Multi-dose biologics contain preservatives that prevent microbial growth. While effective, these antimicrobial preservatives (AMPs) may negatively impact protein stability. Most biologics use one or a combination of the following AMPs: m-cresol, phenol, benzyl alcohol, and chlorobutanol. However, there is no validated method currently available to simultaneously evaluate the four preservatives together. The purpose of this study was to develop a validated GC-MS headspace (HS) method to quantify the AMPs in in-house model teriparatide peptide formulations and marketed products. This method has been used to test the concentration in biologics and studies are in progress to determine the correlation between protein stability and the AMPs with respect to concentration and time.<br/>An Agilent GC7890A/MS5977B interfaced with an Agilent HS7697A analyzer was employed for the method development, validation, and implementation phases of the study. The system was equipped with a split/splitless injector (5:1 or 10:1 split ratio, 220 °C). Separation was achieved on a Supelco Equity-1 fused silica capillary column (30 m x 0.25 mm x 1.0 µm). Teriparatide control formulations were prepared with one or a combination of AMPs. The parenteral teriparatide control formulations (50 µL) were placed in a 20 mL GC HS vial with the external standard (phenol-d6, 5 µL) and p-xylene (30 µL) were extracted by the HS analyzer equilibrated at 125 °C for 10 min.<br/>A GC-MS HS analytical method was developed and validated according to USP &lt;1225&gt; to quantitate the AMPs, with phenol-d6 as the external standard. The system suitability test based on USP &lt;621&gt; was performed where the analyte retention time, peak area, USP tailing factor, and resolution were evaluated based on USP specifications and met acceptance criteria. The method was specific and the average retention time for m-cresol was 9.294 min, % RSD 0.010, phenol was 8.221 min, % RSD 0.009, benzyl alcohol was 8.971 min, % RSD 0.004, and chlorobutanol was 8.521 min, % RSD 0.009 with N=12. The analytical ranges were 1.5–90 ppm (phenol, m-cresol), 30–240 ppm (benzyl alcohol), a0pnd 30–300 ppm (chlorobutanol).<br/>The method was successfully applied to four biologics to quantify AMPs. Product quality studies are in progress to determine if there is a correlation between the protein stability and the AMPs.

Development and validation of a GC-MS headspace method for the simultaneous evaluation of commonly used preservatives in peptide and protein formulations

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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