A Mechanistic Understanding of Monoclonal Antibody Interfacial Protection by Hydrolytically Degraded Polysorbate 20 and 80 under IV Bag Conditions.

PURPOSE: Polysorbates (PS) contain polyoxyethylene (POE) sorbitan/isosorbide fatty acid esters that can partially hydrolyze over time in liquid drug products to generate degradants and a remaining intact PS fraction with a modified ester distribution. The degradants are composed of free fatty acids (FFAs) --primarily lauric acid for PS20 and oleic acid for PS80-- and POE head groups. We previously demonstrated that under IV bag agitation conditions, mAb1 (a surface-active IgG4) aggregation increased with increasing amounts of degradants for PS20 but not for PS80. The purpose of this work is to understand the mechanism behind this observation. METHODS: The surface tension of the remaining intact PS fraction without degradants was modeled and compared with that of enzymatically degraded PS solutions. Next, mAb1 aggregation in saline was measured in the presence of laurate and oleate salts during static storage. Lastly, colloidal and conformational stability of mAb1 in the presence of these salts was investigated through differential scanning fluorimetry and dynamic light scattering under IV bag solution conditions. RESULTS: The surface tension was primarily influenced by FFAs rather than the modified ester distribution of the remaining intact PS. MAb1 bulk aggregation increased in the presence of laurate but not oleate salts. Both salt types increased the melting temperature of mAb1 indicating FFA-mAb1 interactions. However, only laurate salt increased mAb1 self-association potentially explaining the higher aggregation propensity in its presence. CONCLUSION: Our results help explain the observed differences between hydrolytically degraded PS20 and PS80 in affecting mAb1 aggregation under IV bag agitation conditions.

Evaluating a Modified High Purity Polysorbate 20 Designed to Reduce the Risk of Free Fatty Acid Particle Formation.

PURPOSE: To evaluate a modified high purity polysorbate 20 (RO HP PS20)-with lower levels of stearate, palmitate and myristate esters than the non-modified HP PS20-as a surfactant in biopharmaceutical drug products (DP). RO HP PS20 was designed to provide functional equivalence as a surfactant while delaying the onset of free fatty acid (FFA) particle formation upon hydrolytic degradation relative to HP PS20. METHODS: Analytical characterization of RO HP PS20 raw material included fatty acid ester (FAE) distribution, higher order ester (HOE) fraction, FFA levels and trace metals. Functional assessments included 1) vial and intravenous bag agitation; 2) oxidation via a placebo and methionine surrogate study; and 3) hydrolytic PS20 degradation studies to evaluate FFA particle formation with and without metal nucleation. RESULTS: Interfacial protection and oxidation propensity were comparable between the two polysorbates. Upon hydrolytic degradation, FFA particle onset was delayed in RO HP PS20. The delay was more pronounced when HOEs of PS20 were preferentially degraded. Furthermore, the hydrolytic degradants of RO HP PS20 formed fewer particles in the presence of spiked aluminum. CONCLUSION: This work highlights the criticality of having tighter control on long chain FAE levels of PS20 to reduce the occurrence of FFA particle formation upon hydrolytic degradation and lower the variability in its onset. By simultaneously meeting compendial PS20 specifications while narrowing the allowable range for each FAE and shifting its composition towards the shorter carbon chain species, RO HP PS20 provides a promising alternative to HP PS20 for biopharmaceutical DPs.

Characterization of Polysorbate Ester Fractions and Implications in Protein Drug Product Stability.

Polysorbates (PS) are commonly used surfactants in biopharmaceutical protein formulations. However, they are susceptible to a variety of degradation pathways, including chemical hydrolysis, oxidation, and enzymatic hydrolysis. Polysorbates are also heterogeneous mixtures, and it has been observed that the patterns of degradation can be strikingly different between the different pathways. Polysorbates (PS20 and PS80) were fractionated, and the fractions were characterized for their physicochemical properties, such as surface tension, micelle size, critical micelle concentration (CMC), and agitation protection for a monoclonal antibody (mAb). This report seeks to use this information to inform how these properties might change in polysorbates as they degrade in biopharmaceutical formulations. The physicochemical properties examined shed light on some of the differences between PS types and the different chemical components of polysorbates. Differences in physicochemical properties for fractionated polysorbates could help inform biopharmaceutical formulations that use PS surfactants. Importantly, they show that subspecies of PS20 are far more distinct from each other than those of PS80. Fractions of PS20 showed highly different critical micelle concentrations and effects on equilibrium surface tension. These differences, and possibly other untested parameters, led to vastly different protective effects for a model mAb under agitation stress. Additionally, the propensity of various PS fractions to form micelles can impact both polysorbate quantitation measurements, some of which rely on micellization, and the effective solubility of hydrophobic compounds (e.g., fatty acids) in the surfactant solution.

Improving Prediction of Free Fatty Acid Particle Formation in Biopharmaceutical Drug Products: Incorporating Ester Distribution during Polysorbate 20 Degradation.

Polysorbate 20 (PS20) is a commonly used surfactant in biopharmaceutical formulations. It is a heterogeneous surfactant containing a distribution of fatty acid esters, which are subject to hydrolytic degradation, generating free fatty acids (FFAs). The FFAs can form visible or subvisible particles in drug product on stability. A previous FFA solubility model, developed by our group, predicts solubility limits for the three most prevalent FFA degradation products of PS20: lauric, myristic, and palmitic acid. The model takes into account two formulation parameters, pH and PS20 concentration, and their effect on FFA solubility. This work identifies a third parameter that has an impact on FFA solubility: PS20 ester distribution. When PS20 is hydrolytically degraded, the ester distribution of the remaining surfactant changes on stability. Ester distribution is known to influence the critical micelle concentration (CMC) of PS20 such that the monoesters have a much higher CMC compared to the higher-order esters (HOE). We hypothesize that as PS20 degrades, the CMC changes, affecting the proportion of PS20 that is present in micelles and capable of sequestering and solubilizing FFAs in these micelles. Here, PS20 was separated into monoester, HOE, and polyol fractions. The monoester and HOE fractions were mixed together to generate the mock degradation profiles of hydrolytically degraded PS20. FFA solubility was measured as a function of the concentration of these mock-degraded (MD) PS20s. The results indicate that ester distribution does have an impact on FFA solubility, especially at higher MD PS20 concentrations. HOEs solubilize up to 30 µg/mL more lauric acid than an equivalent amount of monoesters at a MD PS20 level of 0.06% w/v. With the addition of % HOE peak area fraction as a third parameter representing the ester distribution of PS20, the refined FFA solubility model more accurately predicts FFA solubility in protein formulations at 5 °C. The refined model suggests that drug products containing trace levels of host cell proteins (HCPs) that preferentially degrade HOEs of PS20 are at a higher risk of particle formation.

Exogenous delivery of chaperonin subunit fragment ApiCCT1 modulates mutant Huntingtin cellular phenotypes.

Aggregation of misfolded proteins is characteristic of a number of neurodegenerative diseases, including Huntington disease (HD). The CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring) chaperonin complex can inhibit aggregation and cellular toxicity induced by expanded repeat Huntingtin (mHtt) fragments. The substrate-binding apical domain of CCT/TRiC subunit CCT1, ApiCCT1, is sufficient to inhibit aggregation of expanded repeat mHtt fragments in vitro, providing therapeutic promise for HD. However, a key hurdle in considering ApiCCT1 as a potential treatment is in delivery. Because ApiCCT1 has a region of similarity to the HIV Tat protein cell-transduction domain, we tested whether recombinant ApiCCT1 (ApiCCT1(r)) protein could enter cells following exogenous delivery and modulate an established panel of mHtt-mediated cell-based phenotypes. Cell fractionation studies demonstrate that exogenous ApiCCT1(r) can penetrate cell membranes and can localize to the nucleus, consistent with a strategy that can target both cytosolic and nuclear pathogenic events in HD. ApiCCT1(r) application does indeed modulate HD cellular phenotypes by decreasing formation of visible inclusions, fibrillar oligomers, and insoluble mHtt derived from expression of a truncated mHtt exon 1 fragment. ApiCCT1(r) also delays the onset of inclusion body formation as visualized via live imaging. ApiCCT1(r) reduces mHtt-mediated toxicity in immortalized striatal cells derived from full-length knock-in HD mice, suggesting that therapeutic benefit may extend beyond effects on aggregation. These studies provide the basis for a potentially robust and unique therapeutic strategy to target mHtt-mediated protein pathogenesis.

Polysorbate 20 Degradation in Biopharmaceutical Formulations: Quantification of Free Fatty Acids, Characterization of Particulates, and Insights into the Degradation Mechanism.

Polysorbate 20 (PS20), a commonly used surfactant in biopharmaceuticals, showed degradation upon long-term (∼18-36 months) storage of two monoclonal antibody (mAb, mAb-A, and mAb-B) drug products at 2-8 °C. The PS20 degradation resulted in the accumulation of free fatty acids (FFA), which ultimately precipitated to form particles upon long-term storage. This study documents the development, qualification, and application of a method for FFA quantification in soluble and insoluble fraction of protein formulation. The method was applied to the quantification of capric acid, lauric acid, myristic acid, palmitic/oleic acid, and stearic acid in placebo as well as active protein formulations on stability. Quantification of FFA in both the soluble and insoluble fraction of mAb-A and mAb-B provided a better mechanistic understanding of PS20 degradation and the dynamics of subsequent fatty acid particle formation. Additionally, the use of this method for monitoring and quantitation of the FFA on real time storage stability appears to aid in identifying batches with higher probability for particulate formation upon extended storage at 5 °C.

Characterization of Recombinantly-Expressed Hydrolytic Enzymes from Chinese Hamster Ovary Cells: Identification of Host Cell Proteins that Degrade Polysorbate.

Enzymatic hydrolysis of polysorbate in drug products is a major challenge for the biopharmaceutical industry. Polysorbate hydrolysis caused by host cell proteins (HCPs) co-purified during bioprocessing can reduce the protective effects of the surfactant for the active pharmaceutical ingredient and cause the accumulation of low-solubility degradation products over the long-term storage. The identities of such HCPs are elusive due to their extremely low concentrations after the efficient purification processes of most biopharmaceuticals. In this work, 20 enzymes-selected for their known or putative hydrolytic activity and potential to degrade polysorbate-were recombinantly expressed, purified, and characterized via orthogonal methods. First, these recombinant HCPs were assessed for hydrolytic activity against a fluorogenic esterase substrate in a recently-developed, high-throughput assay. Second, these HCPs were screened for hydrolytic activity against polysorbate in a representative mAb formulation. Third, HCPs that displayed hydrolytic activities in the first two assays were subjected to more detailed characterization of their enzyme kinetics against polysorbates. Finally, these HCPs were evaluated for substrate specificity towards different sub-species of polysorbates. This work provides critical new insights for targeted LC-MS/MS approaches for identification of relevant polysorbate-degrading enzymes and supports improvements to remove such HCPs, including knockouts or targeted removal during purification.

Extended Characterization and Impact of Visible Fatty Acid Particles - A Case Study With a mAb Product.

In recent years, there has been increased scrutiny on the presence and formation of product-related particles in biopharmaceutical formulations. These types of particles, originating from the degradation of the active pharmaceutical ingredient or the excipients, can be challenging to identify and characterize due to their fragility. Additionally, the mechanisms of their formation as well as the impact of their presence on drug product safety can be complicated to elucidate. In this work, a case study is presented in which multiple batches of one formulated monoclonal antibody (mAb-A) were analyzed at different batch ages to better understand the formation of visible particles resulting from degradation of the surfactant polysorbate 20. The particle identity was determined by Raman spectroscopy as free fatty acid (FFA) and the particle composition over time was monitored by mass spectrometry. Further experimental work includes the counts and morphologies of subvisible particles by flow imaging microscopy. Finally, we evaluated the consequences of saline and human plasma exposure to the visible particles to better understand their fate upon dilution and/or administration which is routinely performed in the clinical setting. The experiments performed in this work can be used to support risk assessments of visible product-related particles.

Identification and Characterization of Polysorbate-Degrading Enzymes in a Monoclonal Antibody Formulation.

Degradation of polysorbate (PS) by hydrolytically active host cell proteins (HCPs) in drug products may impair the protein-stabilizing properties of PS and lead to the formation of particles due to the accumulation of poorly soluble free fatty acids upon long-term storage. The identification of the causative enzymes is challenging due to their low-abundance even when using state-of-the-art instrumentation and workflows. To overcome these challenges, we developed a rigorous enrichment strategy for HCPs, utilizing both Protein A and anti-HCP affinity chromatography, which facilitated the in-depth characterization of the HCP population in a monoclonal antibody formulation prone to PS hydrolysis. Based on the HCPs identified by liquid chromatography coupled to tandem mass spectrometry, a number of enzymes annotated as hydrolases were recombinantly expressed and characterized in terms of polysorbate degradation. Among the selected candidates, Lipoprotein Lipase, Lysosomal Acid Lipase (LIPA) and Palmitoyl-Protein Thioesterase 1 (PPT1) exhibited notable activity towards PS. To our knowledge, this is the first report to identify LIPA and PPT1 as residual HCPs that can contribute to PS degradation in a biological product.

Micellar Morphology of Polysorbate 20 and 80 and Their Ester Fractions in Solution via Small-Angle Neutron Scattering.

Surfactants are commonly used in therapeutic protein formulations in biopharmaceuticals to impart protein stability; however, their solution morphology and the role of the individual components in these structurally heterogeneous commercial grade surfactants at physiologically and pharmaceutically relevant temperatures have not been investigated systematically. The micellar morphologies of Polysorbate 20 and Polysorbate 80 and their primary components monoester fractions, as well as the diester fractions, are evaluated at 4, 22°C, 40°C, and 50°C using small-angle neutron scattering to determine the aggregation number, radius of gyration, core radius, critical micelle concentration, shell thickness, and shell hydration. The sizes and aggregation numbers of the diester fractions of PS20 above 80°C and PS80 above 50°C exhibit significant changes in shape. The analysis of the small-angle neutron scattering data of PS20 confirms that the critical micellar concentration of the monoester fraction is significantly higher at 4°C compared to the diester fraction and their original material, all-laurate PS20. Overall, these experiments identify the dominant components responsible for the temperature-dependent behavior of these surfactants in pharmaceutical protein formulations.

A Rapid High-Sensitivity Reversed-Phase Ultra High Performance Liquid Chromatography Mass Spectrometry Method for Assessing Polysorbate 20 Degradation in Protein Therapeutics.

Polysorbate 20 (PS20), a widely used surfactant in protein therapeutics, has been reported to undergo hydrolytic degradation during product storage, causing the release of free fatty acids. The accumulation of free fatty acids in protein therapeutics was found to result in the formation of particles due to their limited aqueous solubility at 2°C-8°C. Quantitation of free fatty acids originating from PS20 degradation is thus important during bioprocess optimization and stability testing in formulation development to ensure optimum PS20 stability as well as product and process consistency in final drug products. This work reports the development of a simple and robust, high-throughput, reversed-phase ultra high performance liquid chromatography mass spectrometry method for high-sensitivity quantitation of lauric acid and myristic acid by using isotope-labeled fatty acid internal standards. The high sensitivity (<100 ng/mL for lauric acid) and suitable precision (intermediate precision relative standard deviation of 11%) of this method enable accurate detection of lauric acid produced from the degradation of less than 1% of PS20 in a 0.2-mg/mL formulation. Using accelerated thermal stability testing, this method identifies processes that exhibit fast PS20 degradation within only days and consequently allows faster iterative optimization of the process.

Novel markers to track oxidative polysorbate degradation in pharmaceutical formulations.

Polysorbates can undergo oxidative degradation in pharmaceutical formulations resulting in both soluble and insoluble degradation products. The insoluble degradants may precipitate to form subvisible and visible particulates, which are undesirable in liquid parenteral products. To date, no oxidation byproduct has been identified as an established marker to track Polysorbate 20 oxidation. Herein, we identified the aldehyde derivative of free fatty acid esters as a byproduct of polysorbate oxidation that can be derivatized using 2,4-dinitrophenylhydrazine and tracked analytically to monitor oxidative polysorbate degradation in pharmaceutical formulations.

An information theoretic framework reveals a tunable allosteric network in group II chaperonins.

ATP-dependent allosteric regulation of the ring-shaped group II chaperonins remains ill defined, in part because their complex oligomeric topology has limited the success of structural techniques in suggesting allosteric determinants. Further, their high sequence conservation has hindered the prediction of allosteric networks using mathematical covariation approaches. Here, we develop an information theoretic strategy that is robust to residue conservation and apply it to group II chaperonins. We identify a contiguous network of covarying residues that connects all nucleotide-binding pockets within each chaperonin ring. An interfacial residue between the networks of neighboring subunits controls positive cooperativity by communicating nucleotide occupancy within each ring. Strikingly, chaperonin allostery is tunable through single mutations at this position. Naturally occurring variants at this position that double the extent of positive cooperativity are less prevalent in nature. We propose that being less cooperative than attainable allows chaperonins to support robust folding over a wider range of metabolic conditions.

Prenatal corticosteroid therapy for elevated liver enzyme/low platelet count syndrome: a case report.

BACKGROUND: Early preterm onset of elevated liver enzymes/low platelet count (ELLP) syndrome poses a significant management problem. CASE: Antepartum methylprednisolone, 40 mg/d intravenously was employed to stabilize ELLP syndrome, to achieve fetal lung maturity and to postpone delivery at 25 weeks and 5 days' gestation. Normalization of the liver transaminases and platelet count occurred with the use of corticosteroids, but sudden fetal death occurred at 28 weeks and 2 days' gestation. CONCLUSION: Prenatal corticosteroids may improve the biochemical and hematologic parameters of ELLP syndrome but may result in intrauterine death.