6th Science and Standards SymposiumJanuary 16th, 2013Istanbul, Turkey

Quality Attributes of Monoclonal Antibodies

Tina S. Morris, Ph.D., Vice President Biologics & Biotechnology USP-NF

Quality Control for Biotechnology Products - ICH

ICH Guideline Q6B - Test Procedure and Acceptance Criteria for Biotechnology/ Biologic Products

Quality Attributes

– Identity

– Purity

– Impurity profile

– Potency

– Strength

– Safety

Q6B on Analytical Characterization

Product Type versus Molecule Class – Platform Accessibility

Product Type Molecule Class Source Legacy Products in this Class?

Accessible to analytical platform approach?

Cell Therapy Whole Cell Patient-autologous No No

Clotting Factor Protein/Enzyme Plasma and recombinant

Yes to some extent

Cytokine or growth factor

Peptide or small protein

Mostly recombinant Yes - hormones

Yes

Enzyme Protein Both recombinant and naturally derived

Yes Yes

Monoclonal antibody

Protein, IgG1 Recombinant No Yes

Polyclonal antibody

Protein, Ig mixture Plasma-derived Yes to some extent

Toxin Protein Both recombinant and naturally derived

Yes Yes

Vaccine Various Both recombinant and naturally derived

Yes to some extent

Other – what about heparin?

Polydisperse glycosaminoglycan

Naturally derived Yes Yes

Which Quality Attributes to Consider?

Biological characteristics Physico-chemical characteristics

Fab

Fc

Antigen binding

Effector functionscomplement interactionFc recepter interaction

N-terminal heterogeneitypyroglutamate formationOther modifications

AA modificationsdeamidation, oxidation, glycation, isomerization

FragmentationCleavage in hinge region, Asp-Pro

OligosaccharidesFucosylation, sialyation, galactosylation…

Disulfide bondsFree thiols, disulfide shuffling, thioether

C-terminal heterogeneityLysine processing, proline amidation

Monoclonal Antibodies and Platform Assays

Quality Control Assays for Monoclonal Antibodies

Product Class Analytical Methods - platform assays

– Examples

• Size exclusion chromatography

• Isoelectric Focusing

• Oligosaccharide assay

• Peptide Mapping

• Process Related Impurity assaysoHost cell proteinoDNAoProtein A

• Compendial Assays; Endotoxin, pH, Conductivity, Sterility

<129> Analytical Procedures for Recombinant Therapeutic Monoclonal Antibodies

Will contain a collection of validated compendial procedures with established system suitability criteria for therapeutic MAbs

Will be accompanied by USP MAb System Suitability RS

Will not contain product or class specific acceptance criteria

Will be supported by multiple >1000 Information Chapters that discuss quality attributes, manufacturing and quality control aspects for MAbs

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Capturing Platform Assays in a Compendial Chapter

Product Scope:

Murine, chimeric, and humanized IgG isotype Mabs and subtypes (e.g. IgG1 and IgG2)

MAbs for therapeutic, prophylactic and in vitro diagnostic use

EXCLUDES: MAbs used as manufacturing reagents or process materials

Included Procedures:

Size Exclusion Chromatography (SEC) Capillary SDS Electrophoresis (reduced and non-reduced) Oligosacchride Analysis Sialic Acid Analysis

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<129> Current Chapter Scope

Purpose:Evaluate the proposed Size Variant and CE proceduresTest the proposed USP Monoclonal IgG system suitability standardGather batch data on MAbs currently made in commercial

manufacturing as well as in development (clinical and non-clinical)

Study Logistics and Time Line:Study materials were dispatched in at the end of November, 2012,

deadline for data submission is March 30th, 2013Large international study participation of 30 laboratories

Chapter Time Line:Will appear in PF39(3) with a comment deadline of July 31st, 2013

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Round Robin Study in Support of <129>

Compendial use – Needed for system suitability for proposed SEC

and CE-SDS USP procedures

Material Description and Source– IgG1 MAb– Lyophilization protocol available

Will be developed and distributed as a USP reference standard in lyophilized presentation

USP Monoclonal IgG System Suitability Standard

SEC Profile of IgG System Suitability Standard

Antibody Glycosylation Analysis – fit for Common Assay(s)?

Mab Glycan standard

Glycans from polyclonal human IgG

G2F+1 NeuAc

Data courtesy of <129>/<1260> expert panel

CE Analysis of Released Oligosaccharides

Glycosylation and Bioactivity Correlation

0 0.5 1 1.5 2

Bio

acti

vity

Moles GalactoseMole Heavy Chain

galactosidase treated

50

100

150

200

Bio

acti

vity

0 0.5 1 1.5 2

Bioactivity Correlates with Galactose Content

Galactosylation Profile of hIgG Glycans

0

10

20

30

40

50

60

3 6 8 9 12 14 15 20 21 26 34 35 37 39 41 43 47 49 51 52 5 33 40 47 52 13 17 22 23 46 47 52 4 27 31 48

% o

f gly

can

Lab

Galactosylation profile of hIgG N-glycans

0Gal 1Gal 2Gal

Fluorophore - HPLC HPAEC MS methods F-CE

Product-Specific Quality Attributes of MAbs

Several Quality Attributes of MAbs can be highly product specific and should be addressed at the monograph level.

Examples Charge heterogeneity, analyzed by IEX

chromatography or cIEF

Hydrophobic Interaction Chromatography

Ligand binding, e.g. by ELISA

Cell-based potency assay

Quality Control for Biotechnology Products

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

10 20 30 40 50 60

mA

u

Time (min)

23

4

1

53a2b2a1b

1a

pEpEpE

pE

Peak 1

QpE

pEpE

Peak 2 QpEpE Q

Peak 4

QpE

pEQ

Peak 5

QpE

pE

Peak 3

pE

Ion-Exchange Profile of an Intact Antibody

Quality Control for Biotechnology Products

K K K

pEpE

QpE

QpE

QQ

0

20

40

60

80

100

0 12 24 36 48 60

mA

u

Time (min)

A

B

CD F G

H

I

JK

LMN O PE

Ion-Exchange Profile of a Papain Digested Antibody

+ +

Fc F(ab)

Papain

<1260> Therapeutic Monoclonal Antibodies – General Considerations

Product Class Overview

General Manufacturing and Quality Considerations

<1260.1> Analytical Control Strategies for Recombinant Monoclonal Antibodies

Quality Attributes, their determination, control and measurement

General considerations and analytical considerations for quality attributes that are product specific

Comparability and post-approval quality issues

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Information Chapter(s) in Development

USP Monoclonal Antibody Expert Panel Chair: Dr. Anthony Mire-Sluis

USP Biologics & Biotechnology Monographs 1 Expert Committee

Chair: Dr. Michael Mulkerrin

USP Staff EC and EP Liaison: Dr. Anita Szajek

Reference Standard Scientist: Dale Schmidt

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Acknowledgements

USP Headquarters, Rockville, Maryland