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.

Prediction of long-term polysorbate degradation according to short-term degradation kinetics.

Polysorbates (PSs) are a class of surfactants commonly used in the formulation of protein therapeutic agents to provide protection against denaturation and aggregation. When the PS in these drug formulations degrades, loss of stabilization of the protein therapeutic and formulation may occur, resulting in particulate formation or other undesirable changes in product critical quality attributes. Here, we present a simplified platform to predict long-term PS20 and PS80 degradation for monoclonal antibody drugs containing the PS-degrading enzyme lysosomal acid lipase. The platform was based on a temperature-dependent equation derived from existing PS20 degradation stability data. Accurate prediction of both PS20 and PS80 hydrolysis for as long as 2 years was achieved through short-term kinetics studies performed within 2 weeks. This platform substantially shortens the time required to determine the long-term stability of PS degradation and therefore can be used to guide the purification process and optimization of antibody formulations.

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.

Development of a novel affinity chromatography resin for platform purification of bispecific antibodies with modified protein a binding avidity.

There is strong interest in the production of bispecific monoclonal antibodies that can simultaneously bind two distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Regeneron's bispecific technology, based upon a standard IgG, consists of a heterodimer of two different heavy chains, and a common light chain. Coexpression of two heavy chains leads to the formation of two parental IgG impurities, the removal of which is facilitated by a dipeptide substitution in the Fc portion of one of the heavy chains that ablates Fc Protein A binding. Therefore, the affinity capture (Protein A) step of the purification process must perform both bulk capture and high resolution of these mAb impurities, a task current commercially available resins are not designed for. Resolution can be further impaired by the ability of Protein A to bind some antibodies in the variable region of the heavy chain (VH ). This article details development of a novel Protein A resin. This resin combines an alkali stable ligand with a base matrix exhibiting excellent mass transfer properties to allow high capacity single step capture and resolution of bispecific antibodies (bsAbs) with high yields. The developed resin, named MabSelect SuRe™ pcc, is implemented in GMP production processes for several bsAbs. © 2018 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:650-658, 2018.