Residual Host Cell Protein Promotes Polysorbate 20 Degradation in a Sulfatase Drug Product Leading to Free Fatty Acid Particles
Nitin Dixit, Nazila Salamat-Miller, Paul A. Salinas, Katherine D. Taylor, and Sujit K. Basu
Supplementary Figure 1: LC-MS analysis in negative-ion mode - Using single ion monitoring, chromatograms were generated that are specific for each fatty acid (lauric m/z 199.2, myristic m/z 227.2, palmitic m/z 255.2, and stearic m/z 283.2) present in the free fatty acid mixture, vehicle, and sulfatase drug product (DP). For the vehicle and sulfatase DP, the m/z for each fatty acid is extracted together. The free fatty acids eluted prior to the intact PS20 species, demonstrating that the free fatty acids observed eluting with the intact PS20 peaks are the result of post-column fragmentation (i.e., degradation during the ionization and detection process). In this figure, intact PS20 species are missing in DP with a concomitant increase in free fatty acids. Vehicle contains intact PS20 while free fatty acids are not observed.
Supplementary Figure 2: Representative chromatograms from the LC-CAD measurements for PS20 in the drug product sample at time 0 (T0) and upon storage for 6 months (6m) at 30 oC.
Supplementary Table 1: Summarized stability results for the quality attributes for three different sulfatase drug product lots upon 5±3 oC storage for 36 months (compared to baseline samples).
Assay / Results (all results met specifications)Activity / Maintained within the specified range of 50 to 140 U/mg
SE-HPLC / Main peak area was measured to be ≥99%
SDS-PAGE / Conformed to reference standard with no new bands
Peptide map / All of the identified marker peaks with oxidation prone residues had the following relative peak areas of the corresponding peak in reference standard: 94%-104% (baseline);
99%-106% (after 36 months storage at 2-8 oC)
Particulates* / Particulate count changed from 5 to 100 for particles/container ≥10 µm and from 1 to 5 for particles/container ≥25 µm
*Results presented for particulates are from one drug product lot
Supplementary Table 2: Peak retention times observed in Figure 1 and the identified fatty acid species observed for the peak.
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