Standards and Best Practices for Cell, Gene and Tissue ... · Raw and Ancillary materials – USP ...

Standards and Best Practices for

Cell, Gene and Tissue-based Therapies

Rebecca Potts, Ph.D.

July 18, 2017

Introduction- USP

USP <1046> Cellular and Tissue-based Products

– Quality Systems, Qualification of Materials

– Manufacturing of Cell and Tissue-based Products

– Release Tests for Cell and Tissue-based Products

USP <1047> Gene Therapy Products

– Manufacturing Gene Therapy Products, Safety Concerns

– Analytical Methods for assessing gene therapy product quality

Raw and Ancillary materials

– USP <1043> Ancillary Materials for Cell, Gene and Tissue-

Engineered Products

– Risk-based categories of Ancillary Materials

Reference Standards - Examples

Conclusions2

Outline

3

U.S. Pharmacopeial Convention (USP)

Work with more than 900

scientists, practitioners

and regulators to develop

standards that help

protect public health

Internationally recognized

and globally focused

Headquarters in Rockville,

MD

Laboratory facilities

in U.S., India, China,

Brazil and Ghana

Offices in Switzerland,

Ethiopia, Indonesia, the

Philippines and Nigeria

Founded in 1820,

nonprofit, private,

independent and

self-funded

Values-driven

organization focused

on quality standards to

protect the public’s health

More than 1,000

employees worldwide

4

Ensuring Quality Medicines

Suppliers

Manufacturers

Wholesale/

Distributors

Pharmacies/

Hospitals

Healthcare

Patients

USP STANDARDS

General Chapters

Nomenclature and Labeling

Monographs

Reference Standards

USP HEALTHCARE

QUALITY STANDARDS

Nomenclature and Labeling

Compounding – Sterile and Nonsterile

Model Guidelines for Formularies

Safe Medication Use

Prescription Labeling

Hazardous Drugs – Practitioner Handling

Introduction- USP










Engineered Products



Conclusions

5

Outline

Cellular product: living human or animal cells or tissue

that have been manipulated or are used in ways that result in

their regulation as somatic cellular therapies as defined by the

U.S. Food and Drug Administration (FDA)

Tissue-based product: human tissues that do not require

pre-market approval by FDA and only need to comply with good

tissue practices (GTP regulations)

Combination product: cells combined with medical

devices such as natural or synthetic scaffold

* <1046> is undergoing revision

6

Cell, Gene and Tissue-based Products

Definitions— USP <1046>

Source Material for Cell and Tissue-based products: human

and animal derived cells

– Procurement of HCT/Ps for medical use in adherence with donor eligibility

requirements

Isolation

– Solid organs or tissues are usually dissected to expose desired

region

– Enzymatic digestion of extracellular connective tissue is another

common method for dissecting tissue.

Selection

– Varying force during centrifugation selectively enrich cell

populations

– Monoclonal antibodies directed against specific cell-surface

proteins used for both negative and positive selection

7

Source Material for Cell and Tissue-Based Products

Manufacturers of cellular or tissue-based products:

– Ensure that all components used in manufacturing are

appropriately qualified

– Components used in production: source cells and tissues,

natural or synthetic biomaterials, ancillary material and excipients

used in formulation products

Qualification: process of acquiring and evaluating data to establish

the source, identity, purity, biological safety and overall suitability of a

specific component

Material qualification more comprehensive as the product

progresses in development.

Testing for key quality attributes is critical

Cell-based therapies regulated as biological products must comply

with applicable sections of 21 CFR 211 and 21 CFR 610

8

Quality Systems for Components in Cellular and

Tissue-based Products

Analytical methods are used to establish in-process controls

and product release criteria.

Quality specifications for cell and tissue products confirm the

product’s quality, safety and potency.

Specifications are established through characterization of the

product during development phase.

Terminal sterilization is not possible for a living cell-based

product. Cell-based products are required to meet

acceptance criteria for product tests such as sterility,

mycoplasma and endotoxin.

In-process controls and specifications should be anchored by

the use of an appropriate reference standard.

9

Analytical Methods for Cell and Tissue-based

Products

Identity

– Lot release testing for cell-based products must include an

identity test

– Identity test serves to unequivocally identify the product

• Differential surface markers are frequently used to ascertain

product identity

• Cell morphology may also be used to distinguish specific cell types

Purity

– Purity methods specifically quantify the intended active product

components

– Testing for impurities as part of lot release should reflect the

safety risks associated with the impurity and the ability of the

process to remove the impurity

10

Final Product Release Testing for Cell and

Tissue-base Products

Potency

– With dose, potency defines the biological activity of each lot.

– Potency may be assessed by in vitro or in vivo bioassays, or a

combination of the two

• Assays require a well-defined reference material that can serve as

a positive control for the assay. The positive control serves to

qualify the performance of an individual assay

Dose-Defining Assays

– An assay that precisely measures the amount of product

– Cell therapy products may be dosed on the basis of

enumeration of one or more cell populations

11

Final Product Release Testing for Cell and

Tissue-base Products (Continued)

12

Overview of Final Product Release Testing for Cell and Tissue-based

Products, from USP Chapter <1046>

Release Test Examples Criteria

Sterility USP ⟨71⟩ Negative

Mycoplasma Direct and indirect culture method (FDA Points to Consider) Negative; not detected

Endotoxin USP ⟨85⟩ <5 EU/kg (<0.2 EU/kg intrathecal)

Identity • Surface marker determination

• Isoenzyme analysis

• Genetic fingerprint

• Morphology

• Bioassay

• Biochemical marker

Product specific

Purity • Percentage of viable cells

• Percentage of cells expressing specific marker(s)

• Limits on undesired cell types

• Limits on process contaminants (e.g., serum)

Product specific

Potency • Viable cell number, Colony-formation assay, Change in expression of specific genes,

Secretion of desired macromolecule,

• Induction of secondary effect (e.g., human leukocyte antigen (HLA))

• Evidence of metabolic activity

• Evidence of cell function

Product specific

Dose • Viable cell number

• Enumeration of specific cell population

• Total DNA

• Total protein

Product specific

Others • Appearance

• Morphology

• Size

Product specific

Stability of cell or tissue-based products and the

components used to create them will vary depending on

the nature of the product, the intended clinical use,

specific attributes, storage, packaging and shipping

conditions.

Stability studies must verify that the storage conditions

maintain the quality attributes of the product

Where feasible, stability testing should be carried out in

accordance with the principles described in ICH

guideline Q5C.

13

Stability of Cell or Tissue-based Products

Introduction- Background










Engineered Products



Conclusions14

Outline

Gene therapy product: the administration of nucleic acids to

modify the genetic material of cells

– Delivery of nucleic acids can be through

• viral vectors, nucleic acid in simple formulation (naked DNA) or

nucleic acids formulated with agents that enhance the ability to

penetrate the cell

– Genetic material introduced can either cause expression of new genes

or inhibit expression of already expressed genes and products

Cell Therapy products can be modified by treatment with

integrating or non-integrating genetic materials (DNA, RNA,

siRNA), so that the pattern of gene expression is changed.

– Typically cells are taken from the patient and modified outside the body

before they are returned to the patient

– Regulatory bodies consider ex-vivo gene modified cellular product to be

a gene therapy product.

15

Gene Therapy Products

Manufacturing of gene therapy products requires:

Choosing a vector:

– The vector will include: the vector backbone, a promoter, the

therapeutic gene and a polyadenylation signal

Targeting Transduction

– Vector must find and transduce the target cell

– Viruses have a natural host range that is influenced by the

expression of cell-surface receptors, the cell cycle and the route

of administration

– Barrier to transduction is humoral immune response to the vector

16

Manufacturing Gene Therapy Products

Safety Testing focuses on 3 issues:

– Detecting contamination from adventitious sources during product

processing

– Preventing the use of packaging cell lines and plasmids that permit

genetic recombination between packaging cell lines or plasmids

– Testing the final product to ensure a safe level of undesired genetic

and/or structural variants or other viruses used in processing

Viral Gene Therapy Products

– Occurrence of undesired genetic variants

Non-Viral Gene Therapy Products

– Safety testing should be performed on non-viral formulated material

– Focuses on methods similar to those described in USP <88>

Biological Reactivity Tests, In Vivo and <71> Sterility

17

Safety Concerns with Gene Therapy Products

Approved gene therapy products must comply with applicable

sections of 21 CFR 211 and 610 to ensure identity, dose, potency,

purity and safety

Samples must be retained after product release testing is completed.

In-Process Controls

– Manufacturing processes should have well-defined decision criteria that

are established for key in-process manufacturing steps

Specifications

– Specifications for gene therapy product should be chosen to ensure the

safety and efficacy of the product before use

– Development and setting of specifications for cell and gene therapies

should follow the principles outlined in ICH Q6B

– Procedures in a specification for a product should be anchored by

appropriate reference standards

18

Analytical Methods for Assessing Product Quality

of Gene Therapy Products

Dose Defining Assays

– Precisely measures the amount of product

– Example: particle concentration is a commonly used measure

for viral vector dose

Potency

– Defined as the therapeutic activity of the drug product. Dose

and potency define the biological activity of each lot

– Bioassays to measure the potency of viral and non-viral gene

therapies involve infection or transfection of susceptible cell line

in vitro, followed by a measurement of the expressed gene of

interest

19

Tests for Gene Therapy Products

Purity

– Process related impurities for gene therapy products include

residual production medium components (ie FBS, antibiotics,

cytokines, E.coli chromosomal DNA in a plasmid product)

– Product related impurities are specific for each type of gene

therapy product (viral or nonviral)

– Viral gene therapy product-related impurities include aggregates and

defective immature particles that may be produced during the

manufacture or purification of the recombinant vector

– Nonviral gene therapy product-related impurities include nicked

plasmid forms

Identity

– Lot release testing for gene therapy products must include an identity

test

– Identity test clearly identifies the product and confirms that

inadvertent substitution with another product has not occurred.

20

Tests for Gene Therapy Products (continued)

21

Overview of Analytical Tests for Cell and Gene Therapy Biological

Products from USP <1047> Gene Therapy Products

TestGene-Modified Cellular

Gene Therapy Product

Gene Therapy Products

Viral Nonviral

Identity of

Biological

Substance

•Surface marker determination

•Species

• Morphology

•Bioassay

•Biochemical Marker

• Restriction enzyme map

• PCR

• Immunoassay for expressed

gene

• Sequencing

• Restriction enzyme map

• PCR

• Immunoassay for expressed gene

• Sequencing

Dose •Viable cell number

•Enumeration of specific cell population

•Total DNA

•Total protein

• Particle number

• Transducing units (DNA hybridi-

zation assay)

• Total protein

• HPLC assay using authenticated

reference standard

• Plasmid–DNA weight

• Formulated-complex weight HPLC

or capillary electrophoresis assay

using authenticated reference

standard

Potency •Viable cell number (cells intended for structural repair)

•Bioassays

- Colony-formation assay

- Function of expressed gene

- Induction of secondary effect (e.g., human leukocyte

antigen (HLA) induction, secretion of cytokines, and

up-regulation of surface marker)

• Function of expressed gene

(induction of secondary effect and

other bioassays)

• Function of expressed gene

(induction of secondary effect and

other bioassays)

Purity •Percentage of viable cells

•Percentage of transduced cells

•Percentage of cells with specific surface marker

•Process contaminants (e.g., serum)

• Residual host-cell DNA

• Process contaminants (e.g.,

serum and cesium chloride)

• Residual helper virus

• Optical density ratio

• Residual host-cell proteins

• Viral protein profile (HPLC assay

for defective or immature particles)

• Residual RNA

• Percentage of specific physical form

(e.g., percentage super- coiled)

• Residual host-cell DNA

• Residual RNA

• Residual host-cell proteins

• Residual solvents

• Optical density ratio

• Process contaminants (e.g., cesium

chloride)

Safety •Mycoplasma

•Sterility

•Pyrogen and endotoxins

•Adventitious viruses

•Residual virus

•Replication-competent vector

• General safety

• Sterility

• Pyrogen and endotoxins

• Adventitious viruses

• RCV

• Mycoplasma

• Sterility

• Pyrogen and endotoxins

Stability studies verify that the storage conditions

maintain the purity and potency of the product for a

defined period so the product administered to the

patient is still capable of meeting the stability

specifications

Stability assessment should include evaluation of

product functionality (potency)

Stability studies analogous to accelerated aging

studies typically used in pharmaceutical stability

monitoring programs

22

Stability of Gene Therapy Products

23

Outline

Introduction- Background










Engineered Products



Conclusions

Raw and Ancillary Materials are used in Manufacturing

Cell, Gene Therapy and Tissue-based Products

Raw materials may or may not remain in the final

therapeutic product as active substances or as excipients

Ancillary materials are a subset of raw materials.

– Ancillary products may exert an effect on a therapeutic

substance (e.g. a cytokine may activate a population of

cells), but they are not intended to be in final formulation

Some components are more critical than others. Risk

assessment strategies are required to ensure quality

– A critical material will come in contact with cells with a

potential to alter either the growth characteristics of the cells

or the ability of the cell culture to meet lot release

specifications.

Ancillary materials for Cell Therapy/Tissue-based products

– Culture media, buffers, growth factors, cytokines, cultivation and

processing components, monoclonal antibodies and cell-

separation devices

– Residual amounts of ancillary materials used in the manufacturing

process may be antigenic, so the removal must be assessed and

limits on the amount remaining must be set in some cases.

Ancillary materials for Gene Therapy products

– Raw materials used in manufacturing: cells, tissues, biological

fluids, growth factors and monoclonal antibodies

– Quality of the ancillary materials can affect the safety, potency and

purity of the product.

– Qualification of raw and ancillary materials used in manufacturing

Gene Therapy products is necessary

25

Ancillary Materials Used in Cell and Tissue-based

Products and Gene Therapy Products

Raw Materials: Quality Approaches

Reliance on Suppliers

– Specifications: lot to lot consistency and materials for multiple use

– Test methods: are these validated?

– Certificate of Analysis (CoA)

– Pharmacopeial procedures and which pharmacopeia?

– Testing beyond the CoA

Risk-based approaches and critical raw materials

– Use of multi-suppliers materials

– Impact of materials on final product (quality attributes, residuals)

– Define failures mode -use of quantitative tools- and mitigate risk

– Example: USP <1043>, Ancillary Materials (AMs) used in cell

therapy manufacturing

27

Risk-based Approach USP Chapter <1043>Revision available online July 3, 2017

Criteria that define the level of risk

Tier 1:

Low risk

Intended for use as licensed drugs, biologics or medical devices▪

• Suitability for use as a manufacturing component is required

• Quality aspects of these materials may change after use in the manufacturing

process.

Tier 2:

Low risk

Intended to be used as ancillary materials.

Well-characterized

Produced under quality systems well-suited for biological manufacturing,

Material is not a licensed medical product.

Many are produced specifically for the manufacture of biological products.

Tier 3:

Moderate risk

These are research-grade materials not intended for use in biological

manufacturing

• Sometimes approved by regulatory agencies as part of an in vitro diagnostic

device.

More qualification than Tier 1 or Tier 2 materials.

Tier 4:

High risk

Produced as industrial or research-grade materials and may contain harmful

impurities.

May also contain animal- or human-derived components with potential

contaminants.

Requires extensive qualification before use as component in biological product

manufacturing.

<1043> Ancillary Materials: Risk-based Qualification of AMs and Risk Reduction Activities

Elements of Qualification and/or Risk Reduction Activities Tier 1 Tier 2 Tier 3 Tier 4

Drug Master File cross reference X X X X

Certificate of analysis X X X X

Assess lot-to-lot effect on process performance X X X X

Assess removal from finished product X X X X

Assess AM stability as stored and used in CGT product manufacturing X X X X

Confirm Certificate Analysis test results that are critical to product identity, purity

or potencyX X X

Vendor Audit X X X

Upgrade Manufacturing to GMP level X X

Develop internal specifications X X

Lot to lot comparability may be needed X X

Testing for adventitious agents may be needed X X

Safety testing for residuals in / CGT product X

Adventitious agent testing for animal source-relevant viruses X

Level of Risk

USP’s Ancillary Materials Standards

<1043> Ancillary Materials (AMs) for Cell, Gene, and Tissue-based Products

Specific AM Chapters

<90> FBS Quality Attributes

<130> Protein A

<92> Cytokines and Growth Factors<89> Enzymes

Reference Standards:

- FBS- Protein A- Interleukin-4- Trypsin- Collagenase I and II

General

Information

Chapter

Ancillary Material

Requirements for

Specific AMs

Ancillary Materials

Reference

Standards

Introduction- USP










Engineered Products



Conclusions30

Outline

USP Standards for Fetal Bovine Serum (FBS)

FBS Standard Tests

– Identification: Radial Immunodiffusion (RID): species ID, IgG levels

– Functionality Assays (Growth Curve and Clonal Assay)

– Osmolality: 280-360 mOsm/Kg

– Total Protein: 30-45 mg/mL

– pH: 7.00 - 8.00

– Endotoxin: Less Than 10 units/mL

– Hemoglobin level: Less than 30 mg/dL

– Sterility <71>, meets requirements

Associated Reference Standard (RS)

32

Enumeration of CD34+ Stem Cells USP CD34+ Cells Reference Standard

• Stem cells products from bone marrow, peripheral blood and cord blood are

used in treatment of a number of conditions: Leukemia, Hodgkin’s disease,

non- Hodgkin’s lymphomas…

• No licensed products, investigational exemptions

• USP standard at this early stage will support product development and

consistent CD34+ enumeration, a good predictor of success of apheresis

and engraftment potential

Manufacturers of cell, gene and tissue-based products must

ensure that all components used in manufacturing are

appropriately qualified

Quality specifications for cell, gene and tissue products should

confirm the product’s quality, safety and potency

Standardization of ancillary materials used in manufacturing

allows to control consistency in manufacturing of finished

products

Pharmacopeial standards provide tools for the control of

products development and testing strategies, the standards

can also be used to demonstrate compliance with regulatory

requirements

USP is working on a strategies to include additional standards

for ancillary materials in USP-NF

33

Conclusions

Standards and Best Practices for Cell, Gene and Tissue ... · Raw and Ancillary materials – USP ...

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