Kurt Brorson, Ph.D.

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Kurt Brorson, Ph.D.

• Division of Monoclonal Antibodies
• OPS/CDER

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Biopharmaceuticals are complex- more potential
heterogeneity than small molecule drugs
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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

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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)

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Antibody Heterogeneity- Major role of
glycosylation, C-terminal lys
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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 Doesnt seem to impact potency or safety
profile
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Monoclonal antibodiesUnacceptable, stress
induced heterogeneity
Detectable by various methods Manufacturers set
stability specifications Can compromise potency
if OOS
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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

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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

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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

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Strategies assessment of impact

• If heterogeneity cant 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