Storage duration without VPs observed for mAb X DP as a function of [mAb]. The 24 formulations (listed in Table 1) represent unique combinations of [mAb], initial [PS20], and pH. At the end of the DP stability study (24 weeks at 5 °C), only one formulation (F24 as shown in Table 1) remained free from VPs

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... formation was evaluated using two different approaches: Ph. Eur./USP method for VPs and HIAC liquid particle counter for monitoring cumulative SVP counts. PS20 degradation was evaluated using three orthogonal analytical methods to quantify: (1) intact PS20; (2) FFAs, the main degradants from PS20 hydrolysis; and (3) PS20 ester distribution. (Fig. 2). These observations are consistent with previously observed directionality of [mAb] (Labrenz 2014) and initial [PS20] ( Roy et al. 2021) on FFA generation rates and consequently FFA particle formation. The VP results were similar between pH 5 and pH 6 samples ( Fig. 2 and Table 1), with a marginal benefit at pH 6, consistent with our ...

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... the main degradants from PS20 hydrolysis; and (3) PS20 ester distribution. (Fig. 2). These observations are consistent with previously observed directionality of [mAb] (Labrenz 2014) and initial [PS20] ( Roy et al. 2021) on FFA generation rates and consequently FFA particle formation. The VP results were similar between pH 5 and pH 6 samples ( Fig. 2 and Table 1), with a marginal benefit at pH 6, consistent with our projections based on the minimal predicted positive impact of higher pH in this range on PA solubility using our previously published FFA model ( Doshi et al. 2015Doshi et al. , ...

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... (Fig. 4), similar to the directionality of their effect on the storage duration with no VPs (Fig. 2). By contrast, formulation pH did not show a notable impact on the SVP count profiles ( Fig. 3; Supplementary Figs. S1, S2, and S3) or on the duration with low SVP counts (Fig. ...

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... would increase the FFA solubility limit ( Doshi et al. 2015). Over time, the calculated FFA solubility limits are projected to decline because of the corresponding PS20 degradation that lowers the FFAsolubilizing capacity of the formulation. The more rapid decline in FFA solubility limit at the highest [mAb] relative to the lowest [mAb] (Fig. 12) is consistent with the more rapid decline in [PS20] at higher [mAb] (Figs. 6 and ...

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... predictions from the FFA solubility model were compared against the empirical observations for each formulation (Fig. 12). In the top three panels, the blue hollow circles (and associated blue lines) denote the calculated solubility limits for LA, MA, and PA, while the red filled circles (and associated red lines) denote the measured concentrations of these three FFAs. For a given formulation, the intersection of the blue and red lines represents the time ...

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... (and associated red lines) denote the measured concentrations of these three FFAs. For a given formulation, the intersection of the blue and red lines represents the time point where the measured FFA concentration reached its calculated solubility limit and the FFA is expected to thereafter fall out of solution and precipitate as FFA particles (Fig. 12, top 3 panels). The expected VP onset from the FFA solubility model for LA, MA, and PA corroborated the general VP and SVP trends observed in this longitudinal DOE study: (1) lower [mAb] extended VP-free duration; (2) higher initial [PS20] extended VP-free duration; (3) pH had minimal ...

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... solubility for LA, MA, and PA decreases in this order, in an inverse relationship to their chain lengths ( Doshi et al. 2020). Therefore, PA has the lowest calculated solubility limit of the three FFAs. PA is also projected to accumulate beyond its solubility limit at earlier time points than LA and MA and thereby precipitate earlier. In Fig. 12, the intersections of the blue and red lines for PA (third panel from top) occur at earlier time points relative to LA (top panel) and MA (second panel from top), thereby indicating that the long-chain FFAs like PA are likely the root cause for the onset of particles. In the bottom panel of Fig. 12, the first time points where VPs were ...

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... LA and MA and thereby precipitate earlier. In Fig. 12, the intersections of the blue and red lines for PA (third panel from top) occur at earlier time points relative to LA (top panel) and MA (second panel from top), thereby indicating that the long-chain FFAs like PA are likely the root cause for the onset of particles. In the bottom panel of Fig. 12, the first time points where VPs were observed are denoted by green asterisks and the rise The pH of the formulation impacts the LA solubility limits-pH 6 has a higher predicted LA solubility relative to pH 5 (Fig. 12). By contrast, pH has minimal impact on the predicted solubility limits of MA and PA. The higher LA solubility limit at ...

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... thereby indicating that the long-chain FFAs like PA are likely the root cause for the onset of particles. In the bottom panel of Fig. 12, the first time points where VPs were observed are denoted by green asterisks and the rise The pH of the formulation impacts the LA solubility limits-pH 6 has a higher predicted LA solubility relative to pH 5 (Fig. 12). By contrast, pH has minimal impact on the predicted solubility limits of MA and PA. The higher LA solubility limit at pH 6 relative to pH 5 is attributed to the increased proportion of ionized LA-which is significantly more soluble than its non-ionized form-in the pH 6 formulations ( Doshi et al. 2015). Owing to their high pKas (> 7), ...

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... mAb X DP. Based on the longitudinal model, the effect estimates for the interaction between [mAb] and initial [PS20] exceeded the effect estimates for pH on (Table 2). The [mAb] × initial [PS20] interaction is also manifested in the observed differences in slopes at the different initial [PS20] for the following four outputs: (1) VP-free duration (Fig. 2); (2) low SVP duration (Fig. 4); (3) (Fig. 7); and (4) d [LA]/dt with [mAb] (Fig. 9). By contrast, the slopes for pH 5 and pH 6 formulations across these four outputs were relatively similar within the same initial [PS20] (Figs. 2, 4, 7, and 9), consistent with the relatively small effect estimates for pH and its interaction with the ...

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... observed differences in slopes at the different initial [PS20] for the following four outputs: (1) VP-free duration (Fig. 2); (2) low SVP duration (Fig. 4); (3) (Fig. 7); and (4) d [LA]/dt with [mAb] (Fig. 9). By contrast, the slopes for pH 5 and pH 6 formulations across these four outputs were relatively similar within the same initial [PS20] (Figs. 2, 4, 7, and 9), consistent with the relatively small effect estimates for pH and its interaction with the other formulation parameters on particle formation and PS20 degradation (Table ...

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... identity of the recombinant protein or the type of polysorbate (PS20 or PS80). The concentration of residual HCPs in a therapeutic protein product is expected to increase with protein concentration if no bioprocess changes (apart from concentrating the product) are made to the upstream and downstream steps for protein production and purification. Fig. 12 Comparison of calculated FFA solubility limits (blue hollow circles) with corresponding FFA measurements (red filled circles), SVP counts (black filled diamonds), and VP observations (green asterisks) in mAb X DP stored at 5 °C. FFA solubility limits at the different time points for each formulation were calculated using the updated ...