applying prior knowledge to streamline early-stage analytical … · 2018. 4. 2. · biogen |...

1Biogen | Confidential and Proprietary

Applying Prior Knowledge to Streamline Early-stage Analytical Development of Monoclonal Antibody

Li ZangAnalytical Development, Biogen

CMC Strategy Forum 2018


Pre-IND mAb Development

Developability

Cell Line Development

Cell Bank Prep/Testing

Upstream/Downstream Development

DS Mfg DP Mfg

Pilot 1 Pilot 2 Pilot 3 Release/Label/Ship

Development Stability

IND

IND Tox Recovery/final rpt


Analytics Support the Pre-IND Development

How to drive an efficient pre-IND mAb analytical development?

• Analytical Method Development,

• Method qualification and transfer to QC;

• Testing for process development;

• Characterization of critical batches;

• Stability testing; • Reference standard


Use of Prior Knowledge

Critical quality attributes

Analytical method capability

Current process capability and impact/risk on PQs

Historical data from multiple mAbs: • Process

development;• Manufacturing;• Structure/function

relationship study• Stability study

Goals:• Select right set of PQs

to monitor• Accelerate method

development • Perform value-added

method qualification• Design high-throughput

assays monitoring multiple attributes


Case study 1Cell line development testing strategy


General Cell Line Development Workflow

Transfection, Recovery

and Selection

Enrichment, Cloning,

and Scaleup

Pool Fed Batch

(24DW)20-40 pools

Clone Fed Batch (96DW)

600-1000 clones

Ambr Evaluation 23 clones +

pool of clones(duplicates)

3L ConfirmationLead and Backup

(duplicates)

2 Clones 3L volume

PQ at time course

23 Clones 15 ml

volume

PQ at three time points

600-1000 Clones

1 ml volume

PQ at harvest only

20-40 Pools 5 ml volume

PQ on Harvest only

What PQs to measure?


Cell Line Development and Analytical Testing Strategy

~400 Clones24-DW Plate

~800 Clones96-Well plate

12-24 ClonesAMBR

Time Course

Final ClonePilot Scale

Time Course

TiterHarvest Day Only

TiterHarvest Day Only

~48 Clones96-DW PlateHarvest Day

12-24 Clones AMBR

Product quality testing

Question 1: What are the PQs to monitor?1. Sequence variant2. N-glycan3. PTMs4. Charge variants5. Purity and impurities6. Functions

Question 2: What types of PQs are they?1. Molecule-dependent2. Cell line-dependent3. Process-dependent


Historical Data for 5 mAbs were Analyzed

Measures of DispersionIQR = Q3 – Q1MADM = 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑥𝑥𝑖𝑖 − 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑥𝑥Standard Deviation (σ)CV = 𝜎𝜎

𝜇𝜇

IQR correlated to MADM (R2>0.8)CV is scale invariant and skews high for sets with lower mean values

Only IQR, SD used for analysesHigh IQR

High SD

• Assays with a higher IQR or SD provide results with a greater variability • Higher variability PQs are valuable as they provides stronger screening of cell lines

mAb1

mAb2

mAb3

mAb4


0

2

4

6

8

10

0 2 4 6 8 10

SD

IQR

0

2

4

6

8

10

12

14

16

18

20

0 5 10 15 20

SD

IQR

Total HMW (%)

Total LMW (%)

Total** impurity%

Basic Variant

Main Variant

Acidic Variant (SUM)

Total afucsose

Total fucosylation

Total HM

Total sialylated forms

Total charged glycans

%Gal

Assessment of All PQ ResultsPQs with the highest IQR or SD:1. %Gal2. %Acidic variant3. %Main variant4. %Purity5. %Afucosyl6. %Sialylated

% purity

%Gal

• Data from top 36 clones from the 96 DW plate were analyzed from 5 mAbs

• Assays located in the top right quadrant (N-glycan, GXII charge variant) provide more data for clone rejection than those in the bottom left (SEC)


mAb 1 PTM from Peptide Maps of 36 Clones

K glycation

N-glycan, A2G0F

N-glycan, A2G1F

W oxidationN-glycan, Man5 K glycation

W oxidation


mAb 2 PTM from Peptide Maps of 36 Clones

N-glycan, A2G0F

N-glycan, Man5

N-glycan, A1G0F


Molecule-dependent PQ Cell line-dependent PQ Process-dependent PQK glycationW oxidation

N-glycan (Man5, Galactosylation, afucosylation)PurityCharge heterogeneitySequence variantOxidative stress tolerance (carbonylation)

To Answer the Two Earlier Questions

Assays that can cover the above PQs:1. Multiple-attribute method (intact mass or peptide map)2. Chip-based CE-SDS using GXII3. iCIEF4. NGS5. SEC-UV/FL for carbonylation


Intact Mass Multi-Attribute Method for N-glycans

Day-4

Day-14HC+G1F

HC+G2F

HC+G0FHC+Man5HC+G0

HC Agly HC+G0F-GlcNAc

HC+G1F

HC+G2F

HC+G0F

0.0%

1.0%

2.0%

3.0%

4.0%

5.0%

0%

10%

20%

30%

40%

0 2 4 6 8 10 12 14 16

Abun

danc

e

Days

%BG%M…

Man5


Intact mass MAM for Afuc, Agly, Glycation and OthersDay-14

Glucoronidated HC*Degly-HC

HC+1GlycationAgly-HC

Afuc-HC

0%

1%

2%

3%

4%

5%

0 2 4 6 8 10 12 14 16

Abun

danc

e

Days

%Afuc%Agly%Glucoronidation

HC+1Glycation


Carbonylation Assay for Oxidative Stress Tolerance(SEC-UV/FL with fluorescence labeling)


Transfection, Recovery and

Selection

Enrichment, Cloning, and

Scale up

Pool Fed Batch

(24DW)20-40 pools

Clone Fed Batch (96DW)

600-1000 clones

Ambr Evaluation 23 clones +

pool of clones(duplicates)

3L ConfirmationLead and Backup

(duplicates)

All: Titer - OctetTop 10-15:

SSPSEC: HMW

GXII: ImpurityIntact mass: Glycan and

sequenceEndoS2+R intact mass:

agly and afuc

All:Titer - Octet

Top 36: SSP

GXII: ImpurityiCIEF: charge variantIntact mass: Glycan

and sequenceEndoS2+R intact

mass: agly and afuc

All: Timecourse Titer – BioHT (768)

Harvest Titer – ProG (48)NGS

Top 12-18 timecourse (3 pts~45):

SSPSEC: HMW

GXII: ImpurityiCIEF: charge variant

Carbonylation assay: oxidative stress tolerance

Intact Mass (R, degly and gly)

Lead and Backup2 step Purification

Binding Assay

All: Titer - ProG

Time Course : SSP

SEC: HMWGXII: Impurity

iCIEF: charge variantIntact Mass (R, degly and

gly)

Harvest:SSP

Binding AssayDetailed Peptide map

UPLC: Glycan

Cell Line Development Testing Strategy


Case study 2Perform value-added SEC method qualification


Qualification of Platform SEC Method for mAbs

• Goal: Make method qualification activities predictable and value-added

• Minimize experiments that need to be done for method qualification to increase efficiency and speed of method qualification

• Ensure that AD is focusing on value-added experiments

• Define phase-appropriate qualification requirements (AD)

• Create qualification “recipes” for platform methods to ensure qualification is performed consistently

• Define standard timeline for method qualification and transition to QC


Phase-appropriate Method Qualification Leveraging Historical Data

Start with platform methods for early phase antibodies

Lots of historical data available

Make up the bulk of new programs transitioning into QC

Assess method by method to:

Keep experiments that evaluate molecule-specific attributes

Ensure experiments being done provide data that is needed by QC to perform release / stability testing

Eliminate experiments that measure method parameters that are a function of physical laws (e.g. Beer’s law)

Eliminate experiments that have shown consistent results across all molecules

Document justification for removing parameter assessments based on:

Historical qualification and Phase I validation data

Potential impact of not assessing parameter during qualification• Can the impact be mitigated

if parameter is assessed during Phase I validation?


• “Verification consists of assessing selected analytical performance characteristics, such as those that are described in chapter <1225>, to generate appropriate, relevant data rather than repeating the validation process.”

• “Only those characteristics that are considered to be appropriate for the verification of the particular procedure need to be evaluated.”

• The verification process for compendial test procedures is the assessment of whether the procedure can be used for its intended purpose, under the actual conditions of use for a specified drug substance and/or drug product matrix.

Inspiration from USP <1226>:Verification of Compendial Procedures

platform method ≈ internal “compendial” methodQualification ≈ Verification per USP <1226>

• Familiarity and experience with technique, harmonized equipment across sites

• Successfully qualified for > 7 mAbs• Validated (Phase I, III, and commercial) for >

3 mAbs• Historical qualification and validation data

confirm method performance over time, enable good understanding of method capabilities

Platform UPLC SEC method


Platform UPLC SEC

Historical Precision

Repeatability ≤ 1.0% RSDIntermediate Precision ≤ 3.7% RSD

0.0%0.5%1.0%1.5%2.0%2.5%3.0%3.5%4.0%

RSD

repeatability (RSD) intermediate precision (RSD)

Platform UPLC SEC

Historical LOQ

0.04% 0.04%0.02%

0.08%

0.01%0.03% 0.02%

0.00%

0.05%

0.10%

mAb 1 mAb 2 mAb 3 mAb 4 mAb 5 mAb 6 mAb 7

LOQ at target mass load of…

0.0300 0.02950.016

0.058

0.006

0.0230.0172

0.00

0.02

0.04

0.06

0.08

mAb 1 mAb 2 mAb 3 mAb 4 mAb 5 mAb 6 mAb 7

LOQ in ug

LOQ differences are explained by different acquisition rates and baseline noise, not molecule-specific differences in detector response


Linearity and load Injection volume

ProjectEvaluated mass load range (µg)

Linear range (µg)

Target load (µg)

Injection volumes

evaluated (µL)

Acceptable injection volumes

(µL)

Routine injection volume

(µL)

mAb 1 22.5-120 22.5-120 75 1-50 1-10 1

mAb 2 15-150 15-150 75 1-50 1-30 1

mAb 3 22.5-120 22.5-120 75 1-50 1-10 1

mAb 4 22.5-120 22.5-120 75 1-50 1-10 1

mAb 5 1.5-150 6-90 75 1-50 1-25 1

mAb 6 22.5-120 22.5-120 75 1-50 1-25 1

mAb 7 22.5-120 22.5-120 75 1-50 1-10 1

Platform UPLC SEC: Historical Linearity


Benefits

•Ensures that AD does the “just right” amount of work in qualification•Focus on value-added activities

•Reduces amount of rework required•No surprises or requests for additional work after the fact.

•Reduces “additional” experiments that need to be done in QC•Leverage QC “must do” experiments to reduce number of experiments required in AD

Method qualifications are done consistently and meet QC needs

•Don’t need to recreate the wheel for every program•Aligned and visible expectations across departments

Defined timelines make work more predictable


• Two cases studies were discussed to streamline analytical development activities utilizing historical data to

• Select related PQs to monitor at the right step of the process development

• Develop assays that minimize the assay number to cover the necessary PQs

• Develop new assay to meet new learnings

• Perform value-added method qualification activities for platform methods

Summary


Case study 1: Cell line development analytical strategy

Case study 2: UPLC SEC method qualification

Christina AlvesMaxim UsdinRicha SarinJames Lambropoulos

Ruth FrenkelErin Tulip

Acknowledgement

applying prior knowledge to streamline early-stage analytical … · 2018. 4. 2. · biogen |...

Documents