Module 9 2021

24/03/2021

Bioassay Validation

Include stability indicating samples Rehder, et al Biochemistry 2008, 47, 2518-2530 Monoclonal antibody • Decreased in vitro potency when stored under certain conditions  Not due to aggregation or proteolysis, no change in MW RP-HPLC: increased peak preceding light chain peak, both same MW • Peptide maps: additional peptide, same MW as control • Sequence analysis + Asp-N enzymatic digestion: early eluting peptide has isomerization of Asp-92  Potency decreases with increase in iso-Asp at position 92

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Bioassay Validation

• Example format (2 x 96 well plates/assay)

Assay Plate 1

Assay Plate 2

Run

Analyst

Cell bank

TA1

TA2

TA3

TA4 100

1 2 3 4 5 6 7 8 9

1 2 1 2 1 2 1 2

71

71

50 50

200

141

50

1

200 141 100

200 141

71

Specificity

100

100

50 50

71

71

50

141

200

2

Specificity

71

200 141

200 141

100

100

1 or 2

1 or 2

Stability Stability Stability Stability

• Different target potency levels tested and compared to Reference (=100%).

The Organisation for Professionals in Regulatory Affairs

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