(T0930-08-48) Development of a Novel Stabilizer for Biotherapeutic Antibody Formulations with Residual Lipase Activity

Purpose: Polysorbates (PS-20 and PS-80) are used in the majority of US FDA-approved mAb formulations as surfactants to protect the drug substance from agitation induced stress and surface adsorption (1). However, some mAbs contain trace amounts of host cell proteins (HCPs), including lipases and esterases, that may be difficult to remove during purification. Trace quantities of these enzymes can result in polysorbate hydrolysis, potentially leading to formation of free fatty acid particles when concentrations of free fatty acids are above their solubility thresholds (2,3). Hydrolysis-resistant alternatives for polysorbates are needed for such cases. We evaluated polyoxyethylene sorbitan (POES) as a potential stabilizer for mAb formulations with residual lipase activity and showed that, unlike polysorbates, POES is resistant to lipase-mediated hydrolysis and protects mAbs from agitation stress. Free fatty particle generation was not observed during thermal stability testing of lipase-containing mAb formulations with POES. In combination with low levels of PS-80, POES also protected mAbs against adsorption to hydrophobic surfaces. Further development of POES as a stabilizer for mAb formulations with lipase and/or esterase activity is ongoing.

Methods: POES was prepared by hydrolysis of PS-20 and purified by reverse phase chromatography. Agitation studies with mAb A formulations (150 mg/mL mAb A, pH 5.9) comprising of different concentrations (0.01-0.2% w/v) of polysorbates or POES were performed by orbital shaking at 250 rpm for 24 or 48 hours in 6R vials. Thermal stability of mAb A (150 mg/mL mAb A, pH 5.9) in the presence of 0.1% of PS-80 or POES was assessed at 5, 25, and 40°C for up to 6 months. Effect of POES on mAb surface binding was analyzed with QCM-D (quartz crystal microbalance with dissipation monitoring) at 0.05 mg/mL mAb A (pH 6.0) and with a filter binding assay. Filter binding assay was performed by filtering 5 mL of mAb A formulation (2 mg/mL mAb A, pH 5.9) thru the 0.22 µm filter and collecting 1 mL fractions. mAb concentrations in the fractions were measured.

Results: Agitation studies showed that in the presence of 0.05% of either POES or PS-80 agitation stress didn’t have an impact on quality attributes of mAb A. Combinations of POES with 0.01% or less of PS-80 were significantly more effective resulting in no impact on quality attributes of mAb A from agitation stress at 0.01% POES and 0.005-0.01% PS-80 (Fig. 1). Complete hydrolysis of PS-80 at concentrations up to 0.01% will result in free fatty acid concentrations below their solubility limits and thus will not result in particle formation (3). Thermal stability data of mAb A in the presence of 0.1-0.2% of polysorbates or POES showed no detrimental effect (Fig. 2). Decreased surfactant concentration and increased free fatty particle generation were observed during thermal stability studies of lipase-containing mAb A formulations with polysorbates alone but not with POES (4). Filter binding (Fig. 3) and QCM-D data showed that 0.1% PS-80 significantly decreased mAb A adsorption on hydrophobic surfaces while 0.1% POES was much less effective. Since POES alone was not effective in preventing mAb surface adsorption, a combination of POES with low levels of PS-80 was evaluated. QCM-D data showed that a combination of 0.01% PS-80 with 0.1% POES was as effective as 0.1% PS-80 alone in preventing mAb A adsorption to PVDF surface while neither 0.01% PS-80 or 0.1% POES alone were effective.

Conclusion: Agitation studies showed that in the presence of 0.05% of either POES or PS-80 agitation stress didn’t have an impact on quality attributes of mAb A. Combinations of POES with 0.01% or less of PS-80 were significantly more effective resulting in no impact on quality attributes of mAb A from agitation stress at 0.01% POES and 0.005-0.01% PS-80 (Fig. 1). Complete hydrolysis of PS-80 at concentrations up to 0.01% will result in free fatty acid concentrations below their solubility limits and thus will not result in particle formation (3). Thermal stability data of mAb A in the presence of 0.1-0.2% of polysorbates or POES showed no detrimental effect (Fig. 2). Decreased surfactant concentration and increased free fatty particle generation were observed during thermal stability studies of lipase-containing mAb A formulations with polysorbates alone but not with POES (4). Filter binding (Fig. 3) and QCM-D data showed that 0.1% PS-80 significantly decreased mAb A adsorption on hydrophobic surfaces while 0.1% POES was much less effective. Since POES alone was not effective in preventing mAb surface adsorption, a combination of POES with low levels of PS-80 was evaluated. QCM-D data showed that a combination of 0.01% PS-80 with 0.1% POES was as effective as 0.1% PS-80 alone in preventing mAb A adsorption to PVDF surface while neither 0.01% PS-80 or 0.1% POES alone were effective.

References: 1) Strickley, R. G., & Lambert, W. J. (2021). A review of Formulations of Commercially Available Antibodies. Journal of pharmaceutical sciences, 110(7), 2590–2608.e56.
2) Dwivedi, M., Blech, M., Presser, I., & Garidel, P. (2018). Polysorbate degradation in biotherapeutic formulations: Identification and discussion of current root causes. International journal of pharmaceutics, 552(1-2), 422–436.
3) Patel, M., Wasinger, S., Spritzer, D., Tang, X. (2019). Methods of Reducing Particle Formation and Compositions Formed Thereby. United States Patent Application Publication No. 2019/0083618 A1
4) USP. Particulate Matter in Injections

Figure 1. Protection of mAb A from agitation stress (48h, 250 rpm) by POES (A), PS-80 (C) and their combinations (B, D). Both POES alone and in combinations with trace PS-80 are at least as effective as PS-80 in protecting mAb A from agitation stress.

Figure 2. Thermal stability data at 25 and 40°C for 150 mg/mL mAb A in the presence of PS-80 (0.2%), POES (0.1%) and PS-20 (0.2%). No detrimental effect of POES on mAb stability was observed.

Figure 3. Filter binding assay was performed by filtering 5 mL of mAb A formulations through PVDF syringe filter and collecting 1 mL fractions. PS-80 but not POES prevented adsorption of mAb A to PVDF filter.