Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly

Abstract

Significant progress in the manufacturing of biopharmaceuticals has been made by increasing the overall titers in the USP (upstream processing) titers without raising the cost of the USP. In addition, the development of platform processes led to a higher process robustness. Despite or even due to those achievements, novel challenges are in sight. The higher upstream titers created more complex impurity profiles, both in mass and composition, demanding higher separation capacities and selectivity in downstream processing (DSP). This creates a major shift of costs from USP to DSP. In order to solve this issue, USP and DSP integration approaches can be developed and used for overall process optimization. This study focuses on the characterization and classification of host cell proteins (HCPs) in each unit operation of the DSP (i.e., aqueous two-phase extraction, integrated countercurrent chromatography). The results create a data-driven feedback to the USP, which will serve for media and process optimizations in order to reduce, or even eliminate nascent critical HCPs. This will improve separation efficiency and may lead to a quantitative process understanding. Different HCP species were classified by stringent criteria with regard to DSP separation parameters into “The Good, the Bad, and the Ugly” in terms of pI and MW using 2D-PAGE analysis depending on their positions on the gels. Those spots were identified using LC-MS/MS analysis. HCPs, which are especially difficult to remove and persistent throughout the DSP (i.e., “Bad” or “Ugly”), have to be evaluated by their ability to be separated. In this approach, HCPs, considered “Ugly,” represent proteins with a MW larger than 15 kDa and a pI between 7.30 and 9.30. “Bad” HCPs can likewise be classified using MW (>15 kDa) and pI (4.75–7.30 and 9.30–10.00). HCPs with a MW smaller than 15 kDa and a pI lower than 4.75 and higher than 10.00 are classified as “Good” since their physicochemical properties differ significantly from the product. In order to evaluate this classification scheme, it is of utmost importance to use orthogonal analytical methods such as IEX, HIC, and SEC.

Application of the Bradford Assay for Cell Lysis Quantification: Residual Protein Content in Cell Culture Supernatants

“…However, from the downstream perspective, cell viability is only relevant when the amount of RPC and DNA increases, which only occurs during cell lysis. This could also be of significant relevance for the yield, longevity, and overall performance of the subsequent protein purification strategies . In general, reducing the soluble impurity level can simplify early‐stage downstream processing .…”

“…Viability is a commonly derived key process indicator used to illustrate the vitality status of cell culture processes, but it is dependent on the magnitude of the lysis rate . However, cell lysis and the amount of host cell proteins (HCPs) in the culture broth are often not determined during cultivation although these variables significantly affect further process steps . Consequently, the amount of living, dead, and lysed cells must be considered to achieve a comprehensive picture of the process.…”

Special Issue: Monoclonal Antibodies

“…An article by Schmidt et al [7] describes aqueous two-phase extraction (ATPE) as a method to capture monoclonal antibodies using a combined harvest and capture step during the downstream process. A subsequent article by Kornecki et al focuses on the characterization and classification of host cell proteins (HCPs) and how to categorize and avoid them in the manufacturing process [8]. Finally, propose a process analytical approach allowing for controlled automation of the downstream process by inline concentration measurements based on UV/VIS spectral analysis.…”

“…Finally, Rocha and colleagues generated antibodies directed against novel epitopes of the Dengue nonstructural protein 1 (NS1) which is a multi-functional glycoprotein essential for viral replication and modulation of host innate immune responses and represents a surrogate marker for infection [6]. These antibodies are able to differentiate Dengue and Zika virus infections and may contribute to the development of novel diagnostic tools.In a series of three articles, Strube and colleagues [7][8][9] describe approaches useful for the manufacturing and analytical characterization of monoclonal antibodies. An article by Schmidt et al [7] describes aqueous two-phase extraction (ATPE) as a method to capture monoclonal antibodies using a combined harvest and capture step during the downstream process.…”

“…In a series of three articles, Strube and colleagues [7][8][9] describe approaches useful for the manufacturing and analytical characterization of monoclonal antibodies. An article by Schmidt et al [7] describes aqueous two-phase extraction (ATPE) as a method to capture monoclonal antibodies using a combined harvest and capture step during the downstream process.…”