Example 2
Evaluation of 10 mM acetate, 75 mM L-arginine, 2.4% (w/v) sorbitol, 0.01% (w/v) polysorbate 20 excipients formulations and a 10 mM acetate, 5% (w/v) sorbitol, 0.01% (w/v) polysorbate 20 excipients formulation, each with high concentration (120 mg/mL) denosumab, at a temperature of 37° C. for up to 1 month revealed the effects of pH and amino acid aggregation inhibitor on the rate and extent of HMWS formation. The formulations tested are described in Table 2 below. All buffer and excipient values quoted are for the buffer and excipient concentrations that the antibody is diafiltered against.
To prepare test samples M-Q, a 3 mL aliquot of denosumab at 70 mg/mL in acetate, pH 5.2 was dialyzed against 500 mL of DF buffer described below, with a total of 3 buffer changes to achieve a 1 million fold dilution of the previous formulation to ensure complete buffer exchange. The material was then over-concentrated using centrifuge-concentrator, followed by a dilution to 120 mg/mL and the addition of polysorbate 20 to a final concentration of 0.01%.
TABLE 2
AbbreviationDF Formulation Composition
MAcetate/Arginine/10 mM Acetate, 75 mM L-Arginine HCl,
Sorbitol/PS20/pH 4.52.4% (w/v) Sorbitol, pH 4.5
NAcetate/Arginine/10 mM Acetate, 75 mM L-Arginine HCl,
Sorbitol/PS20/pH 4.82.4% (w/v) Sorbitol, pH 4.8
OAcetate/Arginine/10 mM Acetate, 75 mM L-Arginine HCl,
Sorbitol/PS20/pH 5.22.4% (w/v) Sorbitol, pH 5.2
PAcetate/Sorbitol/10 mM Acetate, 5% (w/v) Sorbitol,
PS20/pH 5.2pH 4.5
QAcetate/Sorbitol/10 mM Acetate, 5% (w/v) Sorbitol,
PS20/pH 5.3pH 4.8
FIG. 2 shows the percent HMWS monitored by SE-UHPLC as a function of formulation and time at 37° C. FIG. 3 shows size exclusion chromatograms as a function of formulation following storage at 37° C. for 1 month.
As the solution pH decreased, there was an increase in formation of large aggregates. At pH below 4.8, and especially 4.5, large aggregates were the dominant HWMS, with a dramatic increase for the test formulation at pH 4.5. As shown in FIG. 3, formulations P and Q had the lowest amount of higher order HWMS (retention time about 6 minutes), followed by comparative formulations 0, N, and M having decreasing pH values.
However, as the pH was increased, there was generally a resulting increase in the dimer species. As shown in FIG. 3, formulation N had the lowest amount of dimer species (retention time about 6.8 minutes), followed by formulations M, O, P and Q.
The presence of arginine in formulation O at a concentration of 75 mM resulted in approximately 0.3% and 25% reductions in the amounts of the dimer species and its kinetic rate of formation, respectively, after 1 month at 37° C. when compared to formulation P having the same pH, but without arginine.
