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Ancillary Materials ISCT Technical Session Fouad Atouf, Ph.D. Senior Scientific Liaison Biologics and Biotechnology Email: [email protected]

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Page 1: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

Ancillary Materials ISCT Technical Session

Fouad Atouf, Ph.D.

Senior Scientific Liaison

Biologics and Biotechnology

Email: [email protected]

Page 2: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

USP’s Founding

Page 3: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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.

Page 4: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 5: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 6: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

2010–2015 Expert Committees

Page 7: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 8: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 9: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 10: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 11: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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)

Page 12: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 13: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 14: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

<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

Page 15: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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>)

Page 16: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 17: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 18: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 19: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

• 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>

Page 20: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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)

Page 21: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 22: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 23: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 24: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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)

Page 25: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 26: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 27: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 28: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

The Future?

Zheng et al., Biotechnology Progress, 22(5) Sept-Oct 2006

Page 29: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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

Page 30: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological

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>

Page 31: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological
Page 32: Ancillary Materials - c.ymcdn.com · Ancillary Materials (AMs) •Definitions: –Ancillary Materials also called Ancillary Reagents, Ancillary Products, Process reagents; are –Biological