CQA Assessment for Glycosylation and Oxidation of a Monoclonal Antibody:An FDA OBP Pilot Program Case Study

Andrew Weiskopf, Ph.D.

2010 WCBP CMC Strategy Forum19 July 2010

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Biogen Idec & The FDA Pilot Program

• Biogen Idec a participant in the FDA’s OBP Pilot Program for QbD– Meetings with OBP senior staff and reviewers to discuss approach to product and

process risk assessments, design space development, and control strategy

• Case Study for Pilot Program: Antibody X (AbX)– Monoclonal IgG1, standard Fc N-glycosylation– Oncology drug entering late-stage clinical development– Mechanism-of-Action

• CDR of AbX binds to cellular receptor of target cells• Fc of AbX is involved in promoting apoptosis, but Fc-binding entity in vivo is unknown

– In vitro assessment indicates ADCC capability is modest– CDC is not involved– FcRIIIa binding used as metric of Fc integrity

• Risk Assessment Examples– Glycosylation: Galactosylation & Fucosylation– Methionine Oxidation

Isotype control

Antibody X

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Product Risk Assessment

• Risk-ranking & Filtering– Impact & Uncertainty

• Impact– Review each attribute and

determine severity associated with failure to control

– Consider effects on potency, PK, immunogenicity, safety

• SAR studies• Nonclinical studies• Clinical exposure history• Toxicology reports • Data from related IgGs

• Uncertainty– Evaluate quantity & relevancy of

body of data– Reliance on in vitro / in vivo data– Relevance of related molecules– Range of clinical exposure– Process additives: body of

toxicology data and history of use

• Final Risk Priority Number (RPN)– RPN > 24: CQA

– RPN ≤ 24: Non-CQA

• Considerations of process operation excluded from product risk assessment

– Keeping process capability out of the assessment makes CQA assessment more “modular”

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Product Risk Assessment:Galactosylation

• Potency– SAR studies to assess impact on CDR and FcRIIIa binding

– No effect observed on CDR binding from 36 – 90% G0– Modest but clear effect on FcRIIIa binding over the same range

• Consistent with literature report: fully galactosylated glycoform of an IgG exhibits 2-fold higher FcRIIIa binding affinity than agalactosyl form

0

20

40

60

80

100

120

20 40 60 80 100

% G0

% C

DR

Bin

din

g A

cti

vit

y

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Product Risk Assessment:Galactosylation

• Pharmacokinetics– Literature: Fc glycans of IgG molecules not involved in FcRn binding, thus not expected to impact clearance of

AbX

• Nonclinical & Clinical Experience– Wide range of terminal galactosylation exposure established with AbX in both nonclinical and clinical

development history (36 – 75% G0)– AbX Fc glycosylation variants are typical of human IgG’s, and are not novel structures

• Conclusion: CQA– Impact = Moderate (12)– Uncertainty = Low-to-Moderate (3)– Risk Priority Number: 12 x 3 = 36

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Product Risk Assessment:Fucosylation

• Potency– SAR study with afucosylated AbX prepared by fucosyltransferase knockout cell line

– No effect on CDR binding, but substantial impact on FcRIIIa binding• Consistent with numerous literature reports

• Pharmacokinetics– Fc glycans not expected to impact half-life

• Nonclinical & Clinical Experience– Limited: no detectable levels of afucosyl-AbX observed in DS

• Conclusion: CQA– Very high Impact (20) x low-moderate Uncertainty (3) = 60

CDR Binding FcRIIIa Binding

AbX DS (no afucosyl) 100% 100%

2% afucosyl AbX co-mix 103% 191%

100% afucosyl AbX 98% >250%

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Product Risk Assessment:Methionine Oxidation

• Determination of susceptible residues– Site(s) of methionines prone to facile

oxidation govern approach and rationale for risk assessment

• Forced oxidation of Antibody X by treatment with H2O2

– Five potential sites for oxidation: one on light chain, four on heavy chain

– Peptide mapping by LC-MS

• Results– Two Lys-C peptides (KH15 and KH30)

found to contain hotspots for methionine oxidation

• Both hotspots located in Fc region: Met253 & Met429

Site-Specific Forced Oxidation Profile for AbX

0%

10%

20%

30%

40%

50%

60%

0.00% 0.01% 0.02% 0.03% 0.04% 0.05% 0.06%

Percent Hydrogen PeroxideP

erce

nt

Oxi

dat

ion

KH30 KH15

KL1 KH4

KH2

• Oxidation susceptibility is limited to Fc methionines, conserved among IgGs

– Can leverage knowledge gained from other IgGs for risk assessment

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Product Risk Assessment:Methionine Oxidation

• Potency– SAR study conducted with oxidized

preparations of AbX– No effect on activity observed with either

CDR or FcRIIIa binding assays– Literature: Fc methionines reside outside

of FcR binding region, no impact observed with other IgGs

• Pharmacokinetics– In-house studies with another IgG– Highly-oxidized Fc methionines (67% at

Met253, 41% at Met429) yield several-fold decrease in FcRn binding affinity

• Literature: highly oxidized Fc Mets exhibit similar reductions in FcRn binding affinity

– Fc mutants demonstrating several-fold reduction in FcRn binding affinity showed no correlation with IgG half-life in rats

• Literature: changes in FcRn affinity do not necessarily correlate to impact on half-life

CDR Binding (FRET)

FcRIIIa Binding

(AlphaScreen)

AbX Control 105% 83%

“25% Oxidized”

23% M253, 11% M429101% 95%

“50% Oxidized”

47% M253, 23% M42999% 89%

“100% Oxidized”

100% M253, 100% M42997% 100%

0

1

2

3

4

5

6

0.0 1.0 2.0 3.0 4.0 5.0 6.0

Ratio of FcRn Binding Affinity, Mutant:WT

Hal

f-L

ife

(day

s)

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Product Risk Assessment:Methionine Oxidation

• Nonclinical & Clinical Experience– 3-8% oxidized Met253 in clinical lots of AbX

– No adverse effects traced to differences– Wider range of experience with other IgG products

• Other Considerations– Oxidation-induced increase in aggregation rates not studied in isolation

for AbX, though reported in literature

• Conclusion: Non-CQA– Low-Moderate Impact (8) x Low Uncertainty (2) = 16

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Product Risk Assessment:Attribute Criticality & Control Strategy

• Galactosylation– Classified as a CQA, due to modest impact on FcRIIIa binding– Only critical control parameters linked to galactosylation are readily controlled

• Cell culture duration, pH, and temperature– Primary controls achievable through process, justifying in-process testing

• Incorporate into comparability assesssments

• Fucosylation– Classified as a CQA, due to high impact on FcRIII binding activity– No critical control parameters identified within the design space– Monitor by proxy through release test for FcRIIIa binding activity

• Variations will be evident due to sensitivity of assay to afucosyl content• Afucosyl levels controlled indirectly by FcRIIIa binding activity specification• FcRIIIa binding activity evaluated in comparability assessment

• Methionine Oxidation– Classified as a non-CQA, due to low impact on potency & PK– Comparability assessment, selected process monitoring

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Take-Home Messages From the Pilot Program

• Keep process considerations separate from product risk assessment– Process changes shouldn’t have to trigger review of attribute criticality– Maintains CQA assessment as an evaluation of the biology, efficacy, and safety

of the product

• Certainty scoring for CQAs is helpful, but take care with implementation– Scoring system should not overweigh certainty at expense of impact– Don’t mathematically de-risk a critical attribute on the basis of having data– RPNs should accurately reflect degree of criticality and convince one of the risk

• Attributes reside on a “continuum of criticality”– “CQA” and “Non-CQA” are useful terms, but developing an appropriate control

strategy looks beyond the name • How critical is the attribute?• What process controls can be implemented to control the attribute?• How readily controlled are those parameters?

– Control strategy should be proportional to overall degree of risk• Not all CQAs need to be controlled through release testing

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Acknowledgments

• Research– Graham Farrington– Ann MacLaren– Nuzhat Pathan

• Bioprocess Development– Lynn Conley– Eric Cunningham– Victoria Delerue– Tia Estey– John Pieracci– Valerie Tsang– Michael Villacorta– Matt Westoby– Helena Yusuf-Makagiansar

• QbD Project Team– Vince Narbut– Stephen Notarnicola– Suzanne Stella

• Testing Support– Joseph Siemiatkoski– Michael Molony– Martha Berardino– Namrata Chansarkar– Tongjun Lu– Christine Poliks– Molly Roudabush

• Bioassay Development– Svetlana Bergelson– Yucai Peng– Aeona Wasserman

• Method Development / Characterization

– Yelena Lyubarskaya– Zoran Sosic– Richard Strong– Li Zang– Andy Blum– Tyler Carlage– Hans Fajardo– Damian Houde– Amy Diggle Moran– Samnang Tep

• AD CMC Management Group– Damian Cunningham– Mia Kiistala– Maria LacapKazumi Kobayashi

Helena Madden

Rohin Mhatre

Analytical Development