This presentation from IVT Network's Method Validation Conference covers required and suggested regulations and guidances for biological process specifications. It also covers dosage form considerations and specifications for other components.

1. Setting Biological Product
Specifications
David Lin, Ph.D.
Senior Consultant
Biologics Consulting Group
IVT Method Validation
October 15, 2008
2010 by Biologics Consulting Group. All rights reserved. Reproduction in part or in whole without written permission is strictly prohibited.

2. Biological vs. Chemical
Pharmaceutical Products
Protein
Products
Chemical
Products

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

4. Guidances/Guidelines
 ICH Q5A Viral Safety Evaluation of Biotechnology Products
Derived From Cell Lines of Human or Animal Origin, Sep
1998
 ICH Q5B Quality of Biotechnological Products: Analysis of
the Expression Construct in Cells Used for Production of r-
DNA Derived Protein Products, Feb 1996
 ICH Q5C Quality of Biotechnological Products: Stability
Testing of Biotechnological/Biological Products, Jul 1996
 ICH Q5D Quality of Biotechnological/Biological Products:
Derivation and Characterization of Cell Substrates Used for
Production of Biotechnological/Biological Products, Sep
1998
 ICH Q5E Comparability of Biotechnological/
Biological Products Subject to Changes in Their
Manufacturing Process, Jun 2005

5. Guidances/Guidelines
 ICH Q6A Guidance on Q6A Specifications: Test Procedures and
Acceptance Criteria for New Drug Substances and New Drug
Products: Chemical Substances, Dec 2000
 ICH Q6B Specifications: Test Procedures and Acceptance Criteria
for Biotechnological/
Biological Products, Aug 1999
 ICH Q8 Pharmaceutical Development, May 2006
 ICH Q8(R) Pharmaceutical Development Revision, Jun 2009
 ICH Q9 Quality Risk Management, Jun 2006
 Q10 Pharmaceutical Quality System, Apr 2009

6. FDA Guidances Withdrawn
in May-June 2006
 Submission of CMC Information for Synthetic Peptides, Nov 1994
 Format and Content of the CMC Section of an Application, Feb 1987
 Submitting Documentation for the Stability of Human Drugs and
Biologics, Feb 1987
 Stability Testing of Drug Substances and Drug Products, Jun 1998
 Analytical Procedures and Method Validation - CMC Documentation,
Aug 2000
 BACPAC I: CMC Documentation, Feb 2001
 Drug Product: CMC Information, Jan 2003
 Drug Substance: CMC Information, Jan 2004

7. Definition of Specification
“A list of tests, references to analytical procedures,
and appropriate acceptance criteria which are
numerical limits, ranges, or other criteria for the
tests described. It establishes the set of criteria to
which a drug substance, drug product, or materials
at other stages of its manufacture should conform
to be considered acceptable for its intended use.
Conformance to specification means that the drug
substance and drug product, when tested
according to the listed analytical procedures, will
meet the acceptance criteria. Specifications are
critical quality standards that are proposed and
justified by the manufacturer and approved by
regulatory authorities as conditions of approval.”
ICH Q6B Guidance, Aug. 1999

8. Definition of Acceptance Criteria
“Numerical limits, ranges, or other
suitable measures for acceptance of the
results of analytical procedures which
the drug substance or drug product or
materials at other stages of manufacture
should meet.”
ICH Q6B Guidance, Aug. 1999

9. Specifications
 Which guidance is most relevant, Q6A
or Q6B?
 If synthetic, follow Q6A
 Low MW
 Ifbiologically source (e.g.
recombinant), follow Q6B
 Higher MW

10. Specifications
 Q6A allows for skip testing
 Q6A allow for parametric release
 Monitor sterilization cycle instead of
performing sterility testing
 Sterilization process validation and
demonstration of control critical

11. Specifications
 Q6A references Q3A for establishing
impurity levels in peptide drug
substance, Q3C for residual solvents
and Q3B for the drug product
 Q6A does not discuss bioassay but
Q6B discusses potency as a measure
of biological activity

12. Product attributes
Lead to the specifications
Lead to the method development
requirements

13. Purpose of Specifications
 One part of control strategy to ensure quality
and consistency
 Subset of product characterization and
serves as surrogate of full product
characteristics
 Complements product understanding derived
from process control, in-process testing, raw
material control, stability testing

14. Establishing Relevant
Specifications
 Characterization
 Physicochemical properties
 Biological activity
 Immunochemical properties
 Purity
 Impurities
 Datafrom development and after
process changes

15. Product Attributes
 Chemical
 Physical
 Microbiological
 Biological
 Performance
 Relate to:
 Efficacy
 Safety
 Quality

16. Method Measures Detects
Cell-based potency Biological Activity Overall integrity of the molecule
Ligand binding
SDS-PAGE Molecular Weight Subunit molecular mass
Reduced and non- Fragmentation
reduced Covalent crosslinking
MALDI-TOF
Used with Permission from Jay Schrier, 2004
RP-HPLC, HIC-HPLC Surface Hydrophobicity Chemical and conformational variants
IEF Net Surface Charge Charge variants
ELISA Antibody Recognition Epitope binding site integrity
RIA Specific contaminants
Western Blot Immunogenicity
Surface Plasmon Antigenicity
Resonance
Peptide Map Composition Primary Structure variants
Amino Acid Composition Post-translational Primary structure integrity
N and C terminal modifications Integrity of PTMs
sequence
CD (Far UV) Optical Activity Secondary Structure
UV Absorbance Aromatic Amino Acids Tertiary Structure
Fluorescence Side Chains
CD (Near UV)
SEC HPLC Hydrodynamic Radius Molecular size
Analytical Conformational changes
Ultracentrifugation Aggregation/dissociation
Light Scattering Radius of Gyration Molecular size
Aggregation/dissociation
NMR Nuclear magnetic energy Complete molecular structure
X-ray Cystallography Transitions
X-ray diffraction

17. Protein Structural Elements
Primary = Covalent or chemical structure.
Completely defined by the amino acid sequence
plus any disulfide bonds, and any post-translational
modifications (e.g. glycosylation, phosphorylation)
Secondary = Periodic structures within the overall
conformation (e.g. alpha helix, various beta-
structures)
Tertiary = Total folding pattern of the polypeptide
chain(s)
Quaternary = Association of subunits of polypeptide
chains

18. Functional Characterization
 Potency provides a different level of analytical information on
the structural integrity of the molecular entity
 Assessment of product potency typically requires in vitro
and/or in vivo bioassays
 A bioassay may directly reflect the mechanism of action of the
product, or it may be a surrogate functional assessment (e.g. if
MOA is not known, or is not able to be replicated in a potency
assay)
 R&D likely used a bioassay to facilitate discovery of the
molecular entity; it may serve as a starting point for
development of a potency assay

19. Bioassay
 To assess the activity/potency of the
protein molecule
 To serve as measurement of the
biological activity and structural
integrity (e.g., correct conformation) of
the protein molecule

20. What is a Bioassay?
Ana T. Menendez, Ph.D.
Director of Bioassays and Biosafety Testing
Cardinal Health
 Any biological activity that can demonstrate that the product
that was expressed by the host cell or organism is going to
perform as it should.
 The activity can be cellular, for example:
 can the product kill cancer cells?
 can the product make cells grow?
 can the product stimulate cells to produce cytokines?
 The activity can be microbiological, for example:
 can the product cause an immune response?
 can the product lyse bacteria?
 can the product prevent virus from replicating in cells?

21. Types of Bioassays
Ana T. Menendez, Ph.D.
Director of Bioassays and Biosafety Testing
Cardinal Health
 Cellular:
 Cell growth or inhibition
 Cell lysis
 Angiogenesis induction or inhibition
 Induction of cytokines
 Differentiation
 Bacterial
 Immunogenic response
 Cell lysis or inhibition
 Viral
 Enzymatic
 Immunological

22. Why are Cell-Based Bioassays Different
than other Analytical CMC Assays?
Ana T. Menendez, Ph.D.
Director of Bioassays and Biosafety Testing
Cardinal Health
 Involve recurring consistent source of live
organisms
 Need Master and Working Cell Banks
 Cell lines require characterization
 Biological reagents require qualification
 Results are mostly calculated on 4-parameter
curve
 CVs and %Accuracy are less stringent than in
analytical techniques

23. IND Development
 FDA Guidance for Phase I Studies (11/1995)
 “Validation data and established specifications ordinarily
need not be submitted at the initial stage of drug
development”
 “However, for well characterized biologicals preliminary
specifications and additional validation data may be
needed….. to ensure safety in Phase I”
 FDAGuidance for Phase 2 and Phase 3
Studies, CMC Information (5/2003)
 Acceptance criteria refined based on batch analyses
 Limits on new impurities based on manufacturing
experience, stability data and safety
 Data from stress studies crucial

24. In-Process Specifications
 Confirm consistency by measuring at critical
process steps
 Action limits or acceptance criteria?
 Safety attributes such as adventitous agents
should be based on acceptance criteria
 Testing results could serve to eliminate
testing of drug substance or drug product
 Internal action limits serves as indicator of
potential consistency issue or control issue

25. Raw Materials and Excipients
 Meet quality standards for intended use
 Raw materials for production require
determination of adventitious agents
 Extractables/leachables from purification
resin materials
 Minimum standards conform to
pharmacopoeia

26. Containers and Closures
 Extractables/leachables not just from the
primary container and closure, but also any
delivery system required for administration
 Product formulation specific evaluation is
needed
 Equivalent materials based on
pharmacopoeia standards might not be
adequate for specific formulation

27. Why Worry About Extractables
and Leachables?
27

28. Areas of Concern
Toxicity
Carcinogenicity
Immunogenicity
Product quality
28

29. Required by Regulation
21 CFR 211.65(a) – Equipment
21 CFR 600.11(b) - Equipment
21 CFR 211.94(a) - Drug product
container closures
21 CFR 600.11(h) – Containers and
closures
29

30. Regulatory Citations
 Evans Vaccine (2003)
 The inspection noted the lack of filter extractable validation
studies on filtered […..] monovalent and trivalent bulks
 Similasan AG (August 2005)
 “Further it is unclear to us whether you have conducted filter
extractable and leachable testing with product. If you have this
data, provide it to us. If you do not, let us know when you will
be able to provide it to us.”
30

31. Regulatory Citations
 Wyeth (2006)
 “Your previous investigation into various unknown peaks
occurring in your drug products had identified phenol as a
packaging extractable originating from ink used to print
package inserts. However your firm later identified the
unknown peak as Caprolactarn, an extractable that
potentially originated from Nylon components used to pack
the drug”
 GTC Biotherapeutics (2009)
 “There were no leachable and extractable testing performed
for --b(4)--- materials used in buffer preparation. “
31

32. Public Health Notifications
 PVCdevices containing plasticizer
DEHP (2002)
 http://www.fda.gov/MedicalDevices/Safety/
AlertsandNotices/PublicHealthNotifications
/UCM062182
 IV bags, blood bags, infusion tubing, etc.
 BPA in food (2010)
 http://www.fda.gov/NewsEvents/PublicHeal
thFocus/ucm064437.htm
32

33. Differences in Safety
Consideration for Biologics
 Proteins are large molecules with complex
configurations that are affected by E&Ls
 Larger surface for interactions with E&Ls
 Product administered in high dose so total
E&L exposure is higher
 Lower molar concentration of protein in
product
 Exposure to different materials during
manufacturing
33

34. FDA Container-Closure Data
Requirements for Biological Products
Information to support the container and closure
packaging used with bulk biological products is
required to be included in the FDA submission
(rather than simply referenced, as with traditional
drugs) because there is a greater potential for
adverse effects on the identity, strength, quality,
purity and potency of biologics and protein
products during storage or shipping.
FDA Guidance: Container-Closures for Packaging Human Drugs and
Biologics, Questions/Answers (2002)

35. What Tests to Perform?
35

36. Primary Considerations
 Information from supplier
 Has supplier intended use been modified
 Consider existing databases
 Understand chemistry of materials
 Start with compendial tests
 Need to justify if relevant to specific use
 Don’tfocus on just organics
 Equipment reuse (i.e., column resins, filters, etc.)
Be realistic!!!
36

37. Tests for Protection
 USP <671> Container – Performance
Testing
 Light Transmission
 Water Vapor Permeation
 Dye Ingression Studies
 Microbial Ingression Studies
37

38. USP <660> Containers - Glass
 Water Attack
 Extraction for release of alkali
 Arsenic
 1 ppm
38

39. USP <661> Containers - Plastics
 Extraction
 Nonvolatileresidue
 Residue on Ignition
 Heavy Metals
 Buffering Capacity (for liquid products)
 Total Terephthaloyl Moieties (for PET &
PETG)
 Ethylene Glycol (for PET & PETG)
39

40. USP <381> Elastomeric
Closures for Injections
 Extraction
 Turbidity
 Heavy Metals
 Reducing Agents
 pH Change
 Total Extractables
40

41. Program Approach
 Extract
 Identify
 Assess the risk
 Develop the Method
 Validate the Method in Drug Product
 Perform Leachable Study (Stability)
41

42. Identify
 Analytical testing
 TOC
 UV
 GC/FID, GC/MS, GC/IR
 LC/UV, LC/MS, LC/NMR
 FTIR
 pH
 Conductivity
 ICP/MS
 Work with vendor
42

43. Risk Assessment
 Literaturesearch
 Biological testing
 In-vitro - USP <87>
 In-vivo - USP <88>
 Effect on release/stability methods
43

44. 44
From BioPharm International, Dec. 2002, Miller et.al.

45. “OVERVIEW OF EXTRACTABLES AND LEACHABLES IN
PROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASE
STUDIES”
PROBLEMS ASSOCIATED WITH LEACHABLES
 Increase in drug product impurities
 Interaction with active ingredient, vehicle or
excipients
 May cause toxicity of a drug product
 Interference with drug product assays
 Interference with medical diagnostic tests
Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)

46. “OVERVIEW OF EXTRACTABLES AND LEACHABLES IN
PROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASE
STUDIES”
FDA - CASE STUDY #1
 Process Change: Lyophilized to liquid formulation
 Source: release of divalent metal cation from rubber
stopper
 Mechanism: activation of a contaminating
metalloproteinase in the product caused product
degradation
 Impact: Increase in protein degradation
 Resolution: chelator added to formulation buffer
Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)

47. “OVERVIEW OF EXTRACTABLES AND LEACHABLES IN
PROTEIN THERAPEUTICS: SOURCES, METHODS, AND
CASE STUDIES”
FDA - CASE STUDY #2
 Container closure: prefilled syringe
 Tungsten wires are used perforate the syringe
barrel during syringe manufacturing
 Source: release of tungsten oxide from the syringe
into the product
 Impact:
increase in protein oxidation followed by
aggregation
 Resolution:switch to tungsten-free wires to perforate
syringe barrels
Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)

48. “OVERVIEW OF EXTRACTABLES AND LEACHABLES IN
PROTEIN THERAPEUTICS: SOURCES, METHODS, AND
CASE STUDIES”
FDA - CASE STUDY #3
 Process Change: lyophilized product changed to a
lower dosage form
 Impact:decrease in protein stability at room
temperature after reconstitution
 Hypothesis: leachables from rubber stopper at
increased ratio of leachables to the protein cause for
instability
 Resolution: product storage temperature changed
from controlled room temperature to 2-8 C
Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)

49. “OVERVIEW OF EXTRACTABLES AND LEACHABLES IN
PROTEIN THERAPEUTICS: SOURCES, METHODS, AND
CASE STUDIES”
FDA - CASE STUDY #4
 Process Change: from vials to prefilled syringes
 Source: solvent from partially dried epoxy glue
used for needle attachment to syringe barrel
leached into the product
 Outcome: increase in protein oxidation followed by
aggregation
 Resolution:syringe barrels allowed to dry for 6
months prior to use
Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)

50. “OVERVIEW OF EXTRACTABLES AND LEACHABLES IN
PROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASE
STUDIES”
FDA - LESSONS LEARNED
 Leachables can have a great impact on the
quality and safety of protein therapeutics
 Compendial tests often do not provide adequate
sensitivity and specificity (e.g., did not detect
tungsten oxide)
 Important to monitor leachables over time (e.g.,
extended time points reflective of product dating
period should be included)
Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)

51. Particulate Matter Definition
 USP <788> for Injections states that in
“particulate matter in injections and parenteral
infusions consists of extraneous mobile
undissolved particles, other than gas bubbles,
unintentionally present in the solutions”
 Harmonized with Ph.Eur. and JP
51

52. USP <788> Criteria
 Volume < 100 mL
 NMT 3000 > 10 µm
 NMT 300 > 25 µm
 Volume > 100 mL
 NMT 12/mL > 10 µm
 NMT 2/mL > 25 µm
52

53. Size Range of Particulate Matter
Particulate Analysis
Aggregate Analysis Visible Particles
Subvisible Particles
Soluble Aggregates
Monomer
0.001 0.01 0.1 1 10 100 1000
Size (µm)
53

54. Protein Aggregates
Particulate Analysis
Visible Particles
Oligomers: 10 nm- 0.1 µm Subvisible Particles
Soluble Aggregates Visible: > 125 µm
Micron: 1-125 µm
Monomer
Submicron: 0.1- 1 µm
0.001 0.01 0.1 1 10 100 1000
Size (µm)
54

55. What is Known
 Many biologics form particulates
 Particulates are generally undesirable
 Particulate formation is not well
understood
 Consequences of particulates are not
well understood
 Visible particulates are difficult to
measure objectively
55

56. Why the Interest
 Safety
 Quality
 Guidelines and Regulations
56

57. EMA Guideline
 Guidelineon Development, Production,
Characterisation and Specifications for Monoclonal
Antibodies and Related Products
 Effective July 1, 2009
 “The formation of aggregates, subvisible and visible particulates in
the drug product is important and should be investigated and closely
monitored on batch release and during stability studies. In addition
to the pharmacopoeial tests for particulate matter, other orthogonal
analytical methods…”
 “Visible and sub-visible particulate matter in drug product should
comply with the requirements set forth in the European
Pharmacopoeia”
57

58. Dosage Form Specifications
 Compendial requirements
 Microbiological
 Content uniformity
 Volume in container
 Particulate matter

59. Dosage Form Specifications
 Product specific
 Water content for lyophilized dosage form
 Preservative content for multi-use dosage
form
 Antioxidant content
 Osmolality
 pH
 Container closure integrity during stability
testing
» Replaces sterility?
» Dye ingress, microbial ingress?

60. Preservatives
 Minimize the content in the product
 Need to justify use
 Proof of effectiveness

61. Antioxidants
 Justifyamount by demonstrating lack
of or less degradation

62. Antimicrobial Effectiveness
Testing
Indicator organisms
 E. coli
 P. aeruginosa
 S. aureus
 C. albicans
 A. niger
Environmental isolates

63. Antimicrobial Preservatives
 Evaluate antimicrobial properties during
storage
 Determine preservative content and
degradation during storage
 Confirm antimicrobial effectiveness at lower
limit of preservative specification

64. Antimicrobial Effectiveness
Testing-USP
USP <51> Description Inoculum Acceptance Criteria
Category
Log reduction
1  Injectables, other parenterals 105-106 Bacteria: 7 d NLT 1.0
 Sterile nasal products CFU/mL 14 d NLT 3.0
 Aqueous based ophthalmic 28 d NI from 14
products Yeast & molds: NI
2  Aqueous based topical 105-106 Bacteria: 14 d NLT 2.0
products
CFU/mL 28 d NI from 14
 Nonsterile nasal products Yeast & molds: NI
3  Aqueous based oral 105-106 Bacteria: 14 d NLT 1.0
products
CFU/mL 28 d NI from 14
Yeast & molds: NI
4 Aqueous based antacids 103-104 Bacteria, yeasts & molds: NI
CFU/mL

65. Antimicrobial Effectiveness
Testing-EP
EP Description Inoculum Acceptance Criteria
Log reduction
 Parenterals 105-106 CFU/mL Bacteria: 6 h NLT 2
24 h NLT 3
 Aqueous based
28 d No recovery
ophthalmic
Fungi: 7 d NLT 2
products
28 d NI
Topical products 105-106 CFU/mL Bacteria: 2 d NLT 2
7 d NLT 2
28 d NI
Fungi: 14 d NLT 2
28 d NI
Oral products 105-106 CFU/mL Bacteria: 14 d NLT 3
28 d NI from 14
Fungi: 14 d NLT 1
28 d NI

66. Common Formulation Excipients
for Biotechnology-Based Products
Osmotic Agents (salts)
Chelators (EDTA, citrate)
Cations
Sugars (mannose, maltose, dextrose)
Amino Acids (arginine, glycine, glutamic acid)
Redox Agents (ascorbate, reducing sugars)
Solubilizers (Tween, Deoxycholate)
Stabilizers (albumin, lipids)
Solvents (aqueous, nonaqueous)
Several of these compounds interfere with
analytical technologies used for biotech products

67. Hidden Sources of Variability:
Assay Materials and Reagents
Potentially “Critical” Assay Reagents for
Biotech Methods:
 Complex molecules, often biologically derived
 Demonstrated to be a key assay component
 Sensitive to operational or assay conditions
 Selected characteristics may vary from lot to lot
 Limited concurrent availability of multiple lots
 Single-source product manufacturer
Ritter, N and Wiebe, M (2001) Validating Critical Reagents Used in cGMP Analytical
Testing, BioPharm 14:5, pp 12-20.

68. Potentially Critical Assay Components
HPLC - columns (resins and packing procedures), unique mobile phase components
Capillary electrophoresis - capillaries, electrode buffers, prepared kit components
Gel electrophoresis - gel matrix components, unique buffers, precast gels, stains, dyes
Immunoassays - immunoreagents, detection agents, unique blocking materials
Peptide maps - reduction/alkylation reagents, digestion enzymes, HPLC columns
Colormetric methods - commercial standards, chromogenic agents, prepared assay kits
Amino acid analysis - hydrolysis reagents, derivatization reagents
Protein sequencing - coupling, cleavage and conversion reagents; de-blocking enzymes
Bioactivity assays - substrates, cofactors, ligands, cell cultures, media components
Sample preparation - unique buffer components, filters, membranes, culture plates,
vials and stoppers
Ritter, N and Wiebe, M (2001) Validating Critical Reagents Used in cGMP Analytical Testing,
BioPharm 14:5, pp 12-20.

69. Quality by Design Initiative
 ICH Guidances Q8, Q9 & Q10
 Does this affect how specifications are
established or used?
 Specifications are linked to manufacturing
process
 Specifications should account for stability
 Specifications are linked to preclinical and
clinical studies

70. Conclusions
 Specifications contains two components, the
test method and the acceptance criterion
 Specifications are established as surrogates
of characterization tests
 Specifications selected to ensure quality of
material for safety and efficacy
 Specifications based on manufacturing
process, stability and preclinical/clinical data
 Limited batch data can be compensated for
by more thorough understanding of
manufacturing process and link between
quality attributes and clinical outcome

71. Thank You!!

72. Questions or Advice
David Lin
Senior Consultant
Biologics Consulting Group, Inc.
www.biologicsconsulting.com