1Biogen | Confidential and Proprietary
Applying Prior Knowledge to Streamline Early-stage Analytical Development of Monoclonal Antibody
Li ZangAnalytical Development, Biogen
CMC Strategy Forum 2018
2Biogen | Confidential and Proprietary
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
3Biogen | Confidential and Proprietary
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
4Biogen | Confidential and Proprietary
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
5Biogen | Confidential and Proprietary
Case study 1Cell line development testing strategy
6Biogen | Confidential and Proprietary
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?
7Biogen | Confidential and Proprietary
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
8Biogen | Confidential and Proprietary
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
9Biogen | Confidential and Proprietary
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)
10Biogen | Confidential and Proprietary
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
11Biogen | Confidential and Proprietary
mAb 2 PTM from Peptide Maps of 36 Clones
N-glycan, A2G0F
N-glycan, Man5
N-glycan, A1G0F
12Biogen | Confidential and Proprietary
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
13Biogen | Confidential and Proprietary
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
14Biogen | Confidential and Proprietary
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
15Biogen | Confidential and Proprietary
Carbonylation Assay for Oxidative Stress Tolerance(SEC-UV/FL with fluorescence labeling)
16Biogen | Confidential and Proprietary
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
17Biogen | Confidential and Proprietary
Case study 2Perform value-added SEC method qualification
18Biogen | Confidential and Proprietary
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
19Biogen | Confidential and Proprietary
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?
20Biogen | Confidential and Proprietary
• “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
21Biogen | Confidential and Proprietary
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
22Biogen | Confidential and Proprietary
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
24Biogen | Confidential and Proprietary
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
25Biogen | Confidential and Proprietary
• 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
26Biogen | Confidential and Proprietary
Case study 1: Cell line development analytical strategy
Case study 2: UPLC SEC method qualification
Christina AlvesMaxim UsdinRicha SarinJames Lambropoulos
Ruth FrenkelErin Tulip
Acknowledgement