Daniel Hewitt

Characterization and Stability Study of Polysorbate 20 in Therapeutic Monoclonal Antibody Formulation by Multidimensional Ultrahigh-Performance Liquid Chromatography–Charged Aerosol Detection–Mass Spectrometry

Polysorbate 20 is a nonionic surfactant commonly used in the formulation of therapeutic monoclonal antibodies (mAb) to prevent protein denaturation and aggregation. It is critical to understand the molecular heterogeneity and stability of polysorbate 20 in mAb formulations as polysorbate can gradually degrade in aqueous solution over time by multiple pathways losing surfactant functions and leading to protein aggregation. The molecular heterogeneity of polysorbate and the interference from proteins and the excipient in the formulation matrix make it a challenge to study polysorbate in protein formulations. In this work, the characterization and stability study of polysorbate 20 in the presence of mAb formulation sample matrix is first reported using two-dimensional liquid chromatography (2DLC) coupled with charged aerosol detection (CAD) and mass spectrometry (MS) detection. A mixed-mode column that has both anion-exchange and reversed-phase properties was used in the first dimension to separate protein and polysorbate in the formulation sample, while polysorbate 20 esters were trapped online and then analyzed using an reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) column in the second dimension to further separate the ester species. The MS served as the third dimension to further resolve as well as to identify the polysorbate ester subspecies. Another 2DLC method using a cation-exchange column in the first dimension and the same RP-UHPLC method in the second dimension was developed to analyze the degradation products of polysorbate 20. Stability samples of a protein drug product were studied using these two 2DLC-CAD-MS methods to separate, identify, and quantify the multiple ester species in polysorbate 20 and also to monitor the change of their corresponding degradants. We found different polysorbate esters degrade at different rates, and importantly, the degradation rates for some esters are different in the protein formulation compared to a placebo that has no protein. The multidimensional UHPLC-CAD-MS approach provides insights into the heterogeneous stability behaviors of polysorbate 20 subspecies in real-time stability samples of a mAb formulation.

Mechanism of antibody reduction in cell culture production processes

We recently observed a significant disulfide reduction problem during the scale‐up of a manufacturing process for a therapeutic antibody using a CHO expression system. Under certain conditions, extensive reduction of inter‐chain disulfide bonds of an antibody produced by CHO cell culture may occur during the harvest operations and/or the protein A chromatography step, resulting in the observation of antibody fragments (light chain, heavy chain, and various combination of both) in the protein A pools. Although all conditions leading to disulfide reduction have not been completely identified, an excessive amount of mechanical cell lysis generated at the harvest step appears to be an important requirement for antibody reduction (Trexler‐Schmidt et al., 2010 ). We have been able to determine the mechanism by which the antibody is reduced despite the fact that not all requirements for antibody reduction were identified. Here we present data strongly suggesting that the antibody reduction was caused by a thioredoxin system or other reducing enzymes with thioredoxin‐like activity. The intracellular reducing enzymes and their substrates/cofactors apparently were released into the harvest cell culture fluid (HCCF) when cells were exposed to mechanical cell shear during harvest operations. Surprisingly, the reducing activity in the HCCF can last for a long period of time, causing the reduction of inter‐chain disulfide bonds in an antibody. Our findings provide a basis for designing methods to prevent the antibody reduction during the manufacturing process. Biotechnol. Bioeng. 2010;107:622–632.

Mixed-mode and reversed-phase liquid chromatography―tandem mass spectrometry methodologies to study composition and base hydrolysis of polysorbate 20 and 80

Polysorbate 20 (polyoxyethylenesorbitan monolaurate) and polysorbate 80 (polyoxyethylenesorbitan monooleate) used in protein drug formulations are complex mixtures that have been difficult to characterize. Here, two HPLC methods are used with evaporative light scattering detection (ELSD) and mass spectrometry (MS) to characterize polysorbate from commercial vendors. The first HPLC method used a mixed-mode stationary phase (Waters Oasis MAX, mixed-mode anion exchange and reversed-phase sorbent) with a step gradient to quantify both the total polyoxyethylene sorbitan ester and polyoxyethylene sorbitan (POE sorbitan, a non-surfactant) in polysorbate. The results indicated POE sorbitan was present from 16.0 to 27.6 and 11.1 to 14.5% (w/w) in polysorbate 20 and 80, respectively. The second HPLC method used a reversed-phase stationary phase (Zorbax SB-300 C(8)) with a shallow gradient to separate, identify, and quantify the multiple ester species present in polysorbate. For all lots of polysorbate 20 analyzed, only 18-23% of the material was the expected structure, polyoxyethylenesorbitan monolaurate. Up to 40% and 70% (w/w) di- and triesters were found in polysorbate 20 and polysorbate 80 respectively. Likewise, polyoxyethylenesorbitan monooleate accounted for only 20% of polysorbate 80. A variability of 3-5% was observed for each ester species between multiple lots of polysorbate 20. The reversed-phase method was then used to determine the rate of hydrolysis for each polyoxyethylene sorbitan ester of polysorbate 20 in basic solution at room temperature. Increasing rates of hydrolysis were observed with decreasing aliphatic chain lengths in polysorbate 20.

Quantitation of polysorbate 20 in protein solutions using mixed-mode chromatography and evaporative light scattering detection

An HPLC assay requiring no complex sample preparation for the measurement of polysorbate 20 in protein solutions was developed. An on-off chromatography technique was employed involving a mixed-mode stationary phase (Waters Oasis MAX, mixed-mode anion-exchange and reversed-phase sorbent) to quantify polysorbate 20 in solutions containing >100mg/mL of protein. With 2% formic acid mobile phase, proteins are typically positive charged and are not retained because of electrostatic repulsions from the quaternary amine in the mixed-mode resin. Other formulation components elute in void volume because of their hydrophilicity. Hydrophobic polysorbate 20 is retained, eluted with a step gradient and quantified as a single peak using an evaporative light scattering detector. The performance of the assay is evaluated according to International Conference on Harmonisation (ICH) guidelines and shown to be suitable for polysorbate quantitation. Accuracy (96-108%) and repeatability (2.3% RSD) were demonstrated using protein samples spiked with polysorbate 20. This method was used to accurately measure polysorbate 20 in at least 25 different protein solutions spanning a wide range of formulations. Although the majority of the data reported here target polysorbate 20, this methodology can also be used to assay other common non-ionic surfactants such as polysorbate 80, Brij, Igepal, and Triton X-100.

Rapid identification of low level glycation sites in recombinant antibodies by isotopic labeling with 13C6-reducing sugars.

Recombinant antibodies exhibit low levels of glycation from exposure to reducing sugars during production. As the glycation sites are typically distributed across the entire antibody, the levels at any one site are low and it becomes difficult to detect them in the conventional peptide maps. A model antibody was subjected to forced glycation by incubating with a high concentration of a 1:1 mixture of (12)C(6)/(13)C(6) reducing sugars with the assumption that the same sites in the native antibody will be glycated but to a lower extent. This approach simplified the detection of glycated tryptic peptide elution in the LC/MS analysis by giving a unique signature of two molecular ions with equal intensity and differing by 6.018 Da. An in-house developed script automatically processed large data files to generate a list of such peptide mass pairs. The high mass accuracy of the Orbitrap allowed us to assign the sequences unambiguously by comparison with all possible glycated peptide masses. This sequence list was subsequently used to verify their presence/absence in the digest of the native antibody. This work flow enabled rapid and confident identification of site-specific glycation even when levels are below 0.5%. We found the glycation sites to be distributed across the entire antibody studied.

Identification of a single base-pair mutation of TAA (Stop codon) → GAA (Glu) that causes light chain extension in a CHO cell derived IgG1

We describe here the identification of a stop codon TAA (Stop) → GAA (Glu) = Stop221E mutation on the light chain of a recombinant IgG1 antibody expressed in a Chinese hamster ovary (CHO) cell line. The extended light chain variants, which were caused by translation beyond the mutated stop codon to the next alternative in-frame stop codon, were observed by mass spectra analysis. The abnormal peptide peaks present in tryptic and chymotryptic LC–MS peptide mapping were confirmed by N-terminal sequencing as C-terminal light chain extension peptides. Furthermore, LC-MS/MS of Glu-C peptide mapping confirmed the stop221E mutation, which is consistent with a single base-pair mutation in TAA (stop codon) to GAA (Glu). The light chain variants were approximately 13.6% of wild type light chain as estimated by RP-HPLC analysis. DNA sequencing techniques determined a single base pair stop codon mutation, instead of a stop codon read-through, as the cause of this light chain extension. To our knowledge, the stop codon mutation has not been reported for IgGs expressed in CHO cells. These results demonstrate orthogonal techniques should be implemented to characterize recombinant proteins and select appropriate cell lines for production of therapeutic proteins because modifications could occur at unexpected locations.

Quantitation and characterization of process impurities and extractables in protein-containing solutions using proton NMR as a general tool

The ability to detect and quantitate a variety of components in solution has become increasingly important in carrying out efficient and rigorous validation studies for biopharmaceutical manufacturing processes. Here, we demonstrate the general applicability of NMR spectroscopy for the identification and quantitation of leachables and other impurities in protein‐based drugs, at low levels previously unattainable in protein‐containing solutions. With improved NMR technology (i.e., CryoProbes) and the application of a Carr‐Purcell‐Meiboom‐Gill pulse sequence (CPMG) to attenuate protein signals, we have been able to use NMR to quantify impurities in a protein‐based biopharmaceutical product at ∼1 μg mL−1. The data indicate that NMR spectra can be used to quantitate a range of impurities, from small molecule components to higher molecular weight leachables, without removing protein from solution. Furthermore, quantitation of impurities by NMR is reliable and accurate enough for biopharmaceutical process validation, even for high molecular weight extractables whose structures are not precisely known.

The effects of membrane filters used in biopharmaceutical processes on the concentration and composition of polysorbate 20

Polysorbate 20 (PS‐20) is often included in the formulation for therapeutic proteins to reduce protein aggregation and surface adsorption. During the production process of therapeutic proteins, various membrane filters are used to filter product pools containing PS‐20. The purpose of this study is to quantify the effects of these membrane filtration processes on the concentration and composition of PS‐20. A quantitative understanding of this process provides the knowledge base for better controlling the consistency of formulation excipients in drug products. PS‐20 solutions (without protein) were filtered through either 0.2 µm sterilizing filters or membrane filters with 30 kDa MWCO. The concentration of PS‐20 was measured by a mixed‐mode chromatography method and a nuclear magnetic resonance spectroscopy (NMR) assay. The composition of PS‐20 was characterized by 1H‐NMR and a reverse‐phase chromatography method. Non‐specific adsorption of PS‐20 on both the sterilizing filter and 30 kDa MWCO membrane filter was quantified. Composition of PS‐20 was altered after 30 kDa MWCO membrane filtration, possibly because the different interactions between heterogeneous PS‐20 components and the 30 kDa MWCO membrane were not uniform. As a result, the retentate after the 30 kDa MWCO membrane filtration step contains no POE sorbitan and increased amount of POE sorbitan di‐esters and tri‐esters.

Facile quantitation of free thiols in a recombinant monoclonal antibody by reversed-phase high performance liquid chromatography with hydrophobicity-tailored thiol derivatization

Free thiol content, and its consistency, is one of the product quality attributes of interest during technical development of manufactured recombinant monoclonal antibodies (mAbs). We describe a new, mid/high-throughput reversed-phase-high performance liquid chromatography (RP-HPLC) method coupled with derivatization of free thiols, for the determination of total free thiol content in an E. coli-expressed therapeutic monovalent monoclonal antibody mAb1. Initial selection of the derivatization reagent used an hydrophobicity-tailored approach. Maleimide-based thiol-reactive reagents with varying degrees of hydrophobicity were assessed to identify and select one that provided adequate chromatographic resolution and robust quantitation of free thiol-containing mAb1 forms. The method relies on covalent derivatization of free thiols in denatured mAb1 with N-tert-butylmaleimide (NtBM) label, followed by RP-HPLC separation with UV-based quantitation of native (disulfide containing) and labeled (free thiol containing) forms. The method demonstrated good specificity, precision, linearity, accuracy and robustness. Accuracy of the method, for samples with a wide range of free thiol content, was demonstrated using admixtures as well as by comparison to an orthogonal LC-MS peptide mapping method with isotope tagging of free thiols. The developed method has a facile workflow which fits well into both R&D characterization and quality control (QC) testing environments. The hydrophobicity-tailored approach to the selection of free thiol derivatization reagent is easily applied to the rapid development of free thiol quantitation methods for full-length recombinant antibodies.