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Certificate of Analysis for Biologics: Quality Attribute Characterization and Specifications for Biologic Products

Assist. Prof. Dr. Jiraphun JittikoonDepartment of Biochemistry

Faculty of Pharmacy, Mahidol University

Assist. Prof. Dr. Jiraphun JittikoonDepartment of Biochemistry

Faculty of Pharmacy, Mahidol University

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Biologics

� Human-derived Biologics (Plasma-derived Biologics)

� Biotechnology-derived Biologics

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Biologics: Molecular Complexity

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Proteins have expected:

• Size, charge, hydrophobicity

• Correct folding (S-S bonds)

• Subunits

• Glycosylation

• Bioactivity

& Unexpected:

• Aggregation (side effects)

• Incorrect folding

• Amino acid modifications

– ox, deam, cys

• Truncation, proteolysis

Biologics: Molecular Complexity

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Small Molecule Medicines vs. Biological Medicines

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Biological Medicines vs. Biological Medicines

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The following points should be taken into consideration when establishing scientifically justifiable specifications.

� Specifications are linked to a manufacturing process.

� Specifications should account for the stability of drug substance and drug product.

� Specifications are linked to preclinical and clinical studies.

� Specifications are linked to analytical procedures

Specification for Biologic Productswww.mahidol.ac.th

Biologics: Biotechnology-derived Biologics

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Chemistry, Manufacturing and Control Issues in Production of

Therapeutic Biologic Protein Products

Chemistry, Manufacturing and Control Issues in Production of

Therapeutic Biologic Protein Products

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Drug Substance and Drug Product Characterization

Critical Quality Attribute

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Quality control of biologics

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Analytical Methods and Reference Standards www.mahidol.ac.th

Quality control of biologics

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Quality control of biologics

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Quality control of biologics

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Drug Substance Characterization

� Safety� Ensured by the specified limits for bioburden and endotoxin, misc.

process-related contaminants

� Purity & Characterization � process-related impurities (e.g., endogenous viruses, host-cell

proteins, DNA, leachables, anti-foam, antibiotics, toxins, solvents, heavy metals, etc.)

� Product-related impurities (e.g., aggregates, breakdown products, product variants due to: oxidation, deamidation, denaturation, loss of C-term Lys in MAbs etc.)

� Product substances (product variants that are active)

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Drug Substance Characterization� Identity

� Unique for protein of interest, especially relevant for closely related proteins manufactured in the same facility

� Potency� Required to assess biological activity of the product � Assay should be relevant for protein mechanism of action

� Strength� Protein content

� Stability� Drug Substance stability should be demonstrated with appropriate

stability-indicating assays

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Drug Product Characterization

� Safety� Final Drug Product for injection should be sterile within specified limits

for endotoxin � Immunogenicity should be screened and monitored

� Purity & Characterization � Product and process-related impurities & product-related substances

should be within specified limits

� Identity� Unique for protein of interest, especially relevant for closely related

proteins manufactured in the same facility

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� Potency� Assay should be relevant for protein mechanism of action

� Strength� Protein content

� Stability� Drug Product should maintain stability for the duration of the clinical

trial

� Container closure compatibility� Primary function - barrier to microbial ingress � Extractables/Leachables studies

Drug Product Characterization

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Methodology for Characterization

• Safety– LAL test, rabbit pyrogen test, bacterial culture methods

• Purity & Characterization including but not limited to:– Reversed-phase HPLC, Peptide mapping, MS – SDS-PAGE, Western analysis, capillary electrophoresis– SEC, AUC, FFF, light scattering– Ion Exchange Chromatography – Carbohydrate analysis (capillary electrophoresis, HPAEC = high-pH anion-

exchange chromatography, IEF for sialic acid)

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Methodology for Characterization

• Identity– N-terminal sequencing– Peptide mapping – Immunoassays (ELISA, Western blotting)

• Potency– Animal-based assays, cell-based assays, reporter gene, biochemical (e.g.,

enzyme activity)

• Protein content– RIA, ELISA, UV absorbance, Bradford

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PRINCIPLES FOR CONSIDERATION IN SETTING SPECIFICATIONS

� Characterization� Physicochemical properties� Biological activity� Immunochemical properties� Purity, impurities and contaminants� Quantity

� Analytical Considerations� Reference standards and reference materials� Validation of analytical procedures

� Process Controls� Process-related considerations� In-process acceptance criteria and action limits� Raw materials and excipient specifications

� Pharmacopoeial Specifications� Release Limits vs. Shelf-life Limits� Statistical Concepts

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Specification for Biologic Products

� Drug Substance Specification� Appearance and description� Identity� Purity and impurities� Potency� Quantity

� Drug Product Specification� Appearance and description� Identity� Purity and impurities� Potency� Quantity� General tests� Additional testing for unique dosage forms

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