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Kurt Brorson, Ph.D.

Kurt Brorson, Ph.D. Division of Monoclonal Antibodies OPS/CDER. Biopharmaceuticals are complex- more potential heterogeneity than small molecule drugs. Heterogeneity in recombinant products and Monoclonal antibodies. Cell culture related Fermentation is an artificial process

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Kurt Brorson, Ph.D.

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  1. Kurt Brorson, Ph.D. Division of Monoclonal Antibodies OPS/CDER

  2. Biopharmaceuticals are complex- more potential heterogeneity than small molecule drugs

  3. Heterogeneity in recombinant products and Monoclonal antibodies • Cell culture related • Fermentation is an artificial process • vs. plasma or other natural sources • Bioreactor conditions can impact: • Glycosylation • Some charge variants • Amino acid substitutions (leu  norleucine, etc) • The cell line can impact: • Adduct placement • Folding/misfolding • Cysteine pairing

  4. Heterogeneity in recombinant products and Monoclonal antibodies • Stability related • Pure, high concentration protein is an artificial system • vs. proteins in cells or bodily fluids • Plasma and recombinant products face same challenges • Clipping • Aggregation • Deamidation (Asn  Asp; Gln  Glu; loss of e-NH2 on Lys • Oxidation (Met  Met sulfoxide)

  5. Antibody Heterogeneity- Major role of glycosylation, C-terminal lys K K

  6. Expected Heterogeneity-experience with monoclonal antibodies C terminal lysine variability occurs in most monoclonal antibody products Manufacturers set acceptable ranges for each species Can be measured by various techniques, wCEX-HPLC, IEF, others Doesn’t seem to impact potency or safety profile

  7. Monoclonal antibodiesUnacceptable, stress induced heterogeneity Detectable by various methods Manufacturers set stability specifications Can compromise potency if OOS

  8. Heterogeneity-experience with other recombinant products • Case 1: protein terminus heterogeneity • Traced to metaloprotease • Minimal impact on potency • Case 2: product clipping • Minimal impact on potency • Case 3: N-terminal glutamine cyclization • Cyclized form had increased activity

  9. Strategies to maintain product quality • Testing- lot release & stability • Employ a range of assays • IEF, wCEX-HPLC- charge variants • Tryptic peptide mapping, N & C-terminal sqxing- amino acid sequence variants, oxidation, adduct formation • SDS-PAGE, SEC-HPLC- clipping, aggregate formation • Mass spectroscopy- molecular weight changes • Specialized assays- carbohydrate analysis, IsoQuant, others • Set acceptable ranges for quality attributes • Based on clinical & manufacturing experience

  10. Strategies to maintain product quality • Formulation, purification and storage- minimize change over time • Formulation • Lyophilization vs. liquid • pH control • Stabilizers (sugars, polyhydric alcohols) • Surfactants • N2 overlay in vial • Product attributes: • Residual enzymes • Some metals (Cu++, etc.) • Storage • Correct temperature • Minimize O2, bubbles in vial • Protect from light

  11. Strategies: assessment of impact • If heterogeneity can’t be avoided… • Control it • Does heterogeneity impact API potency? • Is it near effector parts of protein? • Assess via potency assay • Does heterogeneity impact bioavailability? • Major glycoform changes • Assess in PK studies • Does heterogeneity impact immunogenicity? • Placement, type, extent of substitutions • Sometimes assess in immunogenicity studies • Case dependent

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