Example 5
To determine the impact of the amino acid variation of romosozumab PARG (SEQ ID NO: 8) variant as compared to the wild type romosozumab on solubility upon subcutaneous (SC) injection, a dialysis solubility assay was performed on both wild type and PARG (SEQ ID NO: 8) C-terminal variant romosozumab in parallel. This screen entails dialyzing a sample of the romosozumab PARG (SEQ ID NO: 8) C-terminal variant and a sample of the wild-type romosozumab into a solution that simulates the pH and ionic strength of the SC space and monitoring the solubility and physical stability of the antibody in these conditions over a short time period. Samples were formulated at ˜63 mg/mL in formulation buffer (pH 5.2). Then each sample was injected into a dialysis cassette and dialyzed into a PBS buffer to mimic the SC space. Visual observations were made 24 hours after initial dialysis. Wild-type romosozumab typically shows precipitation after 24 hours.
The results show that both molecules precipitate in this analysis but the PARG (SEQ ID NO: 8) C-terminal variant precipitates less and at a slower rate. This suggests that the variant is more resistant to precipitation than wild type, although the variant does not abolish precipitation completely.
