Polysorbate, the Good, the Bad and the Ugly

Introduction

Polysorbate (PS) refers to a family of amphipathic, nonionic surfactants that is derived from ethoxylated sorbitan or isosorbide (a derivative of sorbitol) esterified with fatty acids. Polysorbates, specifically polysorbate 20 (PS20) and polysorbate 80 (PS80), are the most widely used surfactants in biopharmaceutical formulations to prevent proteins from denaturation, aggregation, surface adsorption and flocculant formulation during thaw.

Polysorbates, as a protein stabilizer, are chemically diverse mixtures and may degrade through oxidation and hydrolysis pathways, with the hydrolysis pathway being either chemically induced or enzymatically catalyzed. Since the polysorbate degradation may inadvertently affect the quality, efficacy, safety, and stability of the protein formulation, there is increasing scrutiny from health authorities on polysorbate control strategies to assure that polysorbate content remains constant during shelf life of drug products.

Significant progress has been made to improve our current understanding of polysorbate degradation pathways and root cause to enable implementation of an appropriate polysorbate control strategy, as summarized in several excellent reviews.^1-4 However, polysorbate degradation has emerged as one of the major challenges in the development and commercialization of therapeutic protein products in our industry. This article is intended to provide a high-level overview of different aspects of polysorbates but will focus on the challenges that our industry is currently facing.

The Good

 

PS20 and PS80 are used as protein stabilizers in most commercial therapeutic protein formulations. This is due to a combination of: 1) biocompatibility; 2) low toxicity; and 3) an effective protein stabilizing effect. Even at low concentrations, PS20 and PS80 provide sufficient protein stabilization as a result of their high hydrophile-lipophile balance (HLB) value and low critical micelle concentration (CMC).¹

Although it is not fully clear how the polysorbates stabilize the protein, interfacial competition and surfactant-protein complexation have been proposed as the two main mechanisms.⁴

It was generally believed that the PS20 and PS80 stabilize proteins primarily through interfacial competition.⁵ PS20 and PS80 have much higher surface activities than typical therapeutic proteins, such as monoclonal antibodies (mAbs), and as a result, they can competitively block the interface and inhibit the adsorption of proteins to the air-liquid interface.⁶ This characteristic effectively prevents protein unfolding at the interface during the manufacturing process, sample handling and storage, including mixing, fi ltration, pumping, shaking, agitation, and freeze-thaw.⁴ Similarly, it can also prevent protein adsorption and subsequent loss at the product contacting surfaces, such as filters, primary container/closures, and IV administration tubing, playing a critical role to assure accurate dose delivery to patients.⁴

PS20 and PS80 may also stabilize protein via direct interaction and thereby increase the protein’s colloidal stability.⁷ Therapeutic protein may self-associate via its hydrophobic patches and form aggregate. Polysorbate may interact with the hydrophobic patches on protein surfaces through hydrophobic interaction and thus prevent proteins from aggregation and further unfolding. However, the direct surfactant-protein interaction and subsequent improvement of protein’s colloidal stability may be protein specific and not broadly applicable.^5,8 For example, a thermodynamic study demonstrated that polysorbates bind to human serum albumin, however, the binding of the surfactant to the three investigated immunoglobulins was found to be quite low and negligible.⁸

The Bad

Commercially available PS20 and PS80 are chemically diverse mixtures,² and the expected structure for PS20 (polyoxyethylene (20) sorbitan monolaurate) and PS80 (polyoxyethylene (80) sorbitan monooleate) only accounts for 20% of the total polysorbate.⁹ The heterogeneity arises not only from the hydrophilic head group and fatty acid tail, but also from process-related impurities, levels of byproducts etc., as illustrated in Figure 1. The composition of polysorbate may also vary between vendors with lot-to-lot variability likely resulting from diff erent synthesis routes and raw materials used. PS80 is commercially available at multi-compendium (MC) grade and Chinese pharmacopeia (ChP) grade, with the required minimal oleic acid content at 58% and 98%, respectively.

In addition, the polysorbate structure lacks strong chromophore that precludes readily available UV-vis and fluorescence as detection. As a result, a wide variety of methods have been developed to monitor polysorbate content based on diff erent properties of the polysorbate as summarized in Table 1. The basis for each type of PS content method is very different, but in general accurate and comparable PS content can be obtained by diff erent methods for unstressed samples.¹⁰ However, the PS content in stressed samples measured by different methods showed pronounced differences due to substantial differences in the contribution of each subspecies (mono- or poly-ester) to the overall PS content results following degradation.¹⁰

In addition to the PS content method, PS purity method based on reversed phase-based LC in-line with a universal detector such as CAD, ELSD or mass spectrometry are frequently applied to analyze the composition of major PS subspecies. A wide range of methods based on LC, gas chromatography (GC), Nuclear Magnetic Resonance (NMR) or colorimetry have also been developed and applied to detect polysorbate degradant such as small organic molecules, free fatty acid, H2O2, to understand polysorbate degradation pathways. Significant progress has been made to build an analytical toolbox to characterize the polysorbates as summarized in an excellent review paper.² However, it remains an analytical challenge to fully characterize PS20 and PS80 and their degradants.¹ The analytical toolbox may continue to evolve with the advancement of analytical technologies.

The unique physicochemical properties described above have presented unique challenges not only in developing proper polysorbate control strategy from a raw material control and analytical strategy point of view, but also for elucidation of the structure-function relationship of the polysorbate as detailed below.

The role that each type of PS subspecies and byproducts in commercial PS plays as a protein stabilizer remains to be elucidated, but some progress has been made recently. Monoester and diester fractions of PS20 (alllaurate) and PS80 (all-oleate) at ChP grade were enriched and their micellar morphology in solution were examined by small-angle neutron scattering.¹⁹ The micelle size and aggregation number increase with increasing temperature; however, the monoester fraction shows different temperature dependence than the diester fraction. The PS20 and PS80 diester fraction demonstrates a higher micellar aggregation number than that of monoester fraction.¹⁹ In another related study, the CMC of the monoester and diester fractions of PS20 and PS80 was measured.²⁰ Interestingly the PS20 fractions demonstrate very different CMC values, and drastically diff erent stabilizing effects against aggregation and particle formation of a mAb in an agitation study. In contrast, all PS80 fractions had CMC values similar to each other and within ~3-fold range.

The PS20 and PS80 at ChP grade have much less molecular heterogeneity than those at MC grade by teasing out the heterogeneity introduced by fatty acid. It remains to be seen whether the ester fractions of the polysorbates at MC grade have similar physicochemical properties (micellar morphology, CMC, surface tension) as those at ChP grade. Both ChP grade and MC grade PS80 demonstrate comparable stabilizing effect of mAb during mechanical stress.²¹ However, it was found that PS20 and PS80 at ChP grade demonstrated higher propensity towards oxidative degradation, but similar sensitivity towards and enzymatic hydrolysis as those at MC grade.²² Does it imply that the heterogeneity present in MC grade PS20 and PS80 improves its stability against oxidation?

The Ugly

Polysorbates are prone to degradation by oxidation and hydrolysis,³ as illustrated in Figure 2, with hydrolysis being induced either chemically or enzymatically. Polysorbate degradation, root cause and impact are discussed in more details below.

Hydrolysis of the polysorbate involves cleavage of the fatty acid ester bond, the resulting released free fatty acid may form subvisible or visible particles. Polysorbate hydrolysis induced by enzyme was suggested as a major root cause for the visible and subvisible particles formation that aff ected product quality,²³ as polysorbate hydrolysis at typical protein formulation pH is limited.²⁴ Residual host cell proteins that have been reported to induce polysorbate hydrolysis in protein formulations includes group XV lysosomal phospholipase A2 isomer X1 (LPLA2),²⁵ putative phospholipase B-like-2 (PLBL2),26 liver carboxylesterase.²⁷ The co-purified lipases, a subclass of the esterase that catalyzes the hydrolysis of fats or lipids, tend to have similar physicochemical properties as the protein of interest and thus is difficult to remove. Attempts have been made to knock out lipoprotein lipase (LPL) from the Chinese Hamster Ovary (CHO) cell line that reduced LPL expression by more than 95%, but it only reduces polysorbate degradation by 41-57%,²⁸ suggesting maybe other lipases are also at play. A range of carboxylester hydrolase from different species/resources were used to induce PS20 and PS80 hydrolysis.²⁹ It was found the degradation pattern is not only depending on the hydrolase, but also depending on the PS subspecies types such as the orders of the ester, the identity of hydrophilic head-group, and fatty acid chain length. No PS subspecies was completely resistant to enzymatic hydrolysis.²⁹ Recently, putative phospholipase B-like-2 (PLBL2) was ruled out as the root cause of inducing PS hydrolysis in protein formulation through both genetic knock-out and immunedepletion of PLBL2.³⁰ The confl icting reported results are likely due to the lipase that facilitates the hydrolysis of polysorbate is well below detection limit, making it difficult to track, monitor, or correlate which lipase leads to PS hydrolysis.

Polysorbate may also be auto-oxidized by temperature, light or transition trace metals,³¹ and the resulting peroxide formation may induce protein oxidation,^32,33 whereas the acid produced may lead to a decrease in solution pH.³¹ The oxidation of the polysorbate primarily occurs on the polyoxyethylene (POE or PEO) chains,³⁴ which leads to POE esters and other degradants, such as short chain alkanes, ketones, aldehyde and acids etc.^3,35 The oxidation of polysorbate may also occur on fatty acid moiety,³⁶ with unsaturated fatty acids such as oleate and linoleate moiety being preferentially attacked.³ Oxidation on both moieties is viewed as a free radical chain process that is composed of initiation, propagation and termination.^36,37 Interestingly, it was reported recently that oxidized PS80 showed an increase in its surface activity while maintaining its CMC properties, as well as its protective effect against aggregation for the mAb.38 In contrast, PS80 degraded via hydrolysis led to slower surface adsorption rate, and the free fatty acid release from hydrolysis also forms insoluble particles, negatively impacting protein quality and stability.³⁸

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Histidine, a commonly used protein formulation buffer agent, may have a confounding impact on PS degradation. It was reported that L-histidine has dual effect on PS20 stability. L-Histidine serves as scavenger for reactive oxygen species during 2,2’-azobis (2-amidinopropane) hydrochloride (AAPH)-stress study in protecting PS20, whereas it promotes PS20 oxidation during accelerated storage at 40 °C in solution.³⁹ PS oxidation was observed in histidine placebo buffer in another study,²² however, trace metal contaminants in histidine buffer was found to be the root cause as the PS oxidation is suppressed upon the addition of EDTA under otherwise the same condition. Histidine chloride buffer was also reported to facilitate polysorbate hydrolysis in placebo, catalyzed by the imidazole of histidine.⁴⁰ However, the histidine-catalyzed polysorbate hydrolysis was minimized in the presence of therapeutic proteins.

Novel Excipient as PS Alternatives?

Poloxamer 188 (P188) is another excipient used as a protein stabilizer in protein formulations, although not as widely as PS20 and PS80. Adsorption kinetics measurement suggests that P188 inhibits protein adsorption at solid interface by interacting with proteins in solution, whereas PS20 and PS80 stabilizes proteins at interface by competitively binding and blocking protein access to the interface.⁴¹ P188 may degrade in solution mainly through an oxidation pathway, forming oxidized PEO, and small polypropylene oxide (PPO) species.⁴² Both histidine and P188 were found to be oxidized at pharmaceutically relevant conditions in the histidine buffer, and a new species was detected as a result of reaction between the degradant from both histidine and P188.⁴²

A few other novel excipients were also explored as PS alternatives in the past few years, trying to circumvent the polysorbate hydrolysis issue caused by residue lipase.^6,43 Brij-5843 and FM10006 were found to be promising PS alternatives that may be resistant to lipase hydrolysis. However, substantial work will need to be done before any of these novel excipients may be brought into therapeutic protein formulations and to patients.

Conclusion

PS20 and PS80, as effective protein stabilizers, are widely used excipients in protein formulations for parenteral administration approved by health authorities. However, the commercially available PS20 and PS80 are chemically diverse mixtures and the polysorbate structure lacks a strong chromophore. A combination of these unique physicochemical properties presents challenges in developing a polysorbate control strategy and elucidating structure-functional relationship of polysorbate.

PS20 and PS80 may also degrade through the oxidation and hydrolysis pathway, and negatively impact product quality and stability. There is an increasing number of reports on visible/subvisible fatty acid particles formation due to polysorbate hydrolysis catalyzed by residual lipase in the past few years. When the residue lipase likely at play is at very low abundance e.g. ppb level, it presents a tremendous challenge to positively detect and identify the lipase and correlate it to the polysorbate hydrolysis in high protein concentration. To circumvent the challenges associated to detect/identify/remove low abundant lipase that led to PS hydrolysis, novel excipients resistant to lipase hydrolysis have been explored as potential PS alternatives.

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Author Biographies

Dr. Yunyu (Linda) Yi is a Sr. Scientist in the Protein Analytical Development department of Biogen. She has developed her expertise in recombinant therapeutic protein characterization by electrophoresis, liquid chromatography, as well as LC-MS or LC-MS/MS over the last 15 years. She has participated in drug development for monoclonal antibodies, blood coagulation factors, bi-specific antibodies, fusion proteins, protein vaccines, and biosimilars for preclinical and clinical programs.

Dr. Yutong Jin is a Scientist I in the Protein Analytical Development department of Biogen. She joined Biogen in 2019 and works on developing liquid chromatography and mass spectrometry methods for recombinant therapeutic protein separation and characterization. Rashmi Menon completed her MS in Chemistry from Univ. of Missouri, St Louis. She has worked in analytical and formulation development of both large and small molecules and has been at Biogen for the last 8 years.

Dr. Bernice Yeung is the Head of the Biochemical and Chemical Development within the Biogen Analytical Development organization. She has over 20 years of experience in the biopharmaceutical industry in various analytical roles supporting the development and commercialization of protein-based therapeutics, including monoclonal antibodies, fusion proteins, cytokines, and enzymes.