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Ancillary Materials ISCT Technical Session
Fouad Atouf, Ph.D.
Senior Scientific Liaison
Biologics and Biotechnology
Email: [email protected]
USP’s Founding
Recognition of USP Standards in Federal Food Drug and
Cosmetic Act (FDCA)
• Section 501(b) - Adulterated Drugs and Devices – A drug or device shall be deemed to be adulterated if it purports to be or
is represented as a drug the name of which is recognized in an official compendium, and its strength differs from, or its quality or purity falls below, the standards set forth in such compendium.
– Such determination as to strength, quality, or purity shall be made in accordance with the tests or methods of assay set forth in such compendium…
• Section 502(g) Misbranded Drugs and Devices – A drug or device shall be deemed to be misbranded if it purports to be a
drug the name of which is recognized in an official compendium, unless
it is packaged and labeled as prescribed therein.
Provisions of the FD&C Act apply to biologics regulated
under PHS Act
• PHS ACT (42 USC 262)
– 262(j) Application of FDCA:
• The Federal Food, Drug, and Cosmetic Act… …applies to a
biological product subject to regulation under this section, except
that a product for which a license has been approved under
subsection (a) shall not be required to have an approved application
under section 505 of such Act (21 U.S.C. 355)
• Biological products approved under the PHS Act
are subject to the adulteration and misbranding
provisions of FD&C Act
• If an official monograph in USP-NF is available, the
biological product should conform
USP’s Standards
• The United States Pharmacopeia and the National Formulary (USP–NF)
• Food Chemicals Codex
• USP Dietary Supplements Compendium
• Reference Standards
• Other Resources
–Pharmacopeial Forum –FCC Forum –USP Dictionary
2010–2015 Expert Committees
Standards for B&B Product Classes
<xxxx> Overarching Guidance
<XXX> Common Product Class Quality
Attributes
<XXX> Analytical
Procedures
<XXX> Ancillary
Materials
Monograph Monograph Monograph Monograph Monograph
Chapters
– <1043> Ancillary Materials
– <1046> Cell and Tissue Based Products
– <1047> Gene Therapy Products
– <1027> Flow Cytometrty
– <1024> Bovine Serum
– <90> FBS Quality Attributes and Functionality Tests
– <92> Cytokines and Growth Factors Quality Attributes
– Other Biologics and biotechnology related chapters
Monographs
– Monographs for Tissue-based products
– Monographs for Cell therapy products
Reference Standards
– Physical RS associated with AMs Chapters
– Photomicrographs associated with tissue products histology tests
Cell & Gene Therapies Standards
Ancillary Materials (AMs)
• Definitions: – Ancillary Materials also called Ancillary Reagents,
Ancillary Products, Process reagents; are – Biological and biochemical substances used in
processes for manufacturing of cell-based therapies and other therapeutics derived from cell culture (i.e. vaccines, proteins)…these substances are not intended to be in final product.
• Highest quality of AMs are necessary for safety and
efficacy of products, and demanded by regulators
• Quality Challenges- Cell therapies applications: – Manufacturing process does not ensure removal of
ancillary material – Short shelf life product does not allow extensive testing,
need to ensure quality of AM before use
Regulatory expectations? Discuss with specific agency
Source of material
– Animal free vs. animal derived material
– Traceability of animal derived material
Testing is the key to determine if AM is acceptable for use
AMs may become component of finished product, some of
the regulatory concerns are:
– AM should pass test for adventitious agents
– Residuals (solvents and other process related)
AM should pass test specifications
– Functionality: potency/strength
Supplier audit and qualification of the AM (Verify GMP claim)
AMs- Quality Concerns& Approaches
Quality of AMs- Resources
• Source of material ( RUO, GMP) and Certificate of Analysis
‒ Fewer or more tests depending on suppliers.
‒ Additional testing may be needed? Supplier, end user?
• Some of the mechanisms used by U.S. FDA for review of
quality of ancillary materials
‒ During review of IND submission
‒ Inspections (pre-BLA, cGMP compliance, cGTP compliance)
• Standards
– International Standards (WHO) when available
– Pharmacopeial Standards (USP)
Ancillary Materials - Examples
Ancillary Material Typical use
Human Serum Albumin Cell culture supplement
Antibiotics Cell culture
Monoclonal antibodies Negative/positive cell selection
immunomagnetic beads Immunomagnetic cell selection
Density gradient media Cell separation
Recombinant growth factors, cytokines,
and hormones (e.g. insulin, bFGF)
Cell culture
Heparin sodium salt
Cell culture
(e.g. binding of FGF to receptor)
Feeder cell lines Cell culture substrate
Fetal Bovine Serum Cell culture- growth promotion
Proteoloytic enzymes Process enzymes
Ancillary Materials Standards: USP Approach
<1043> Ancillary Materials for Cell-,
Gene-, and Tissue-Engineered
Products
Ancillary Material
Requirements- Specific
Products
Ancillary Material
Reference Standards
Guidance- Information
Chapter
Specific Products Chapters: -<1024> Bovine Serum
-<90> FBS Quality Attributes
-<92> Cytokines and Growth
Factors Quality Attributes
<123> Protein A Quality Attributes
Reference Standards: -FBS
-Interleukin-4
-Protein A
<1043> Ancillary Materials - Chapter Scope
• Quality of AMs can affect stability, safety, potency and
purity of medicinal products
• Validation of manufacturing processes to ensure removal
of ancillary materials from final products
• Residual testing is important (potential immune reaction)
• Risk-based categories of AMs
• Qualification programs for AMs used in cell
manufacturing:
– Identification
– Selection
– Suitability of use
– Characterization
– Vendor qualification
– QA/QC data
Risk-based Classification of Ancillary Materials
• USP chapter <1043> lists four(4) risk-based categories,
with examples for each category
• The tables are provided only as a guide
• Other criteria for risk assessment are the amount and
stage of use during manufacturing as part of a risk
assessment strategy (not addressed in the tables of
chapter <1043>)
Four categories of ancillary materials:
Tier 1 – Low-Risk, Highly Qualified Materials with Intended Use
as Therapeutic Drug or Biologic, Medical Device, or
Implantable Material
Tier 2 – Low-Risk, Well Characterized Materials with Intended
Use as AMs, Produced in Compliance with GMPs
Tier 3 – Moderate-Risk Materials Not Intended for Use as AMs
(frequently produced for in vitro diagnostic use or reagent
grade materials)
Tier 4 – High-Risk Materials, Materials not Produced in
Compliance with cGMPs and materials not intended to be
used in cell manufacturing
Risk-based Classification of Ancillary Materials
AMs Risk Assessment - Examples
Ancillary Material Typical use
Human Serum Albumin Cell culture supplement
Antibiotics Cell culture
Monoclonal antibodies Negative/positive cell selection
immunomagnetic beads Immunomagnetic cell selection
Density gradient media Cell separation
Recombinant growth factors, cytokines,
and hormones (e.g. insulin, bFGF)
Cell culture
Heparin sodium salt
Cell culture
(e.g. binding of FGF to receptor)
Feeder cell lines Cell culture substrate
Fetal Bovine Serum Cell culture- growth promotion
Proteoloytic enzymes Process enzymes
Ancillary Materials Standards: USP Approach
<1043> Ancillary Materials for Cell-,
Gene-, and Tissue-Engineered
Products
Ancillary Material
Requirements- Specific
Products
Ancillary Material
Reference Standards
Guidance- Information
Chapter
Specific Products Chapters: -<1024> Bovine Serum
-<90> FBS Quality Attributes
-<92> Cytokines and Growth
Factors Quality Attributes
<123> Protein A Quality Attributes
Reference Standards: -FBS
-Iinterleukin-4
-Protein A
• Chapter outlines testing requirements for Cytokines and Growth factors used in cell manufacturing
– General Introduction
– Interleukin-4, is first cytokine to be included in in the chapter. Section written in a monograph format.
– FGF2 requirements and test specifications will be added to chapter <92>
• Chapter written with intention to add additional growth factors and cytokines in the form or revisions (Transferrin, GM-CSF, etc..)
Chapter <92>
Chapter <92>
• Interleukin-4 – Interleukin-4 (Human Recombinant)
– Nonglycosylated, 130 amino acid polypeptide expressed in E.coli
– Used ex-vivo to stimulate the proliferation of activated B- and T-
cells
• IL-4 required Tests – Identification
• Amino acid sequence analysis (minimum 8 residues)
• Western blot analysis
– Purity by SDS-PAGE
– Protein content (UV Absorption)
– Bioidentity (bioactivity)
Chapter <92> Revision – FGF2 Draft
• Definition:
• Identification: – N-term Sequencing
– SDS-PAGE/Western Blot
• Purity- – SDS-PAGE
– Host cell DNA content
• Bioidentity: Unit assigment based on cell-based assay (FBHE cell line)
• Microbial limits: Meet the requirements <71>
• Bacterial Endotoxins-<85> NMT100 USP EU per mg
• Specific tests – Protein content
• Additional Requirements: – Packaging and storage
– Labeling
– Reference standard- USP FGF RS
USP <90> FBS Quality Attributes
• Initial Ranges for FBS quality attributes based on data from
suppliers, while trying to harmonize with the European
Pharmacopeia
• Published chapter <90>, quality attributes adjusted based on USP
multi-lab study (USP Lab, 2 Contract Labs, 4 Industry Labs)
– Osmolality: 280-360 mOsm/Kg
– Total Protein: 30-45 mg/mL
– pH: 7.00-8.00
– Endotoxins: <10 Units/mL
– Hemoglobin levels <30 mg/dL
– Identification: Radial ImmunoDiffusion (RID): species ID, IgG levels
– Functionality Assays
• Growth Curve
• Clonal Assay
Quality Attributes- FBS vs. other Bovine Sera
Serum Supplier pH Osmolality Hemoglobin
(mg/dL)
Tot Proteins
(mg/ml)
Newborn Calf Serum A 7.6 295 12.3 5.3
Calf Serum A 7.7 301 5.5 8.2
Fetal Bovine Serum A 7.1 309 14.1 4.38
Bovine Serum B 7.8 250 12.1 6.6
Fetal Bovine Serum (a) B 7.2 298 5.4 4.75
Fetal Bovine Serum (b) B 7.2 299 6.1 3.89
Donor Adult Bovine Serum B 7.9 249 4.6 6.75
Newborn Calf Serum B 7.5 283 3.6 4.59
Horse Serum C 7.1 289 9.1 7.6
Fetal Bovine Serum (a’) C 7.2 253 6.6 2.76
Fetal Bovine Serum (b’) C 7.1 280 10.1 3.26
Newborn Calf Serum C 7.3 276 9.5 7.33
Bovine Serum C 7.2 281 14.4 8.03
Newborn Bovine Serum D 7.0 293 7.1 7.59
Bovine Calf Serum D 7.4 214 4.7 4.84
Fetal Bovine Serum D 6.9 301 10.6 4.28
Adult Bovine Serum D 7.7 289 10.6 8.02
pH, Osmolality, Total Protein: data is mean of 3 replicates
Hemoglobin: data is mean of 2 replicates
Identification: Radial ImmunoDiffusion
Sample Source Average
Bovine IgG Concentration
mg/L
Fetal Bovine Serum A 204.89
Fetal Bovine Serum B 321.82
Fetal Bovine Serum C 292.44
Bovine serum A 24941
Bovine serum B 41782
Bovine serum C 31865
Horse Serum A 0
FBS horse serum Samples: average of duplicates, 2 different concentrations (neat, 1:2)
Bovine Serum Samples: average of duplicates, 2 different concentrations (1:50, 1:100)
Functionality Assays
• The methods for cell growth-supporting assays based on
procedures used by ATCC for the qualification of serum for cell
culture.
– Growth Curve
– Clonal Assay
• The Growth Promotion Curve assay have been verified in the
Biologics and Biotechnology Lab at USP
• Five cell lines listed for use in chapter <90>. Two cell lines (out
of the five) and a 3rd cell line relevant to their application, are
recommended for the verification of functionality by the end
user
Growth Curve- Procedure
Challenges: Cell line, Cell density, Cell counting, days in culture
•Three cell densities, determine viable cell
counts on days 0,1,2,3,4, and 7. Select the
cell density that exhibit a growth curve with
3 phases: Lag, Log, Stationary; and linear
over 3 time points or more
•Use the selected cell density to assess the
test FBS side by side with the reference
standard FBS
•Doubling time is estimated using a growth
curve that is linear over three or more time
points.
•Acceptance Criteria:
R2≥ 0.98
Doubling time of test sample should be no
less than 90% of doubling time of RS
Reference Standard Candidate for FBS
• Challenges:
– Choice of RS Candidate
– Country of origin of RS material
– Small size container/validation
– Irradiation/validation
– Fill and fill site
– Product shelf life/closure system
• FBS will be a mixture of 3 lots, each lot contributed by an
independent supplier
• Official uses of FBS Reference Standard
– Identification: Radial Immunodiffusion
– Functionality Tests: Growth Promotion Curve
The Future?
Zheng et al., Biotechnology Progress, 22(5) Sept-Oct 2006
The Future, Other Challenges
• Reports of FBS immunogenicity :
– Selvaggi TA et al (Blood, 1997)
– Sakamoto N et al (Blood, 2007)
• Need to develop test(s) for residual FBS
The Immediate Future
Collaborate with us:
• Participate in the FBS collaborative study
• Propose new generation of test methods for FBS (e.g.
proteomics-based methods for identification)
• Propose methods for residual testing of FBS in
therapeutic products
• Propose new AM standards to us
• <1043> is being updated – this is your time to comment!
• Comment on the upcoming revision of <92>