The document discusses the analytical requirements for biotechnology and biosimilar products. It notes that biologics have different CMC requirements than traditional small molecule drugs due to significant differences in their raw materials, production processes, physiochemical properties, and other factors. These differences make biologics more complex and risky than traditional drugs. The key regulatory documents that provide guidance on CMC analytical requirements for biologics are identified as FDA regulations, ICH guidelines, and the ICH Common Technical Document, which provides a standardized format for data presentation. Critical elements for establishing reliable product specifications are said to be the capabilities of the manufacturing process, product stability, clinical batches, and analytical method capabilities. A variety of analytical technologies are described that are

1. Analytical CMC Requirements for
Biotech and Biosimilar Products:
More Than Meets the Eye!
Nadine M. Ritter, Ph.D.
President and Analytical Advisor
Nadine.Ritter@GlobalBiotechExperts.com
www.GlobalBiotechExperts.com

2. Why do biotechnology products have
different CMC requirements from traditional
chemical products for comparability,
specifications, expiration dating, and the
development of ‘generic’ forms?

3. Biological/Biotechnological vs.
Chemical Pharmaceutical Products
Chemical
Products
Protein
Products

4. Biological/Biotechnological vs. Chemical
Pharmaceutical Products
Significant
Differences In:
Raw Materials
Production Processes
Handling Conditions
Physiochemical
Characteristics
Formulations
Methods of Analysis
Reference Standards
Stability Profile
Storage Conditions
Expiration Dating
Specifications

5. Why is this more risky for Biopharms?
Oral Dosage Route (Protein)
Gut = Food!
Parenteral Delivery
Oral Dosage Route (Chemical)
Not Destroyed with Ingestion

6. Why are impurities and contaminants
more risky for Biopharms?
Parenteral Dosage Route (Biotech)
Impurities (e.g. host cell proteins)
Contaminants (e.g. microbials)
Oral Dosage Route (Chemical)
5 second rule?

7. Why is degradation more risky
for Biopharms?
Parenteral Dosage Route (Biotech)
Increase in Degradants = Less Safety
BODY’S MEMORY = IMMUNOGENICITY
Oral Dosage Route (Chemical)
Decrease in Active Moiety = Less Efficacy

8. Biological/Biotechnological vs. Chemical
Pharmaceutical
“GENERIC”
Products
“SIMILAR”
“Due to the complexity of biological/biotechnology-derived
products, the generic approach is scientifically not appropriate
these products. The “similar biological medicinal products”
for
approach, based on a comparability exercise, will then have to be
followed.” CHMP/437/04 Guideline on Similar Biological
Medicinal Products Oct 2005

9. What world-wide regulations detail CMC
analytical study requirements for
biotech/biological products?

10. Where is it Written?
U.S. Regulatory Documents (www.fda.gov)
Code of Federal Regulation (CFR)
U.S. FDA quality practices approved by Congress and enforceable
by law (“thou shalt…”).
•
• Guidance for Industry (GFI)
Information from FDA on recommended
CFR requirements (“how to….”)
activities to satisfy specific
• Compliance Policy Guidance (CPG)
Guidance to FDA reviewers/inspectors on what to look for in
product submissions and manufacturing operations (“did they…”)

11. Where is it Written?
International Regulatory Documents (www.ich.org)
International Conference on Harmonization:
U.S., E.U., Japan (Canada, Australia)










Stability (Q1A, Q1B, Q1C, Q1D, Q1E, Q1F)
Test Method Validation (Q2 R1)
Product and Process Impurities (Q3A, Q3B, Q3C)
Test Procedures and Acceptance Criteria (Q6A, Q6B)
GMP for Active Pharmaceutical Ingredients (Q7A)
Pharmaceutical Development (Q8)
Quality Risk Management (Q9)
Pharmaceutical Quality System (Q10)
Development and Manufacture of Drug Substances (Q11)
Content and Format of the Common Technical Document
(M4Q; Module 3 “Quality”)

12. Where is it Written?
ICH Q5 Series = Biotech/Biologic
Q5A = Viral Safety Evaluation of Biotechnology Products Derived
From Cell Lines of Human or Animal Origin
Q5B = Quality of Biotechnological Products: Analysis of Expression


Construct in Cells Used for Production of r-DNA Derived Protein
Products
Q5C = Quality of Biotechnological Products: Stability Testing of
Biotechnological/Biological Products

 Q5D = Derivation and Characterization of Cell Substrates Used for
Production of Biotechnological/Biological Products
Q5E = Comparability of Biotechnological/Biological Products Subject
to Changes in Their Manufacturing Process


13. Current Regulatory Guidance Documents Relevant
to Designated Biotech Product
 Monoclonal Antibodies
 Recombinant Proteins
 Naturally-Derived Products (Allergenic extracts)
 Vaccine Products (Protein, DNA, Conjugate, etc…)
 Cellular and Tissue-Derived Products
 Gene Therapy Products
 Plasma-Derived Biopharmaceuticals
 Plant-Derived Biopharmaceuticals
Transgenically-Derived Biopharmaceuticals
Types
 Synthetic Peptides (Redacted in 2006, but principles still relevant)
 Biosimilars

14. Is there one place where it is written where all of the
required CMC studies and data to be presented in each
regulatory dossier for biotechnology products?

15. ICH Common Technical Document
ICH Guidance M4Q (August 2001)
+ ICH M4Q Q&A (July 2003)
+ ICH M4Q Q&A (December 2004)
(CTD)
Harmonized NDA/BLA content and format for regulatory
reviews in US, EU, Japan, Canada, and Australia
Module
Module
Module
Module
Module
1:
2:
3:
4:
5:
Administrative Information
CTD Module Summaries
Quality (CMC Sections)
Nonclinical Study Reports
Clinical Study Reports
and Prescribing Information

16. 3.2.S. Drug Substance
1. General Information
1.1 Nomenclature
1.2 Structure
1.3 General Properties
2. Manufacture
2.1 Manufacturer
2.2 Manufacturing Process and Controls
2.3 Control of Materials
2.4 Control of Critical Steps and Intermediates
2.5 Process Validation
3. Characterization
3.1 Structure and Characteristics
3.2 Product and Process Impurities
2.6 Process Development
4. Control
4.1 Specifications
4.2 Analytical Procedures
4.3 Validation of Analytical Procedures
4.4 Batch Analyses
4.5 Justification of Specification
5. Reference Standards
7. Stability
6. Container Closure System

17. 3.2.P. Drug Product
1. Description and Composition
2. Pharmaceutical Development
2.1 Components
2.2 Drug Product
2.3 Manufacturing Process Development
2.4 Container Closure System
2.5 Microbiological Attributes
2.6 Compatibility
3. Manufacture
3.1 Manufacturer
3.2 Batch Formula
3.3 Description of Process and Process Controls
3.4 Control of Critical Steps and Intermediates
3.5 Process Validation
4. Control of Excipients
5. Control of Drug Product
4.1 Specifications
4.2 Analytical Procedures
4.3 Validation of Analytical Procedures
4.4 Batch Analyses
4.5 Justification of Specification
6. Reference Standards
7. Container Closure System
8. Stability

18. 3.2.A Appendices
A.1 Facilities and Equipment
A.2 Adventitious Agents Safety Evaluation
A.3 Novel Excipients
3.2.R Regional Information
- Executed Batch Records (USA only)
- Method Validation Packages (USA and ASEAN)
- Comparability Protocols (USA only)
- Process Validation Scheme for Drug Product (EU only)
- Medical Device (EU only)
Biosimilar Dossier: Quality and Safety

19. 3.2.S. Drug Substance
1. General Information
1.1 Nomenclature
1.2 Structure
2. Manufacture
2.1 Manufacturer
2.2 Manufacturing Process and Controls
2.3 Control of Materials
2.5 Process Validation
2.6 Process Development
3. Characterization
3.1 Structure and Characteristics
3.2 Product and Process Impurities
4. Control
4.1 Specifications
4.2 Analytical Procedures
4.3 Validation of Analytical Procedures
4.4 Batch Analyses
5. Reference Standards
6. Container Closure System
7. Stability
1.3 General Properties
2.4 Control of Critical Steps and Intermediates
4.5 Justification of Specification

20. 3.2.P. Drug Product
1. Description and Composition
2. Pharmaceutical Development
2.1 Components
2.2 Drug Product
2.3 Manufacturing Process Development
2.4 Container Closure System
2.5 Microbiological Attributes
2.6 Compatibility
3. Manufacture
3.1 Manufacturer
3.2 Batch Formula
3.3 Description of Process and Process Controls
3.4 Control of Critical Steps and Intermediates
3.5 Process Validation
4. Control of Excipients
5. Control of Drug Product
4.1 Specifications
4.2 Analytical Procedures
4.3 Validation of Analytical Procedures
4.4 Batch Analyses
4.5 Justification of Specification
6. Reference Standards
7. Container Closure System
8. Stability

21. 3.2.A Appendices
A.1 Facilities and Equipment
A.2 Adventitious Agents Safety Evaluation
A.3 Novel Excipients
3.2.R Regional Information
- Executed Batch Records (USA only)
- Method Validation Packages (USA and ASEAN)
- Comparability Protocols (USA only)
- Process Validation Scheme for Drug Product (EU only)
- Medical Device (EU only)
Biosimilar Dossier: Quality and Safety?

22. What CMC characterization, comparability, reference
standards, release specification and stability data
packages are required for a biotechnology-based
product throughout product development and at time
of commercial approval?

23. CMC (Chemistry, Manufacturing, and Controls) Data
Packages for Biopharmaceutics
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Product physiochemical profile (compositional and structural analysis)
Reference material characterization (establish product gold standard)
Test method qualification (linearity, precision, accuracy, specificity)
Test method validation (robustness, stability-indicating capability)
Lot release testing (manufacturing consistency and product quality)
Formulation screening (excipient selection, formulation stability)
Degradation evaluation (forced degradation to assess product, methods)
Stability testing (accelerated and ongoing real-time/real condition)
Extractable/Leachable studies (in parallel with ICH stability studies)
Comparability assessment (pre and post approval process/product changes)
Comparability – Biosimilar products (comparisons to reference licensed drug)

24. How should the required CMC analytical and stability
studies be staged during the product
lifecycle?
development

25. SELECT CHAR-COMP QUALIFY QUALIFIED VALIDATE QC RE-VALIDATE
SELECT QC EVALUTE STABILITY FORCED DEGRADE VALIDATE RE-VALIDATE
DS and DP METHODS* INDICATING METHODS
INITIAL PRODUCT 1ST REFERENCE STD NEW REFERENCE LOT NEW REFERENCE LOT NEW REFERENCE LOT
CHARACTERIZATION CHARACTERIZATION QUALIFICATION CERTIFICATION CERTIFICATION
DP STABILITY STABILITY - DP
STABILITY – INITIAL STABILITY – LEAD STABILITY – FINAL
TOX LOTS 1 ANNUAL LOT
FORMULATION FORMULATION EXT & LEACH FORM or C/C CHANGE
SCREENING STUDY SELECTION STUDY STUDY RE-DO E&L
DS STABILITY STABILITY – STABILITY – STABILITY – STABILITY - DS
TOX LOTS DS CLIN LOTS DS CLIN LOTS DS CLIN LOTS 1 ANNUAL LOT
DS LOTS PHASE II LOTS DS LOTS EACH BATCH
DP LOTS DP LOTS DP LOTS EACH BATCH
TOX - CLIN LOT TOX – CLIN LOT TOC-CLIN LOT PROCESS CHANGE
COMPARABILITY COMPARABILITY COMPARABILITY
CLINICAL STUDY TIMELINES
PRELIMINARY
FORMULATION TOX LOTS
Pre-Clinical Phase 1 Phase 2 Phase 3 Commercial
DS and DP METHODS METHODS* METHODS METHODS QC METHODS
DRUG SUBSTANCE STABILITY METHODS STABILITY METHODS
FORMULATION FORMULATIONS FORMULATION
*VALIDATE
QC SAFETY
METHODS
LOT RELEASE – LOT RELEASE - DS LOT RELEASE – LOT RELEASE - DS
LOT RELEASE – LOT RELEASE – LOT RELEASE – LOT RELEASE - DP
COMPARABILITY
CLINICAL S

26. QUALIFY
METHODS
QUALIFY STABILITY
INDICATING METHODS
NEW REFERENCE LOT
QUALIFICATION
STABILITY – LEAD
FORMULATIONS
FORMULATION
SELECTION STUDY
STABILITY –
DS CLIN LOTS
LOT RELEASE - DS
PHASE II LOTS
LOT RELEASE –
DP LOTS
TOX – CLIN LOT
COMPARABILITY
SELECT CHAR-COMP QUALIFY VALIDATE QC RE-VALIDATE
SELECT QC FORCED DEGRADE VALIDATE RE-VALIDATE
DS and DP METHODS* DRUG SUBSTANCE
INITIAL PRODUCT 1ST REFERENCE STD NEW REFERENCE LOT NEW REFERENCE LOT
CHARACTERIZATION CHARACTERIZATION CERTIFICATION CERTIFICATION
DP STABILITY STABILITY - DP
STABILITY – INITIAL STABILITY – FINAL
TOX LOTS 1 ANNUAL LOT
FORMULATION EXT & LEACH FORM or C/C CHANGE
SCREENING STUDY STUDY RE-DO E&L
DS STABILITY STABILITY – STABILITY – STABILITY - DS
TOX LOTS DS CLIN LOTS DS CLIN LOTS 1 ANNUAL LOT
DS LOTS DS LOTS EACH BATCH
DP LOTS DP LOTS EACH BATCH
TOX - CLIN LOT TOC-CLIN LOT PROCESS CHANGE
COMPARABILITY COMPARABILITY
PRELIMINARY
FORMULATION TOX LOTS
Pre-Clinical Initial Clin Add’l Clin Commercial
DS and DP METHODS METHODS* METHODS QC METHODS
STABILITY METHODS STABILITY METHODS
FORMULATION FORMULATION
*VALIDATE
QC SAFETY
METHODS
LOT RELEASE – LOT RELEASE – LOT RELEASE - DS
LOT RELEASE – LOT RELEASE – LOT RELEASE - DP
COMPARABILITY
ANALYTICAL SIMILARITY
ORIGINATOR
ANALYTICAL SIMILARITY
ORIGINATOR
???
INTERCHANGEABILITY

27. What four main elements are critical
establishing reliable, meaningful
for
product specifications?

28. ICH Q6B: Specifications; Test Procedures and Acceptance Criteria
or Biotechnological/Biological Products (August 1999)
Biotech Product Specifications and Acceptance Criteria
must be derived from:
Capabilities of the manufacturing process
Stability of the bulk substance and final product
Nature of batches used in pre-clinical and clinical
studies
Capabilities of the analytical methods

29. Biotech Product Development CMC Activities
Specifications are proposed by sponsors and accepted by
regulatory authorities as conditions of their approval
use the product in humans.
to
Product Specifications are Your
CONTRACTS
with the Regulatory Authorities
ANALYTICAL METHODS play a
KEY ROLE in this system
OBJECTIVE:
Establishment of Meaningful, Supportable Specifications

30. CHEMICALS VS BIOLOGICALS
= DIFFERENT ANIMALS!
Statin
MW 405
IgG
MW 150,000
BIOLOGIC
DRUG

31. Blinded Single-Feature Elephant Analysis
“All of you are right. The reason every one of you is telling it differently is
because each one of you touched the different part of the elephant. Actually the
elephant has all the features you mentioned.”

32. Protein
Primary
Biomolecular Analysis
Secondary Tertiary
Cozzone, A. J. Proteins: fundamental chemical properties.
in Enc.of Life Sciences (Nature Publ Group, London, 2001)
Protein Tertiary Structure Prediction; D
Xu, Y Xu; in Current Protocols in Protein
Science (May, 2001)
http://psychology.wikia.com/wiki/Amino_acids
Complex Structure
Linda Stannard, Dept of Medical
Microbiology, U of Cape Town
Quaternary
P. Rudd, et. al; J. Immunology, 2004, 173: 6831-6840

33. Dürer, “Rhinoceros”
Albrecht (1514)

34. Analytical Test Method
“TOOL KITS”
CHARACTERIZATION
COMPARABILITY METHODS
(C)
BATCH RELEASE METHODS
(R)
STABILITY METHODS
(S)

35. What are the major analytical technologies
used to assess physiochemical attributes of
biotechnology products?

36. Orthogonal Methods of Analysis
Used with Biopharmaceutical Products
USE
METHOD ATTRIBUTES
Solution hydrogen ion, stability
Moisture, stability
Physical quality, stability
Subvisible Particles, Leachates
Concentration
Identity, purity, stability (aggregation, hydrolysis)
Identity, purity, stability (aggregation, hydrolysis)
Identity, stability (deamidation, oxidation, disulfide scrambling)
Identity, stability (deamidation, oxidation, DS, hydrolysis)
Identity, stability (hydrolysis, desialyation)
Identity, stability (aggregation, hydrolysis)
Process Residuals (proteins)
Process Residuals (nucleic acids)
BioIdentity, potency, stability
BioIdentity, potency, stability
Identity, heterogeneity
Identity, heterogeneity
Identity, composition, concentration
Identity, composition
Identity, composition, stability (deglycosylation)
Identity, composition, stability (desialyation)
Identity, purity, stability (oxidation, hydrolysis, glycation, DS)
Conformation
Conformation
Impurities (Aggregates)
Structure
R, S, C/C
R, S, C/C
R, S, C/C
R, S, C/C
R, S, C/C
R, S, C/C
R, S, C/C
R, S, C/C
R, S, C/C
R, S, C/C
R, S, C/C
R, C/C
R, C/C
R, S, C/C
R, S, C/C
C/C
C/C
C/C
C/C
C/C
C/C
C/C
C/C
C/C
C/C
C/C
pH (liquid)
Karl Fisher (lyophilized)
Appearance (liquid, lyophilized)
Light Obscuration, MFI
UV Absorbance
SDS-PAGE (R/NR)
SEC-HPLC/UPLC
RP-HPLC, HIC-HPLC/UPLC
Peptide Mapping
IEF, CIEF, iCE, IEX-HPLC/UPLC
CE-SDS (R/NR)
Immunoassay, ECL
qPCR
Ligand Binding Assay
Cell-Based Bioassay
N terminal Sequencing
C-terminal Sequencing
Amino Acid Analysis
Monosaccharides (HPAE-PAD)
Oligosaccharide (HILIC/CLIF)
Sialic Acid (HPAE-PAD)
Mass Spectrometry
Circular Dichroism
FTIR
AUC
NMR

37. Current Analytical Benchmarks –
Monoclonal Antibody Products
Analysis and Structure Characterization of
Monoclonal Antibodies
BioProcess International (Feb 2004)
Mark A. Schenerman, Brooks R. Sunday, Steve
Kozlowski, Keith Webber, Hélène Gazzano-
Santoro, and Anthony Mire-Sluis

38. Current Analytical Benchmarks –
Viral Vaccines and Gene Therapy Products
Lot Release and Characterization Testing of Live-Virus–
Based Vaccines and Gene Therapy Products:
Part 1: Factors Influencing Assay Choices
BioProcess International (April 2006)
Part 2: Case Studies
BioProcess International (May 2006)
Jim Gombold, Keith Peden, Denise Gavin, Ziping Wei,
Khandan Baradaran, Anthony Mire-Sluis, and Mark
Schenerman

39. What are the key parameters for release and
stability testing of biotech products?

40. Key Product Parameters (ICH Q6B)
Product-related impurities:
Aggregates, truncated forms, other modified forms (deamidated, isomerized, oxidized,
etc…).
Degradation products are considered product-related impurities; they change in nature
and abundance over time on storage so could affect the safety and efficacy of the
product.
Product-related substances:
Molecular variants of the desired product formed during manufacture and/or storage
which are active and have no deleterious effects on the safety and efficacy of the
product.
Many (most) proteins have defined degree of heterogeneity that is expected; the process
must generate consistent patterns of heterogeneity.

41. Key Product Parameters (ICH Q6B)
Process / product contaminants:
Adventitious agents (viruses, prions), environmental microbes (bacteria, fungi), microbial
fragments (endotoxins/pyrogens), equipment contact surfaces (glass, metal, fibers,
solvents), container or closure leachates or flakes (glass, rubber, lubricants, elastomeric
closure compounds).
Raw materials, equipment, and facilities must be kept as free of contaminants as
possible at all points in production.
Process-related impurities:
Host cell proteins, host cell nucleic acids, cell culture derived components (e.g. serum,
antibiotics), downstream process additives (e.g. enzymes, reducing agents, ligands).
Process impurities must be cleared by process steps to consistently low levels;
specification limits are based on levels present in clinical trial batches.

42. Current Analytical Expectations for Biotechnology
Product and Process Impurities
www.casss.org
Defining Your Product Profile and Maintaining Control Over It, Part 4: Process-
Related Impurities - Aggregates (Brorson and Phillips) BioProcess International
Nov 2005
Defining Your Product Profile and Maintaining Control Over It, Part 3: Product-
Related Impurities (Boerner and Clouse) BioProcess International Oct 2005
Defining Your Product Profile and Maintaining Control Over It, Part 2:
Monitoring Host Cell Proteins (Champion, Madden, Dougherty, and Shacter)
BioProcess International Sept 2005
Defining Your Product Profile and Maintaining Control Over It, Part 1:
Process-Related Impurities (Simmerman and Donnelly) BioProcess
International June 2005

43. What are the specification considerations
acceptable amounts of heterogeneity
for
in biotechnology/biosimilar products?

44. Expected (Allowed) Molecular Heterogeneity of
Biopharmaceutical Products
“Define the pattern of heterogeneity of the desired product, and demonstrate
consistency with that of the lots used in preclinical and clinical studies.” ICHQ6B
Each batch can have all of the heterogeneity you can measure, monitor,
and confirm stays within its established ranges throughout shelf life
Rx Lot
Clin Lot 3
Clin Lot 2
Clin Lot 1
Tox Lot

45. Risks of Aberrant Molecular Heterogeneities
“When process changes and degradation products result in heterogeneity patterns
that differ from those observed in the material used during preclinical and clinical
development, the significance of these alterations should be evaluated.” ICHQ6B
Clin Lot 3
Clin Lot 1
PreClin Lot Rx Lot
Clin Lot 2

46. Biotech Products Comparability Guiding
Principles: Nature of the Process Change
The more upstream of the manufacturing process the change is
made, the greater the potential for impact of the change, and the
greater the body of data required to demonstrate comparability
For each step in manufacturing that is changed, all claims made
for that step (eg removal of a specific impurity) must be confirmed
Biosimilar products present the ultimate process change:
An entirely new expression system, with limited ability for direct
analytical comparability to the original process

47. Comparability Requirements
• Demonstrate no clinically meaningful differences in safety,
purity and potency between products
– Analytical data
– Animal testing
– One or more clinical studies,
deems this not necessary
unless the regulatory body

48. Comparability of
Product-Related Substances
Process/Product A Process/Product B

49. Comparability of Product-Related Impurities
and Process-Related Substances
Some species
below LOD of
analytical
methods, BUT
May not be
below LOD of
-
the
human body
“Fingerprint” Methods
Differences In
Minor Species?

51. Typical Comparability Analytical Test Plan for Glycoprotein
Parameter Test Method Data Type
Tabular Images
General Visual Appearance Visual Inspection X
Acidity or Alkalinity USP Potentiometry <791> X
Subvisible Particulates USP Particulate Matter <788> X
Clarity EP Opalescence <2.2.2> X
Color EP Color <2.2.1> X
Subvisible Particulates Microflow Imaging X X
Concentration Absorbance UV A280 nm (Em = Protein-Specific) X
US/VIS Spectral Scan X X
Identity/
Conformation
Immunoidentity Western Blot with Anti-Product Antibodies X X
N-terminal Sequence Edman Degradation X
Molecular Mass Mass Spectrometry X X
Isoform Pattern cIEF, iCE, IEX-HPLX, IEF Gels X X
Amino Acid Composition Amino Acid Analysis X X
Monosaccharide Composition AA or AB NP-HPLC X X
Sialic Acid Content HPAE-PAD HPLC X X
N-linked Oligosaccharides CE-LIF or HPAE-PAD HPLC X X
Gal 1,3 Gal HPAE-PAD HPLC X X
Secondary/Tertiary Structure Peptide Map (HPLC fingerprint) X X
Peptide Fragments (HPLC + MS) X X
Circular Dichroism X
FTIR X
DSC X
2D NMR X

52. Parameter Test Method Data Type
Tabular Images
Purity/
Product-
Related
Substances/
Degradants
Product-related
substances/impurities
SDS-PAGE reduced and nonreduced, Coomassie
stain; Densitometry
X X
SDS-PAGE reduced and nonreduced Silver stain,
Visual
X X
SEC-HPLC X X
SEC-HPLC + MALS X X
IEX-HPLC, iCE, cIEF, IEF Gels X X
RP-HPLC X X
RP-HPLC +MS X X
HIC-HPLC X X
2D- DIGE (Co-mixed with Ref Std) X X
Potency Cell Binding Cell Based Assay X X
Antigen Binding ELISA X X
Kd/Ka BIACore or ECL X X
Km/Kcat Enzyme Assay X X
Process-
Related
impurities
Residual Media Components Component-Specific Test Method (eg ELISA) X
Process Additives/Leachates Component-Specific Test Method (eg ELISA) X
Host Cell DNA q-PCR X
Host Cell Proteins HCP ELISA (Process-Specific) X X
Process
Contaminants
Endotoxin USP LAL <85> X
Pyrogens USP Rabbit Pyrogen <151> X
Bioburden USP Direct Inoculation <62> X
Sterility USP Sterility <71> X

53. Side-by-Side Chem-Phys Forced
Degradation to Demonstrate Comparability

54. Illustration of Biotech Forced Degradation
“Matrix” Study for Stability-Indicating Methods
A = Appearance B= pH C = SDS-PAGE D= SEC-HPLC
H = Potency
E = RP-HPLC F = IEF G = Peptide Map
Freeze-Thaw
Method A
Method B
Method C
Method D
Method E
Method F
Method G
Method H
Photodegradation
Method A
Agitation
Method A
Method B
Method C
Method D
Method E
Method F
Method G
Method H
T = 0
T = 1
T = 2
T = 3
T = 4
T = 5
T = 0
T = 1
T = 2
T = 3
T = 4
T = 5
T = 0
T = 1
T = 2
T = 3
T = 4
T = 5
Method B
Method C
Method D
Method E
Method F
Method G
Method H
Hydrolysis
Method A
Method B
Method C
Method D
Method E
Method F
Method G
Method H
Oxidation
Method A
Method B
Method C
Method D
Method E
Method F
Method G
Method H
Deamidation
Method A
Method B
Method C
Method D
Method E
Method F
Method G
Method H
T = 0
T = 1
T = 2
T = 3
T = 4
T = 5
T = 0
T = 1
T = 0
T = 1
T = 2
T = 3
T = 4
T = 5
T = 2
T = 3
T = 4
T = 5

55. Hypothetical Stability-Indicating
Method Validation Results
A = Appearance B= pH C = SDS-PAGE
G = Peptide Map
Freeze-Thaw
D= SEC-HPLC
H = Potency
E = RP-HPLC
Agitation
F = IEF
Photodegradation
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Hydrolysis Oxidation Deamidation
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
A B C D E F G H
- - - - - - - -
- - - - - - - -
- - + + - - - -
- - + + - + - +
- - + + + + - +
- - + + + + - +
A B C D E F G H
- - - - - - - -
- - - - - - - -
- - - - - - - -
- - - - - - - -
- - - - - - + -
- - - + + - + -
A B C D E F G H
- - - - - - - -
- - - - - - + -
- - - - - - + -
- - - - - - + -
- + - - - + + -
- + - - - + + -
A B C D E F G H
- - - - - - - -
- - - - - - - -
- - + - - - + -
- - + + - + + -
- - + + + + + +
- - + + + + + +
A B C D E F G H
- - - - - - - -
- - - - + - - -
- - - - + - - -
- - + - + - - -
- - + - + + - -
+ - + + + + - +
A B C D E F G H
- - - - - - - -
- - - + - - - -
- - - + - - - -
- - - + - - - -
- - - + - - - -
+ - - + - - - -

56. Comparable PATTERNS of Force-Degraded Materials
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
A B C D E F G H
- - - - - - - -
- - - - - - - -
- - + - - - - -
- - + - - - + -
- - + + - + + -
- - + + + + + +
A B C D E F G H
- - - - - - - -
- - - - - - - -
- - - - + - - -
- - - - + - - -
- - + - + + - -
- - + + + + - +
A B C D E F G H
- - - - - - - -
- - - - - - - -
- - + + - - - -
- - + + - + - +
- - + + + + - +
- - + + + + - +
A B C D E F G H
- - - - - - - -
- - - - - - - -
- - + + - - - -
- - + + - + - +
- - + + + + - +
- - + + + + - +
A B C D E F G H
- - - - - - - -
- - - - - - - -
- - + - - - + -
- - + + - + + -
- - + + + + + +
- - + + + + + +
A B C D E F G H
- - - - - - - -
- - - - + - - -
- - - - + - - -
- - + - + - - -
- - + - + + - -
+ - + + + + - +
Photolysis
Freeze-Thaw
Hydrolysis

57. What are some of the current CMC ‘hot buttons’ for
biotechnology analytical and stability studies that are of
increasing regulatory concern for chronic deficiencies in
product dossiers?

58. Current CMC ‘Hot Buttons’
Analytical and Stability
Appropriate characterization and comparability of Host Cell Proteins (HCP);
validation of specificity for the host cell proteome of HCP ELISAs
Characterization and quantitation of subvisible particulates in protein solutions;
semi-quantitative visual particle test
Comprehensive, systematic experimental validation via FD of analytical methods
that are claimed to be ‘stability-indicating’
Establishment of the stability profile using appropriate orthogonal stability-
indicating methods
Sufficient assessment of container-closure interactions (extractables/leachates)
under long-term, real-time conditions
Development and validation of methods for immunogenicity screening for anti-
HCP antibodies = CBER plasma products
•
•
•
•
•
•

59. Current CMC ‘Hot Buttons’
Analytical and Stability
Tiered strategy for in-house reference standards; calibrating standards for
potency assays; bridging and stability studies for reference standards
•
• Adequate, complete, traceable Method “Life Cycle” packages - CTD
“method history” requirements; ICHQ10 “Product Knowledge”
• Quality practices in academic and R&D labs; control and documentation of
the analytical methods used by non-GMP laboratories only for
characterization and comparability
• Establishing allowable ranges of product characteristics for an analytical
determination of biosimilarity; selection of regional reference licensed
product; number of batches to include in analytical comparisons

60. What are some useful resources to
help remain current on
biotech/biosimilar CMC issues?