A mass spectrometry-based approach to host cell protein identification and its application in a comparability exercise.

Host cell proteins (HCPs) are process-related impurities present in biopharmaceuticals and are generally considered to be critical quality attributes. Changes in a biopharmaceutical production process may result in qualitative shifts in the HCP population. These shifts are not necessarily detectable when overall HCP levels are measured with traditional approaches such as enzyme-linked immunosorbent assays (ELISAs). Thus, the development of techniques that complement the ELISA's functionality is desirable. Here, a mass spectrometry (MS)-based approach for the analysis of HCP populations in biopharmaceuticals is presented. It consists of (i) the generation of exclusion lists that represent the masses of the active pharmaceutical ingredient (API), (ii) the compilation of inclusion lists based on an HCP catalog derived from the analysis of protein A-purified samples, and (iii) the analysis of purified biopharmaceuticals using the generated exclusion and inclusion lists. With this approach, it was possible to increase sensitivity for HCP detection compared with a standard liquid chromatography tandem MS (LC-MS/MS) run. The workflow was successfully implemented in a comparability exercise assessing HCP populations in drug substance samples before and after a process change. Furthermore, the results suggest that size can be an important factor in the copurification of HCPs and API.

Top-down MS for rapid methionine oxidation site assignment in filgrastim.

Protein therapeutics have emerged as a major new class of pharmaceuticals. One important shelf-life-limiting factor of biopharmaceuticals is methionine oxidation, and therefore, it is important that analytical methods are able to thoroughly characterize all possible oxidized variants. Here, we present a fast and sensitive method to perform online methionine oxidation site assignment using granulocyte colony-stimulating factor (filgrastim) as a model. The method is based on top-down MS using the all-ion fragmentation mode of the Exactive benchtop mass spectrometer. Conditions that provide information on the intact mass of the protein as well as on fragment ions that allow unambiguous site assignment of methionine oxidation in filgrastim variants as low as 0.12% of total peak area in a chromatographic time scale were identified. Using this method, we performed methionine oxidation site assignment in H2O2-stressed filgrastim and in filgrastim which was stored at intended conditions, respectively. We show that the relative abundance of oxidation species observed in filgrastim stored under intended conditions differs strikingly from the oxidized species observed after H2O2 stress. Additionally, we report an oxidized filgrastim variant that has not been previously described in the literature.

The structure-function relationship of disulfide bonds in etanercept.

Etanercept is a TNFα receptor Fc fusion protein used for the treatment of rheumatic disease and psoriasis. Physicochemical and functional investigation of process fractions during development of the etanercept biosimilar GP2015 (Erelzi®) revealed a correlation between reduced potency and incorrect disulfide bridging between specific cysteines in the receptor domain. This novel structure-function relationship was found to be the molecular basis for reduced potency in recent Enbrel® batches, which exhibit higher levels of incorrect disulfide bridging. Interestingly, incorrect disulfide bridging was found to be reversible under serum-like redox conditions, restoring potency to normal levels. This redox dependent reversibility suggests that these variants are likely not relevant for clinical efficacy once the drug enters the bloodstream. Nonetheless, incorrect disulfide bridging in etanercept represents a new quality attribute that is critical for biopharmaceutical functionality and should thus be carefully monitored and controlled to guarantee patient safety.

Identification of Low-Level Product-Related Variants in Filgrastim Products Presently Available in Highly Regulated Markets.

BACKGROUND: Filgrastim is a recombinant, non-glycosylated form of human granulocyte colony-stimulating factor, used to stimulate leukocyte proliferation in patients suffering from neutropenia. Since the expiration of patents associated with Amgen's filgrastim biopharmaceutical, Neupogen(®), in 2006, a number of filgrastim products have been marketed; however, a detailed characterization and comparison of variants associated with these products have not been publically reported. OBJECTIVE: The objective of this study was to identify and quantify product-related variants in filgrastim reference products and biosimilars thereof that are presently available in highly regulated markets. METHODS: In this study, we used intact and top-down mass spectrometry to identify and quantify product-related variants in filgrastim products. Mass spectrometry has become the method of choice for physicochemical characterization of biopharmaceuticals, allowing accurate and sensitive characterization of product-related variants. RESULTS: In addition to modifications ubiquitously present in biopharmaceuticals, such as methionine oxidation and asparagine/glutamine deamidation, we identified six different low-level, product-related variants present in some, but not all, of the tested products. Two variants, an acetylated filgrastim variant and a filgrastim variant containing an additional C-terminal tryptophan extension, are newly identified variants. CONCLUSION: This study demonstrates that filgrastim products already in widespread clinical use in highly regulated markets differ in low-level, product-related variants present at levels mostly below 1 % relative abundance. This study provides a comprehensive catalog of minor differences between filgrastim products and suggests that the filgrastim product-related variants described here are not clinically relevant when present at low abundance.