Transcript Quality Attributes of Biologics

6th Science and Standards Symposium
January 16th, 2013
Istanbul, 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
N-terminal heterogeneity
pyroglutamate formation
Other modifications
AA modifications
deamidation, oxidation, glycation,
isomerization
Fab
Fragmentation
Cleavage in hinge region, Asp-Pro
Oligosaccharides
Fucosylation, sialyation,
galactosylation…
Fc
Effector functions
Disulfide bonds
complement interaction
Fc recepter interaction
Free thiols, disulfide shuffling, thioether
C-terminal heterogeneity
Lysine 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 assays
o Host cell protein
o DNA
o Protein A
• Compendial Assays; Endotoxin, pH, Conductivity, Sterility
Capturing Platform Assays in a Compendial Chapter
 <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
7
<129> Current Chapter Scope
 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
8
Round Robin Study in Support of <129>
 Purpose:
 Evaluate
the proposed Size Variant and CE procedures
 Test the proposed USP Monoclonal IgG system suitability standard
 Gather 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, 2013
 Large international study participation of 30 laboratories
Chapter Time Line:
 Will
appear in PF39(3) with a comment deadline of July 31st, 2013
9
USP Monoclonal IgG System Suitability Standard
 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
SEC Profile of IgG System Suitability Standard
Antibody Glycosylation Analysis – fit for Common Assay(s)?
CE Analysis of Released Oligosaccharides
Mab Glycan standard
G2F+1 NeuAc
Glycans from polyclonal
human IgG
Data courtesy of <129>/<1260> expert panel
Glycosylation and Bioactivity Correlation
Bioactivity Correlates with Galactose Content
Bioactivity
Bioactivity
200
150
100
galactosidase treated
50 0
0
0.5
0.5
11
Moles Galactose
Mole Heavy Chain
1.5
1.5
22
Galactosylation Profile of hIgG Glycans
Fluorophore - HPLC
HPAEC
MS methods
F-CE
Galactosylation profile of hIgG N-glycans
60
0Gal
1Gal
2Gal
% of glycan
50
40
30
20
10
0
3
6
8
9 12 14 15 20 21 26 34 35 37 39 41 43 47 49 51 52
Lab
5 33 40
47 52 13 17 22 23 46 47 52
4 27 31 48
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
Ion-Exchange Profile of an Intact Antibody
pE
pE
pE
pE
0.07
Q
pE
pE
pE
Q
pE
pE
pE
0.06
mAu
0.05
pE
pE
Peak 1
0.04
Peak 2
Q
Q
pE
Q
Peak 3
pE
Q
0.03
0.02
1
Peak 4
1a
0.01
1b
2
Peak 5
3
2a
2b
3a
4
5
0
10
20
30
Time (min)
40
50
60
Quality Control for Biotechnology Products
Ion-Exchange Profile of a Papain Digested Antibody
100
+
+
80
Papain
H
60
F(ab)
mAu
Fc
B
pE
40
Q
Q
Q
20
J
A
C
0
0
12
D
E
I
F G
24
36
Time (min)
K
48
LM N O P
60
Information Chapter(s) in Development
Therapeutic Monoclonal Antibodies – General
Considerations
 <1260>

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
18
Acknowledgements
 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
19
USP Headquarters, Rockville, Maryland