2019

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Definitions

Challenges

Regulatory guidance – specifications

Quality attributes/specifications for peptides

Characterization methods

Qualification/validation requirements

Peptide-related impurities

Origins

Regulatory guidance

Examples of Purity/Related Substances method development

Setting limits for peptide-related impurities

Bioassay/bioidentity considerations

Final thoughts

Agenda

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Protein

“Any alpha amino acid polymer with a specific defined sequence that is greater than 40 amino acids in size” (May be regulated as biological products or drugs, e.g., insulin and human growth hormone)

Chemically synthesized polypeptide

“Any alpha amino acid polymer that is (a) made entirely by chemical synthesis, and (b) is less than 100 amino acids in size”(Regulated as drugs unless they meet the statutory definition of “biological products”)

Peptide

“Any alpha amino acid polymer with specific defined sequence composed of 40 or fewer amino acids” (Regulated as a drugs unless they meets the statutory definition of “biological products”, e.g.

peptide vaccines)

FDA Draft Guidance. Biosimilars: Questions and Answers Regarding Implementation of the Biologics Price Competition and Innovation Act of 2009. February 2012

Definitions

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Biological product

“A virus, therapeutic serum, toxin, antitoxin, vaccine, blood, blood component or derivative, allergenic product, protein (except any chemically synthesized polypeptide), or analogous product, or arsphenamine or derivative of arsphenamine (or any other trivalent organic arsenic compound), applicable to the prevention, treatment, or cure of a disease or condition of human beings”

Proposed Rule: Definition of the Term “Biological Product”. Federal Register, Vol. 83, No. 238.

Wednesday, December 12, 2018

Definitions (Continued)

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Because of improved manufacturing technology, the complexity of synthetic peptides entering clinical development is increasing:

Longer sequences (up to 50 AAs or more)

Macrocyclic/multicyclic structures

Conjugates with e.g. lipids, PEG, proteins, etc.

Characterization of the drug substances and their related impurities becomes correspondingly more challenging

In order to keep pace with the clinical development program, the manufacturing process and the associated analytical methods (in-process and final product) must be developed in parallel

Because of their complexity, a relatively large number of tests is required to adequately describe the quality characteristics of peptides (the specification)

Challenges

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Additional testing is performed during characterization studies (ongoing throughout development)

Meeting regulatory requirements presents some challenges, since synthetic peptides are specifically excluded from most guidance documents, particularly those for product-related impurities

Challenges (Continued)

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ICH Q6A Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances

Specification:

“A specification is defined as a list of tests, references to analytical procedures, and

appropriate acceptance criteria which are numerical limits, ranges, or other criteria for

the tests described. It establishes the set of criteria to which a new drug substance or

new drug product should conform to be considered acceptable for its intended use.”

“The following tests and acceptance criteria are considered generally applicable to all new drug substances:”

Description:

“A qualitative statement about the state (e.g. solid, liquid) and color of the new drug

substance.”

Regulatory Guidance – Specifications

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Identification:

“Identification testing should optimally be able to discriminate between compounds of

closely related structure which are likely to be present. Identification tests should be

specific for the new drug substance, e.g., infrared spectroscopy.”

Assay:

“A specific, stability-indicating procedure should be included to determine the content of

the new drug substance. In many cases it is possible to employ the same procedure

(e.g., HPLC) for both assay of the new drug substance and quantitation of impurities.”

Impurities:

“Organic and inorganic impurities and residual solvents are included in this category.”

Regulatory Guidance – Specifications (Continued)

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Guidance for Industry. CGMP for Phase I Investigational Drugs

(US FDA, 2008)

“You should perform laboratory testing of the phase 1 investigational drug to evaluate

quality attributes including those that define the identity, strength, potency, purity, as

appropriate. Specified attributes should be monitored, and acceptance criteria applied

appropriately. For known safety-related concerns, specifications should be established and

met.”

Guidance for Industry. INDs for Phase 2 and Phase 3 Studies. Chemistry, Manufacturing and Controls Information (US FDA, 2003)

“During the clinical investigation process, the sponsor would establish tentative

acceptance criteria that are continually refined based on data obtained from analysis of

batches of drug substance and new information that becomes available. In the course of

product development, the analytical technology or methodology often evolves parallel to

the clinical investigations. In setting subsequent acceptance criteria, relevant correlations

should be established between data generated during early and late drug development.“

Regulatory Guidance – Specifications (Continued)

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Quality Attribute Method Comments

Appearance Visual inspection

Identification Mass spectral analysis

Amino acid analysis

HPLC comparison with standard

(co-elution)

Peptide mapping

Monoisotopic mass

Hydrolysis and derivatization methods

should be specified

Method may be same as related substances

Only rarely required (for complex

sequences)

Assay HPLC Preferred method if quantitative standard

available

Peptide Content Quantitative amino acid analysis

Nitrogen determination

Hydrolysis method must be validated; only

stable amino acids used for calculation

Elemental analysis preferred; Kjeldahl and

HPLC using chemiluminescence nitrogen

detector (CLND) may also be used

Routine Quality Attributes for Peptide APIs

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Quality Attribute Method Comments

Organic Impurities

Peptide-related Impurities, including aggregates (for more complex sequences)

HPLC methods (RP-HPLC, IEX-HPLC, SEC-HPLC, etc.)

UHPLC methods preferred; must be validated for both process-related and degradation impurities; limits for individual and total impurities should be specified

Non-peptide Impurities:Residual solvents

Residual TFA

Gas chromatography

RP-HPLC, Ion chromatography

May be limited to those used in the final process steps (after validation)Only if used in manufacturing process

Inorganic Impurities

Other anions Ion chromatography Only those used in manufacturing process (e.g., phosphate)

Fluoride Ion selective electrode orion chromatography

Only if used in manufacturing process

Routine Quality Attributes for Peptide APIs (Continued)

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Quality Attribute Method Comments

Inorganic Impurities (Continued)

Elemental impurities ICP-MS Required if metal catalysts used in manufacturing process; justification based on data from development if not tested

Other (Specific) Tests

Counterions, e.g.AcetateChloride

Other anions

Water content

Mass balance

BioburdenEndotoxins

RP-HPLC, ion chromatographyAgNO3 titration or ion chromatographyIon chromatography

Karl Fischer titration

Calculation

USP <61>, <62>USP <85>

Coulometric method most commonly used

Peptide content + counterion content + water contentTAMC and TYMCOften required for drug substances used for manufacture of parenteral products

Routine Quality Attributes for Peptide APIs (Continued)

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Specification:

A list of tests, analytical procedures and numerical limits or ranges which are used to establish the quality of the product

The specification tests must always be executed and results must be within the ranges/limits prior to batch release

Characterization:

A number of characteristics meant to describe the API, independent of process variation (but not necessarily independent of process)

Complete characterization may eliminate the need for tests in the specification (e.g., characterization of chiral impurity levels during development can eliminate need for this test as part of release)

Characterization studies typically start during process development to evaluate attributesof the API without knowing whether they may prove important or not; studies continue throughout development

Impurity understanding and tracking is a part of the characterization which can support process optimization studies

Characterization studies are summarized in section 3.2.S.3.1 of the registration file

Specification versus Characterization

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Method Comments

Sequence AnalysisMS-MS SequencingN-terminal Edman Degradation

May be complicated for longer sequencesNot applicable to N-terminally blocked sequences

Peptide Mapping Mainly for longer, more complex sequences; complementary to MS-MS sequence analysis

NMR Spectroscopy Complex interpretation for longer sequences

Infrared Spectroscopy Limited utility for peptides

Enantiomeric Purity (Chiral Amino Acid Analysis by GC-MS)

Non-routine, can be challenging for certain sequences

Optical Rotation Enantiomeric purity preferred

Higher Order Structure(by CD, NMR, FTIR)

Relevant for longer, more complex sequences

Bioidentity/Bioassay May be required for complex sequences where secondary structure essential for activity; mainly used for drug product

Small Molecule Impurities (from manufacturing process)

Non-peptide impurity limits required to follow ICH Q3A guidelines; potential genotoxic impurities should be evaluated

Examples of Supplementary Characterization Methods

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Requirements for many quality attributes are “generic” and provided in regulatory guidance documents, for example:

Residual solvents (limits provided in USP <467>)

Residual TFA (guidance for PDE calculation provided in USP <467>)

Elemental Impurities (limits provided in USP <232>)

Bioburden (limits for TAMC and TYMC provided in Ph. Eur. 2.6.12)

Bacterial endotoxins (calculation of limits provided in USP <85>)

Limits for product-specific attributes may be set on a theoretical basis and/or process experience, for example:

Counterion content (calculation of theoretical content + “safety factor”)

Water content (based on experience with lyophilized peptides)

Considerations for Development of Specifications

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Specifications for Purity/Related Substances are generally developed and refined throughout development, as the manufacturing process is optimized, analytical methods are improved and impurities are identified and controlled

In general, limits for all parameters should be set as loosely as possible initially, while still providing adequate control of critical variables, e.g. impurity profile (process flexibility) – should be tightened during development, based on data and process capability

Considerations for Development of Specifications (Continued)

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Test Tentative Acceptance Criteria Method

Description

Appearance White to off-white powder Visual inspection

Solubility Determined by formulation

requirements

Visual inspection

Identification

Mass Spectrometry Theoretical monoisotopic mass ± 1-2 amu (depending on MW)

USP <736>

MS/MS Sequence Analysis

(Tandem Mass Spectrometry)

Conforms to sequence USP <736>

Amino Acid Analysis (AAA) Theoretical ratios ±15%, except for

except for Cys, Ser, Trp, Ile, Val and

Pro, whose recovery may be low

USP <1052>; hydrolysis

method 1, analysis method 1

RP-HPLC Co-elutes with standard (if available) Product specific

Preliminary Specifications for Peptide APIs

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Test Tentative Acceptance Criteria Method

Peptide Content Report result Quantitative AAA

Nitrogen Content

Organic Impurities [Related Substances by (U)HPLC]

Peptide Purity ≥95% (by area integration; excluding

pyroglutamic acid or dimer formation,

if applicable)

Product specific

Related Substances ≤5% total

Individual Impurities ≤1%

Other tests

Counterion Content (RP-HPLC

or ion chromatography)

Report result USP <503> or

USP <1065>

Water Content (Karl Fischer) ≤10% USP <921> (method 1c)

Total Mass Balance 90 to 105% Calculation

(peptide content + counterion

content + water content)

Preliminary Specifications for Peptide APIs (Continued)

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Test Tentative Acceptance Criteria Method

Other tests

Residual Organic Solvents Complies with USP <467> limits;

piperidine, diisopropylethylamine:

report results (if applicable)

USP <467>

Residual TFA (RP-HPLC or ion

chromatography)

≤0.25% (justify on basis of PDE

calculation)

USP <503.1> or

USP <1065>

Elemental Impurities Complies with USP <232> limits USP <233>

Bioburden

TAMC

TYMC

<1000 CFU/g

<100 CFU/g

USP <61>

(membrane filtration method)

Bacterial Endotoxins (LAL) To be determined (based on maximum

allowed exposure limit, e.g., 5 EU/kg/hr

for IV)

USP <85>

Preliminary Specifications for Peptide APIs (Continued)

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Compendial methods

Must be shown to be suitable for all phases of development

– Lack of interference

– Recovery studies

Non-compendial methods (mainly related substances)

Phase I/II:

– Appropriate qualification required, particularly with respect to specificity

– Must be shown to be stability-indicating using forced degradation studies, where appropriate, to support ongoing stability studies

Phase III:

– All methods should be validated prior to initiating process validation

In-process methods

– Should be appropriately validated prior to initiating process validation, e.g.:

▪ In-process HPLC methods used during purification should be validated, at a minimum, for

specificity, linearity, robustness and LOQ

Note: Methods are often an adaptation of release methods

Validation Recommendations

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I. Eggen, B. Gregg, H. Rode, A. Swietlow, M. Verlander and A. Szajek (2014). Control Strategies for Synthetic

Therapeutic Peptide APIs. Part III: Manufacturing Process Considerations. Pharm. Tech. May edition.

Formation of Product-related Impurities in Synthetic Peptides

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*Note: possibility of β-Ala containing impurities derived from Fmoc-amino acid derivatives

HPLC = high performance liquid chromatography; LC-MS = liquid chromatography-mass spectrometry;

LC-MS/MS = liquid chromatography-tandem mass spectrometry; AAA = amino acid analysis

Process-derived Related Substances in Synthetic Peptides

Impurities Origin of Impurities Identification MethodHPLC

Detectability

Deletion Synthesis LC-MS or LC-MS/MS +/-

Insertion* Raw material

or Synthesis

LC-MS or LC-MS/MS +/-

Truncation Synthesis LC-MS +

Diastereomer Raw material

or Synthesis

HPLC spiking with synthesized

diastereomeric analogs; chiral GC-MS

+/-

Substitution

(Leu/Ile)

Raw material HPLC spiking with synthesized

analogs or isolation/AAA

-

Functional group

modification

Synthesis

or Stability

LC-MS or LC-MS/MS +/-

Disulfide

modification

Synthesis

or Stability

LC-MS or LC-MS/MS ++

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HPLC = high performance liquid chromatography; LC-MS = liquid chromatography-mass spectrometry; LC-MS/MS =

liquid chromatography-tandem mass spectrometry;

HPLC-IEX = high performance liquid chromatography-ion exchange chromatography;

HPLC-SEC = high performance liquid chromatography-size exclusion chromatography.

Related Substances in Peptides – Degradants

ImpuritiesIdentification

Method

HPLC

Detectability

Deamidation of Gln/Asn/

C-terminus

LC-MS or LC-MS/MS; HPLC-IEX; HPLC

spiking with synthesized analogs

+/-

Acetylation of amino functions LC-MS or LC-MS/MS ++

Oxidation (especially Met sequences) LC-MS ++

β-Asp formation in Asp or Asn sequences LC-MS or LC-MS/MS; (HPLC-IEX) -

Backbone cleavage LC-MS ++

Disulfide modification LC-MS or LC-MS/MS ++

Aggregation HPLC-SEC ++

Modifications caused by excipients (e.g.

degradants of PLGA or PEG)

LC-MS or LC-MS/MS ++

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ICH Q3A Impurities in New Drug Substances

Note: Applies only to new drug substances manufactured by chemical synthesis; peptides and

biological/biotechnology products specifically excluded

Impurity Thresholds in Drug Substances

Maximum

Daily Dose

Reporting

Threshold

Identification

Threshold

Qualification

Threshold

≤2g/day 0.05% 0.10% or 1.0 mg per day

intake (whichever is lower)

0.15% or 1.0 mg per day

intake (whichever is lower)

>2g/day 0.03% 0.05% 0.05%

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EP General Monograph 2034 Substances for Pharmaceutical Use

Table 2: Reporting, identification and qualification of organic impurities in peptides obtained by chemical synthesis

Not accepted by US FDA and some other regulatory agencies which work on a case-by-case

basis and require specification limits to be justified

Impurity Thresholds in Drug Substances (Continued}

Reporting threshold Identification threshold Qualification threshold

≥0.1% >0.5% >1.0%

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Draft Guidance: ANDAs for Certain Highly Purified Synthetic Drug Products That Refer to Listed Drugs of rDNA Origin (US FDA, October 2017)

Scope limited to the following products:

Glucagon, liraglutide, nesiritide, teriparatide and teduglutide

“Given the current state of technology for peptide synthesis and characterization, FDA believes it is now possible for an ANDA applicant to demonstrate that the active ingredient in a proposed generic synthetic peptide drug product (proposed generic synthetic peptide) is the “same” as the active ingredient in a previously approved peptide of rDNA origin.”

Recent Peptide Guidance Document

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Draft Guidance: ANDAs for Certain Highly Purified Synthetic Drug Products That Refer to Listed Drugs of rDNA Origin (US FDA, October 2017)

Impurities :

New impurities ≤0.5%

Common impurities (including degradants) ≤RLD

New impurities ≥0.10% and ≤0.5% must be identified and justified on the basis of lack of

impact on physicochemical properties, biological activity or immunogenicity risk

Use of UHPLC-HRMS methods recommended

Recent Peptide Guidance Document (Continued)

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Regulatory expectations are increasingly focused on the use of UHPLC methods, especially for longer, more complex sequences

It is strongly recommended that method development should start early so that the impact of process changes during manufacturing process development can be assessed (also stability studies)

Multiple, orthogonal methods are recommended in order to minimize therisk of co-elution of impurities with the main peak, or with each other

At least one method should be MS-compatible

Additional characterization methods may be required, e.g.:

–Chiral analysis if stereoisomers are not resolved

–Peak purity analysis by LC-MS (UV peak purity may not be sufficient)

Maintaining resolution: assess method robustness

Expectations for Related Substances Methods

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The suitability of the method(s) should be assessed for process-related impurities and degradation products

Forced degradation studies should be performed to support method suitability

Structural assignments should be confirmed through synthesis where possible

Tox batch should be used as standard for impurity profile

Should be manufactured by same process as clinical batches but of lower purity than

routine production batches

Impact of process changes on impurity profile should be assessed against tox batch

Expectations for Related Substances Methods (Continued)

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Requirements unclear since most general guidance documents specifically exclude synthetic peptides

Likely to be treated on a case-by-case basis by US FDA

Focus of development should be resolution and quantitation of process-related impurities and degradation products from the substance and also from each other, if possible

Should be confirmed using HR-MS studies

May be difficult for complex sequences

May not be possible with a single method

Identification of Related Substances

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2-Dimensional LC-MS can facilitate identification of impurities in non-LC-MS compatible mobile phases

Identification can be used for optimization/control of manufacturing process

Quantitation may be difficult or impossible for co-eluting peaks (i.e., coelution of impurities with the product or co-elution of impurities with each other)

Note: Area percentages expected to reflect wt. %, based on similar UV extinction coefficients of product and impurities (Kuipers B.J.H. and Gruppen H., J. Agric. Food Chem. 2007, 55, 5445)

Identification of Related Substances (Continued)

Examples from Method Development Studies

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Peptides containing Asx-Gly, Asx-Ala, Asx-Ser, and Asx-Phe (and often other Asx dipeptide sequences) are known to undergo the transformations indicated on the scheme below, where R1 denotes the N-terminal part of the sequence, R3 denotes the C-terminal part of the sequence and R2 depends on the amino acid (Gly: R2 = H; Ala: R2 = CH3, Ser: R2 = CH2-OH; Phe: R2 = CH2-Phe). Low pH (<7) typically favors form (2), whereas high pH favors form (3) and/or cleavage (4).

*Asx = Asp, Asn; Y = Gly, Ala, Ser, Phe

Degradation of Peptides Containing Asx-Y Sequences*

R3

NH

O

O

N CH

O

R1 R2

R3

NH

O

O

NH

CH

O

R1 R2

OH

R3

NH

O

O

NH

CH

O

R1 R2OH

R3NH2

CH

O

R2NH

O

O

R1

OH

OH

(1) (2) (3)

+

(4)

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ACTH (1-39): SYSMEHFRWGKPVGKKRRPVKVYPNGAEDELAEAFPLEF

System: TFA* System: TEAP*27 to 47% in 40 min 21 to 41% in 40 minRs = 0.7 Rs = 1.2

System: NaClO4* System: IEX, pH 7.0**31 to 51% in 40 min 5 to 65% in 40 minRs <0.5 Rs = 6.7

*Column: Supelco Discovery HS C18, 150 x 4.6 mm, 3 µm, 120 Å

**Column: Polysulfoethyl A, 200 x 4.6 mm, 5 µm, 200 Å

Analytical Development: ACTH (1-39) vs. [Asp]25-ACTH (1-39)

0 10 20 30

Time (min)

020

040

0

0 10 20 30

Time (min)

010

020

030

040

0

mA

U0 10 20 30

Time (min)

010

020

030

0

mA

U

0 10 20 30

Time (min)

020

4060

8010

012

014

0

mA

U

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GLP-2: HADGSFSDEMNTILDNLAARDFINWLIQTKITD

Mobile Phase A = 0.1% TFA/H20 Mobile Phase A = TEAP, pH 2.3 Mobile Phase A = KPF6

Mobile Phase B = 0.1% TFA/ACN Mobile Phase B = ACN Mobile Phase B = ACNFlow Rate = 1.5 mL/min Flow Rate = 1.5 mL/min Flow Rate = 1.5 mL/minTemperature = Ambient Temperature = Ambient Temperature = AmbientGradient = 35% - 60% B Gradient = 35% - 60% B Gradient = 35% - 60% B Time = 25 min Time = 25 min Time = 25 min

Separation of Degradants in Peptides Containing Asx-Y Sequences

97.82% 93.58% 86.73%

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Column: ACQUITY UPLC Peptide CSH 130 C18, (150 x 2.1 mm) 1.7 µm; flow rate = 0.1 mL/min; run time = 90 min

UHPLC Separation of Impurities During Development

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LOQ of method(s) must be taken into account

For shorter peptides (≤10 AAs), FDA may require ICH Q3A limits for related substances (i.e., identification at the level of 0.10%)

For generic products, limits generally determined by those present in the innovator’s product

Limits for individual impurities may not exceed those for the same impurities in the

innovator’s product (comparison may be difficult since API from innovator usually not

available)

Limits for new impurities (i.e., not present in the innovator’s product) likely to be

negotiated with FDA and may be relatively strict (e.g., 0.1-0.2% for smaller peptides and

up to 0.3-0.5% for longer sequences)

If monograph exists, limits for identified and unidentified impurities usually specified

Setting Limits for Related Substances

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For new, proprietary products limits for identified and specified (e.g., by RRT or MW), unidentified impurities may be set on the basis of levels that have been qualified in tox studies and process capability established during development

Limits for unidentified impurities are likely to be negotiated with FDA on a case-by-case basis

Setting Limits for Related Substances (Continued)

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Bioassay

Traditionally used to measure the potency of products relative to a standard

–reflects the mechanism of action of the product and is defined in “units” of activity, not mass

Generally not a requirement for release testing of synthetic peptide APIs

Bioidentity

Like bioassay, must reflect mechanism of action, maintaining a link to clinical activity

May be similar to a bioassay but generally requires less replicates since the specification is usually “not less than” a specified value

While this may be a requirement for release testing of some peptide APIs manufactured by recombinant techniques (e.g., teriparatide, salmon calcitonin), generally not required for synthetic peptides, since they can be fully characterized by physicochemical techniques (exception: bivalirudin).

Expectation that bioidentity should be included as part of characterization studies, especially for more complex sequences.

Bioassay/Bioidentity considerations

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Peptides are specifically excluded from most guidance documents

To date, the US FDA has not published any general guidance for peptides, specifically for impurities (identification and qualification)

FDA may require ICH Q3A limits for related substances in shorter peptides (≤10 AAs)

Characterization studies should be used throughout development to support the establishment of specifications

Specifications for many quality attributes based on existing guidance

Impurity identification important for manufacturing process optimization

Specifications and associated limits should evolve during development, based on process refinements, development of analytical methods and process experience

The October 2017 Draft Guidance may be indicative of FDA’s current thinking for complex synthetic peptide therapeutics

Final Thoughts

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Send USP your comments on the new General Chapter <1503> Quality Attributes of Synthetic Peptide Drug Substances, online May 1, 2019in the Pharmacopeial Forum 45(3), see: https://www.uspnf.com/pharmacopeial-forum-new

Submit your abstract for a talk or poster (or attend) in the next USP Workshop on Peptide and Oligonucleotide Therapeutics: Regulations, Standards and Quality, November 4-5, 2019, see: https://www.usp.org/events-training/workshops.

Opportunities to Get Involved with USP Standard Setting

Thank You!