Characterization of ionic strength for X-MuLV inactivation by low pH treatment for monoclonal antibody purification.

In the production of monoclonal antibodies (mAbs) intended for use in humans, it is a global regulatory requirement that the manufacturing process includes unit operations that are proven to inactivate or remove adventitious agents to ensure viral safety. Viral inactivation by low pH hold (LPH) is typically used to ensure this viral safety in the purification process of mAbs and other biotherapeutics derived from mammalian cell lines. To ascertain the effectiveness of the LPH step, viral clearance studies have evaluated LPH under worst-case conditions of pH above the manufacturing set point and hold duration at or below the manufacturing minimum. Highly acidic conditions (i.e., pH < 3.60) provide robust and effective enveloped virus inactivation but may lead to reduced product quality of the therapeutic protein. However, when viral inactivation is operated above pH 3.60 to ensure product stability, effective (>4 log10 reduction factor) viral inactivation may not be observed under these worst-case pH conditions in viral clearance studies. A multivariate design of experiments was conducted to further characterize the operating space for low pH viral inactivation of a model retrovirus, xenotropic murine leukemia virus (X-MuLV). The statistically designed experiment evaluated the effect of mAb isotype, pH, temperature, acid titrant, sodium chloride (NaCl) concentration, virus spike timing, and post-spike filtration on X-MuLV inactivation. Data from the characterization study were used to generate predictive models to identify conditions that reliably achieve effective viral inactivation at pH ≥ 3.60. Results of the study demonstrated that NaCl concentration has the greatest effect on virus inactivation in the range studied, and pH has a large effect when the load material has no additional NaCl. Overall, robust and effective inactivation of X-MuLV at pH 3.65-3.80 can be achieved by manipulating either the pH or the NaCl concentration of the load material. This study contributes to the understanding of ionic strength as an influential parameter in low pH viral inactivation studies.

Speed and need: twin development challenges in rapid response for a SARS-CoV-2 antibody cocktail.

Neutralizing antibodies are one available tool for the treatment of infectious diseases. Speed in developing monoclonal antibody treatments is an understood requirement for emerging infectious diseases, and need for COVID-19 treatments during the worldwide pandemic has provided additional urgency. Process development (at Regeneron) and technology transfer (within Regeneron and to Genentech) of casirivimab and imdevimab (REGEN-COV™ or Ronapreve™) manufacturing processes have addressed speed and need with selected purification and cell culture examples provided, respectively, for these two development challenges. This was achieved through three key pillars: (1) Regeneron's proprietary Velocisuite® technologies, (2) deep monoclonal antibody process and manufacturing knowledge at both companies, and (3) Regeneron's and Genentech's commitment to deliver therapeutics to patients in need. Combined with business processes and risk management, these pillars rapidly allowed casirivimab and imdevimab to move to clinical manufacturing and to production at Genentech in a first-time process transfer under compressed timelines between the companies.

Identification of the specific causes of polysorbate 20 degradation in monoclonal antibody formulations containing multiple lipases.

PURPOSE: Polysorbates (PS) are excipients used in the biotech industry to stabilize monoclonal antibody (mAb) protein products. However, PS in drug product formulations can be degraded during storage and lead to particle formation because of the limited solubility of the free fatty acids released through the enzymatic hydrolysis of PS-a process driven by residual host cell proteins, especially lipases, that are co-purified with the drugs. When multiple lipases are present, it is very difficult to know the cause for PS degradation. In this study, we aim to determine the cause of PS degradation from two lipases, lysosomal acid lipase (LAL) and lipoprotein lipase (LPL). METHODS: PS degradation pattern of the drug product was compared with those induced by recombinant lipases. Correlations between the concentration of LPL or LAL and PS20 loss were compared. Specific inhibitors, LAL inhibitor lalistat2 and LPL inhibitor GSK264220A, were used to differentiate their degradation of PS in the drug products. RESULTS: The complete inhibition of PS20 degradation by lalistat2 suggested that LAL, rather than LPL, was responsible for the PS20 degradation. In addition, LAL was more strongly correlated than LPL with the percentage of PS20 degradation. No PS20 degradation was observed for several mAbs containing similar levels of LPL (0.5-1.5 ppm) in the absence of LAL, suggesting that LPL concentrations below 1.5 ppm does not degrade PS20 in drug products. CONCLUSIONS: LAL was determined to be the cause of the PS20 degradation. This study provides a practical strategy to determine the root cause of PS degradation.

Host cell protein profiling of commercial therapeutic protein drugs as a benchmark for monoclonal antibody-based therapeutic protein development.

Therapeutic proteins including monoclonal antibodies (mAbs) are usually produced in engineered host cell lines that also produce thousands of endogenous proteins at varying levels. A critical aspect of the development of biotherapeutics manufacturing processes is the removal of these host cell proteins (HCP) to appropriate levels in order to minimize risk to patient safety and drug efficacy. During the development process and associated analytical characterization, mass spectrometry (MS) has become an increasingly popular tool for HCP analysis due to its ability to provide both relative abundance and identity of individual HCP and because the method does not rely on polyclonal antibodies, which are used in enzyme-linked immunosorbent assays. In this study, HCP from 29 commercially marketed mAb and mAb-based therapeutics were profiled using liquid chromatography (LC)-MS/MS with the identification and relative quantification of 79 individual HCP in total. Excluding an outlier drug, the relative levels of individual HCP determined in the approved therapeutics were generally low, with an average of 20 ppm (µmol HCP/mol drug) measured by LC-MS/MS, and only a few (<7 in average) HCP were identified in each drug analyzed. From this analysis, we also gained knowledge about which HCP are frequently identified in mAb-based products and their typical levels relative to the drugs for the identified individual HCP. In addition, we examined HCP composition from antibodies produced in house and found our current development process brings HCP to levels that are consistent with marketed drugs. Finally, we described a specific case to demonstrate how the HCP information from commercially marketed drugs could inform future HCP analyses.

Putative Phospholipase B-Like 2 is Not Responsible for Polysorbate Degradation in Monoclonal Antibody Drug Products.

Polysorbates (PS) are surfactants commonly added in a therapeutic protein drug product as excipients to protect proteins from denaturation and aggregation during storage, transportation, and delivery. Significant degradation of PS in drug products could lead to shortened drug shelf lives and PS-degrading activity in drug products must be minimized. Identification of lipases that degrade PS could lead to better process control in drug manufacturing. In 2016, phospholipase B-like 2 (PLBD2) was proposed as a residual host cell protein responsible for degrading PS20 in a drug formulation. We have carried out a series of studies to verify the role of PLBD2 in degrading polysorbates in drug products purified from recombinant Chinese Hamster Ovary (CHO) cells. Genetic knock-out and immuno-depletion results showed that when PLBD2 was removed or depleted, the degradation of PS20 or PS80 was neither diminished nor reduced. In addition, a quantitative analysis of PLBD2 and PS20 degradation in multiple formulated mAb products did not establish a correlation between the amount of PLBD2 and the level of PS20 degradation. Collectively these results suggest that PLBD2 is not the primary cause of polysorbate degradation in formulated drug products purified using standard Protein A and ion exchange chromatography.

Retrospective Evaluation of Cycled Resin in Viral Clearance Studies-A Multiple Company Collaboration.

The BioPhorum Development Group Viral Clearance Workstream performed a collaborative retrospective analysis to evaluate packed bed chromatographic resin performance after repeated cycling for two commonly used chromatography steps in biopharmaceutical manufacturing: protein A and anion exchange. Key variables evaluated in the assessment included virus type, resin type, number of reuse cycles, and virus challenge. In this retrospective analysis of viral clearance data on naïve versus cycled resin, powered by the availability of a decade's worth of accumulated industry data, clearance capability was not negatively impacted by resin cycling. This finding is consistent with publications showing that surrogates for viral clearance capabilities could be employed in lieu of testing the viral clearance of cycled resins for protein A and anion exchange chromatography. The rigorous analysis of the retrospective data supports the view that viral clearance studies for cycled resins are not necessary provided that appropriate cleaning methods are applied during repeated use of the chromatography columns.LAY ABSTRACT: The manufacturing processes for biopharmaceutical products often include reusable chromatographic resins that remove process- and product-related impurities as well as potential contaminating viruses. Typically, chromatography resin is "cycled" through repeated steps of resin conditioning, product purification, and resin cleaning. The cycling approach has been evaluated in both small- and full-scale studies that show the performance parameters are maintained. The ability to remove virus is demonstrated separately in a focused small-scale virus-spiking study that is resource-intensive and costly. This paper is a retrospective review of industry data comparing virus removal by naïve and repeatedly cycled resins that summarizes the viral clearance impact of re-using protein A and anion exchange chromatography resins. The key variables evaluated in the assessment included virus type, resin type, number of cycles, and virus challenge. In this retrospective analysis, it was found that the viral clearance capability is not negatively impacted by resin cycling. This finding is consistent with other publications and supports the view that viral clearance studies for cycled resins are not necessary if appropriate cleaning methods are applied during the repeated use of the chromatography columns.Abbreviations: AAV-2, Adeno-associated virus; A-MuLV, Amphotropic murine leukemia virus; AEX, Anion-exchange chromatography; B/E, Bind and elute; BVDV, Bovine viral diarrhea virus; C.P.G., Controlled pore glass; DEAE, Diethylaminoethanol; EMCV, Encephalomyocarditis virus; FT, Flow through; HAV, Hepatitis A virus; HSV-1, Herpes simplex virus type 1; LOD, Limit of detection; LOQ, Limit of quantification; LRF, Log10 reduction factor; mAb, Monoclonal antibody; MVM, Minute virus of mice; NaOH, Sodium hydroxide; PA, Protein A; PPV, Porcine parvovirus; QA, Quaternary amine; QP, Quaternized polyethyleneimine; qPCR, Quantitative polymerase chain reaction; Reo3, Reovirus type 3; SuHV-1, Suid herpesvirus; SV40, Simian virus 40; X-MuLV, Xenotropic murine leukemia virus.

Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity.

There is strong interest in the design of bispecific monoclonal antibodies (bsAbs) that can simultaneously bind 2 distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Multiple bispecific formats have been proposed and are currently under development. Regeneron's bispecific technology is based upon a standard fully human IgG antibody in order to minimize immunogenicity and improve the pharmacokinetic profile. A single common light chain and 2 distinct heavy chains combine to form the bispecific molecule. One of the heavy chains contains a chimeric Fc sequence form (called Fc*) that ablates binding to Protein A via the constant region. As a result of co-expression of the 2 heavy chains and the common light chain, 3 products are created, 2 of which are homodimeric for the heavy chains and one that is the desired heterodimeric bispecific product. The Fc* sequence allows selective purification of the FcFc* bispecific product on commercially available affinity columns, due to intermediate binding affinity for Protein A compared to the high avidity FcFc heavy chain homodimer, or the weakly binding Fc*Fc* homodimer. This platform requires the use of Protein A chromatography in both a capture and polishing modality. Several challenges, including variable region Protein A binding, resin selection, selective elution optimization, and impacts upon subsequent non-affinity downstream unit operations, were addressed to create a robust and selective manufacturing process.

Retrospective Evaluation of Low-pH Viral Inactivation and Viral Filtration Data from a Multiple Company Collaboration.

Considerable resources are spent within the biopharmaceutical industry to perform viral clearance studies, which are conducted for widely used unit operations that are known to have robust and effective retrovirus clearance capability. The collaborative analysis from the members of the BioPhorum Development Group Viral Clearance Working Team considers two common virus reduction steps in biopharmaceutical processes: low-pH viral inactivation and viral filtration. Analysis included eight parameters for viral inactivation and nine for viral filtration. The extensive data set presented in this paper provides the industry with a reference point for establishing robust processes in addition to other protocols available in the literature (e.g., ASTM Std. E2888-12 for low-pH inactivation). In addition, it identifies points of weakness in the existing data set and instructs the design and interpretation of future studies. Included is an abundance of data that would have been difficult to generate individually but collectively will help support modular viral clearance claims.