title: Hydrolysis of Polysorbate 20 and 80 by a Range of Carboxylester Hydrolases.
abstract: Degradation of the surfactant polysorbate (PS) by enzyme impurities has been previously suggested as a mechanism for the formation of visible and subvisible particles that affect product quality. Although chemical degradation pathways of PS, such as oxidation and acid/base hydrolysis, have been previously characterized, enzymatic degradation of PS remains poorly understood. In this report, enzyme-mediated hydrolysis of the major components of PS was monitored using an evaporative light scattering detection-high-performance liquid chromatography method. PS20 and PS80 tested contained 99% of laurate and 98% oleate esters, respectively, were heterogeneous with respect to head group, and contained a distribution of ester types. Carboxylester hydrolases tested included those from Pseudomonas cepacia, Thermomyces lanuginosus, Candida antarctica, rabbit liver, and pig pancreas. PS hydrolysis was monitored by observing the change in the peak area of major PS components over time and quantified using a parameter called t50, which was defined as the time required for each peak to reach 50% of its initial value. Time course experiments suggested that PS hydrolysis was dependent on the order of esters (mono-, di-, or triester), the identity of the hydrophilic head group (sorbitan or isosorbide), and the identity of the fatty acid ester tail (C12 vs C18:1). In addition, the pattern of PS hydrolysis was unique to the type of enzyme used. Importantly, we observed that no PS component was completely resistant to the carboxylester hydrolases tested here. Our results illustrate a potential fingerprint approach that could be useful in verifying enzyme-mediated PS degradation in drug substance and provide an improved understanding of the complexity of PS degradation in the presence of enzymes.
  Degradation of the non-ionic surfactant polysorbate (PS) has been reported to lead to the formation of visible and subvisible particles that affect product quality. Chemical degradation pathways of PS, such as oxidation and acid/base hydrolysis, have been previously studied, but enzymatic degradation of PS remains poorly understood. In this study, enzyme-mediated hydrolysis of the major components in a heterogeneous mixture of PS20 or PS80 was monitored using an evaporative light scattering detection-high-performance liquid chromatography method. Carboxylester hydrolases from a broad range of organisms were tested, including enzymes from Pseudomonas cepacia, Thermomyces lanuginosus, Candida antarctica, rabbit liver, and pig pancreas. Time course experiments suggested that PS hydrolysis was dependent on the order of esters (mono-, di-, or triester), the identity of the hydrophilic head group (sorbitan or isosorbide), the identity of the fatty acid ester tail (C12 vs C18:1), and the identity of the enzyme. Importantly, no PS component was completely resistant to all the carboxylester hydrolases tested here. Our results illustrate a potential fingerprint approach that could be useful in verifying or identifying enzyme-mediated PS degradation in drug substance and provide an improved understanding of the complexity of PS degradation in the presence of enzymes.
authors.affiliation: Department of Late Stage Pharmaceutical Development, Genentech, Inc., South San Francisco, CA 94080; Department of Molecular Biosciences, University of Kansas, Lawrence, KS;
authors.forename: Andrew C
authors.initials: AC
authors.lastname: McShan
authors.affiliation: Department of Early Stage Pharmaceutical Development, Genentech, Inc., South San Francisco, CA 94080;
authors.forename: Pervina
authors.initials: P
authors.lastname: Kei
authors.affiliation: Department of Late Stage Pharmaceutical Development, Genentech, Inc., South San Francisco, CA 94080;
authors.forename: Junyan A
authors.initials: JA
authors.lastname: Ji
authors.affiliation: Department of Late Stage Pharmaceutical Development, Genentech, Inc., South San Francisco, CA 94080; Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, KS.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Kim
authors.affiliation: Department of Late Stage Pharmaceutical Development, Genentech, Inc., South San Francisco, CA 94080; wang.john@gene.com.
authors.forename: Y John
authors.initials: YJ
authors.lastname: Wang
mesh_terms: D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000032:analysis / Q000378:metabolism*; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006868:Hydrolysis; D011136:Polysorbates / Q000032:analysis / Q000378:metabolism*; D011817:Rabbits; D013552:Swine
chemical_list: D011136:Polysorbates; D002265:Carboxylic Ester Hydrolases