Nadine M. Ritter, Ph.D. · Reference Standards and Critical Reagents for Biopharm/Biosimilar...

Reference Standards and Critical Reagents for Biopharm/Biosimilar Products:

The Good, The Bad, and the Ugly (Not Necessarily in That Order)

Nadine M. Ritter, Ph.D.President and Analytical Advisor

[email protected]

www.GlobalBiotechExperts.com

1© 2019 N. Ritter, Ph.D.

What are the Categories of Product Standards?

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What are biopharmaceutical Product-Specific ‘Standards’?

Product Documentary Standards

Written specifications (attributes, methodology, acceptance criteria) to control product quality and stability

Come from:• Approved innovator product regulatory dossier

• Approved biosimilar product regulatory dossier AND• Published pharmacopeial biosimilar product monograph

(except in US)



Product Reference Standards

Physical material used in the QC methodology that is specified in the documentary standards

Come from:• Approved innovator product regulatory dossier

• Approved biosimilar product regulatory dossier AND• Published pharmacopeial product monograph (except US)

• Approved vaccine or plasma product, from designated agencies (eg WHO, CBER)



Product Physical Reference Materials

Biosimilars Only: Reference Licensed Product(s)

Come from• Regionally-authorized Rx batches of the originator drug

product used in:

• Analytical characterization of RLP• Side-by-side similarity studies RLP to biosimilar• Preclinical and clinical head to head studies

ICHQ6B Test Procedures and Acceptance Criteria for Biotechnology Products (1999)

“…distinct reference materials for product-related substances, product-related impurities and process-related impurities, may need to be established. “

Two critical process-specific impurities for biological products:

– Host Cell Proteins (proteome of the expression system)

– Host Cell DNA (genome of the expression system)


What are biopharmaceutical Process-Specific ‘Standards’?

What regulatory guidance is there for biopharm product reference standards?



FDA Biotechnology Inspection Guide (1991)

Standard/Reference Material

Reference standards should be well characterized and documented, properly stored, secured, and utilized during testing.

Potency (Activity) Test

A reference preparation for biological activity should be established and used to determine the bioactivity of the final product.

Note: Where applicable, in-house biological potency standards should be cross-referenced against international (World Health Organization (WHO), National Institute of Biological Standards and Control (NIBSC)) or national (National Institutes of Health (NIH), National Cancer Institute(NCI), Food and Drug Administration (FDA) reference standard preparations, or USP standards.


FDA Biotechnology Inspection Guide (1991)

Host Cell Protein ELISA (Quantitative Standard Curve)

A multiantigen test for unknown residual (host) cellular protein. It requires a reference standard

preparation of host cell protein impurities to serve as an immunogen for preparation of polyclonal

antibodies used for the assay.

Circular Dichroism (Qualitative Spectral Patterns)

With optical rotary dispersion, one of the optical spectrophotometric methods used to determine

secondary structure and to quantitate the specific structure forms (a- helix, B-pleated sheet, and

random coil) within a protein. The resultant spectra are compared to that of the natural protein form

or to the reference standard for the recombinant.

Endotoxin Test (Unit Calibration)

A heat- stable lipopolysaccharide associated with the outer membrane of certain gram- negative

bacteria. It is not secreted and is released only when the cells are disrupted. When injected into

humans, endotoxins produce a febrile response, leading to severe clinical problems, including

death. An endotoxin unit (EU) is defined in comparison to the current USP Reference Standard Lot

EC- 5. One vial of lot EC- 5 contains 10,000 EU. The official test for endotoxin is found in the USP.

ICH Q6B: Specifications; Test Procedures and Acceptance Criteria for Biotechnological/Biological Products (1999)

At the time of the submission, the manufacturer should have established an appropriately characterized in-house primary reference material, prepared from lot(s) representative of production and clinical materials

International reference standards for potency and/or concentrationmay not be suitable as identity and purity/impurity reference standards due to process-specific product characteristics.

Documentation of the manufacturing, purification, characterization, storage conditions, and formulation supportive of each reference material’s stability should be provided.

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BIOTECH PRODUCT REFERENCE STANDARDS

ICH Q7: GMP Guidance for API (2001)

Primary reference standards should be obtained, as appropriate, for the manufacture of APIs. The source of each primary reference standard should be documented.

Records should be maintained of each primary reference standard’s storage and use in accordance with the supplier’s recommendations.

Primary reference standards obtained from an officially recognized source are normally used without testing if stored under conditions consistent with the supplier’s recommendations.

Where a primary reference standard is not available from an officially recognized source, an in-house primary standard should be established. Appropriate testing should be performed to establish fully the identity and purity of the primary reference standard. Appropriate documentation of this testing should be maintained.

Secondary reference standards should be appropriately prepared, identified, tested, approved, and stored. The suitability of each batch of secondary reference standard should be determined prior to first use by comparing against a primary reference standard.

Each batch of secondary reference standard should be periodically requalified in accordance with a written protocol.

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FDA Guidance on Analytical Method Validation (2015)

Primary and secondary reference standards and materials are defined and discussed in the following ICH guidances: ICHQ6B and ICHQ7.

For all standards, you should ensure the suitability for use. You should strictly follow storage and usage conditions and handling instructions for reference standards to avoid modifications and contaminations, which could result in additional impurities and inaccurate analysis.

You should include information supporting any reference standards and materials that you intend to use in the application (dossier).

Information supporting reference standards and materials should include qualification test reports and certificates of analysis (including stability protocols, reports, and relevant known impurity profile information) as applicable.

For biological products under BLAs, qualification of subsequent reference standard lots should be included in annual reports

Reference standards can often be obtained from USP and may also be available through the EP, JP, WHO or NIST. Reference standards for a number of biological products are also available from CBER. For certain biological products marketed in the U.S., reference standards authorized by CBER must be used before the product can be released to the market.

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Reference materials from other sources should be characterized by procedures including routine and beyond routine release testing as described in ICH Q6B.

You should consider orthogonal methods for reference material characterization.

Additional testing could include attributes to determine the suitability of the reference material not necessarily captured by the drug substance or product release tests (e.g., more extensive structural identity and orthogonal techniques for potency, purity and impurities).

A new batch of reference standard material (official or in-house) should be qualified/calibrated against the current reference standard.

For biological reference standards and materials, we recommend that you follow a two-tiered approach when qualifying new reference standards to prevent drift in the quality attributes.

A two-tiered approach involves a comparison of each new reference standard with a primary reference standard so that it is linked to clinical trial material and the current manufacturing process.

What are key issues with the types and sources of reference standards and reference materials for biopharma

products?

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TYPES of Biopharm Reference Standards and Materials

What EXACTLY is being Tested?Product

• Product related substances

• Species endogenous to the active product type

(e.g. Protein, Nucleic acid, Protein mixture, Viral particles, Cellular species)

• Process related impurities

• Species endogenous to the production process

• Upstream (e.g. Host cell proteins, Host cell DNA, Media components)

• Downstream (e.g. Enzymes, Column Leachates)

• Formulation/Filling (e.g. Particles, C/C Leachates)

• Complex Products (eg gene and cell therapy)

• Morphology of 3D structure (eg shape and size; intact or disrupted)

• Histology (eg immunohistochemistry of marker proteins)

Excipients / Raw Materials

• Components added to DS or DP

(e.g. Surfactants, Buffering compounds, Isotonic agents, Bulking agents)

EACH test method should be examined to determine:

(1) What exactly is being measured in the test method? (target analyte)

(2) Is a reference material used to generate the reportable result?

(3) Is a reference material used for system suitability?

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How a Product Ref Std Can Be Used in a Test Method

As a system suitability standard (ie assay control)

• It is prepared and run in the test method like the test samples

• It may have a different analysis order than the test samples

• It has criteria for assuring the method run is valid (eg

precision of replicates)

• Does not directly impact accuracy of reportable results from

test samples

As a comparator for generating qualitative reportable

results


• Test samples are visually compared to the Ref Std run

concurrently

• Directly impacts accuracy of reportable results from test

samples

As a calibrator for generating quantitative reportable results


• Test samples are quantitatively compared to the Ref Std run

concurrently

• Directly impacts accuracy of reportable results from test

samples

Ref Std

Test Sample

Ref Std

Test Sample

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How a Non-Product Ref Material Can Be Used in a Test Method

As a system suitability standard (ie assay control)

• It is prepared and run in the test method like the test

samples

• It may have a different analysis order than the test

samples

• It has criteria for assuring the method run is valid (eg

precision of replicates)

• Does not directly impact accuracy of reportable results

from test samples

SEC MW Standards

As a standard curve for generating quantitative

reportable results

• It is prepared and run in the test method as a series of

concentrations (standard curve)

• Test samples are quantitatively interpolated to the

standard curve run concurrently

• Directly impacts accuracy of reportable results from

test samples

Avg

OD

HCP ng

HCP ELISA

REF STD

HCP ELISA

TEST

SAMPLES

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Who makes and controls the quality of the Reference Material?

Product• Product related substances

• Unique to each DS/DP• Must come from the actual production process for each product

• Process related impurities• Host Cell Proteins (proteome)

• Unique to each process expression system• Must come from the actual production process (by end of Ph 3)

• Host Cell DNA (genome)• Unique to each genome• Some may be commercially available (with appropriate C of A)• If not, must be prepared and tested internally

• Other species (media, column resins, etc..)• Not unique to the process• Usually commercially available (some are vendor-specific)

Excipients / Raw Materials• Used with multiple processes and products• Usually commercially available• Some have quality specifications based on monographs (USP, EP, JP, etc…)• Those monographs would utilize certified reference standards

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Regulatory and Quality Considerations for Reference Standards or Materials

Regulated Reference Standard or Material: ✓ Used in GMP methods in product specifications✓ May be used for system suitability to show run is valid/invalid✓ Always used to generate reportable results from test samples✓ Reported in the method qualification/validation/verification reports ✓ Included in the qual/val/ver method SOPs✓ Presented in the Module 3 method summary✓ Described in Reference Standard section of Module 3 CTD✓ May not make changes in these without prior regulatory approval

Quality System Reference Standard or Material: ✓ Used in GMP methods in product specifications✓ Used only for system suitability to show run is valid/invalid✓ Never used to generate reportable results for test samples✓ Reported in the method qualification/validation/verification reports ✓ Included in the qual/val/ver method SOPs✓ Presented in the Module 3 method summary✓ Not described in the Reference Standard section of Module 3 CTD✓ May make changes in these without prior regulatory approval

How do you create and manage biopharm product in house reference standards throughout the product’s

lifecycle?

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CMC Strategy Forums on Reference Standards

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www.casss.org; tab: Resources, tab: CMC Forum Papers

http://www.casss.org/

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Mire-Sluis, et al: CMC Strategy Forum Reference Standards, BioProcess Intl March 2014

TWO-TIERED APPROACH TO PRODUCT REFERENCE STANDARDS

What are other critical assay reagents besides various types of

reference standards?

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Hidden Sources of Variability: Critical Assay Materials and Reagents

Potentially “Critical” Assay Reagents for Biotech Methods often have one or more of these characteristics:

• 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.

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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) BioPharm 14:5, pp 12-20.

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IV. CONTENT OF ANALYTICAL PROCEDURES

D. Reagents/Standards

The following should be listed where applicable:• Description of reagent or standard• Grade of chemical (e.g., USP/NF, ACS, HPLC, GC) • Source (e.g., USP reference standard, qualified in-house reference material, WHO

International Standard/Reference Material, CBER standard)• Purity (for pure chemicals only)• State (e.g., dried, undried)• Concentration• Potencies (where required by CFR, USP)• Storage conditions• Directions for safe use (as per current Safety Data Sheet)• Validated or documented shelf life

New batches of biological reagents, such as monoclonal antibodies, polyclonal antisera, or cells may need extensive qualification procedures included as part of the analytical procedure.

How can you validate robustness of methods to changes in critical reagents?

• Indicate in SOP that there is no ‘or equivalent’ source or type of the reagent

• Each new lot must be subjected to qualification in assay performance prior to

being used in the validated method

• Generate a written procedure for conducting the performance qualification of

the new lot side by side with the old lot; predetermine which procedural

parameters are allowed to be adjusted to obtain comparable results (e.g.

adjusted dilution range)

• Include documentation of the qualification of each new reagent lot in the

method’s lifecycle package and/or lab tracking documentation for the method

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Aren’t cells critical reagentsin cell based potency assays?


“Best Practices” Recommendations forAnalytical Applications of Cells

BioProcess International 10(1) January 2012 pp 27-40


Perform a Risk Assessment on Use of Cells

How will the data generated from the cell-based material be used?

R&D Methods: Moderate risk• Preliminary research studies for proof-of-principle• Comparison among cell lines for selection

Qualified Methods: Greater risk• Product characterization, comparability (and similarity) studies

Validated Methods: Greatest risk • cGMP product release/stability testing


Then Assess Risks for Source of Cells

Cell Source Risks: Origin, history and documentation

Lower risk:• Good lab quality practices (instruments, materials)• Complete chain of custody handling, storage• Testing performed to confirm identity• Proven uncontaminated (eg mycoplasma negative)• Documentation to prove all of the above

Higher risk:• Limited or no lab quality practices (“trust me…”)• Incomplete or unclear chain of custody• No confirmatory testing (reliance on literature)• Untested for contamination• No documentation to support verbal claims


Cell Bank “Best Practices” Characterization Testing

Mammalian cells❖ Culture contamination

❖Mycoplasma❖Culture sterility (absent antibiotics)

❖Fungus❖ Bacteria (aerobic and anaerobic)

❖ Verification of Function for Intended Use ❖ Bioassay❖ Reagent Production

❖ Culture Purity / Homogeneity❖ Cell Viability❖ Cell Morphology❖ Cell Identity❖ Receptor Expression Analysis (endogenous or exogenous)


Cell Bank “Best Practices” Characterization Testing

Bacterial cells❖ Identity

❖ Species❖ Strain❖Genotype❖ Phenotype

❖ Culture Purity❖ Cell Viability❖ Function in the Intended Assay

❖ Direct Activity❖ Transformation then Activity


Example of Cell Bank Establishment Report

This documentation should go into the method validation package!


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