Physicochemical Comparison of Kolliphor HS 15, ELP, and Conventional Surfactants for Antibody Stabilization in Biopharmaceutical Formulations

Abstract

Stabilization of therapeutic protein formulations with nonionic surfactants such as Polysorbate 20 (PS20), Polysorbate 80 (PS80), or Poloxamer 188 (P188) is imperative to avoid critical loss of the active pharmaceutical ingredient by aggregation or adsorption onto different types of interfaces. In the present work, we hae characterized the interfacial activity of the  aforementioned surfactants, alone and in competition with antibodies, in comparison to two other excipients with approval for use in parenteral applications, Kolliphor HS 15 (HS15) and Kolliphor ELP (ELP). To this end, we applied a comprehensive suite of experimental techniques, including tensiometry, interfacial rheology, and quartz-crystal microbalance with dissipation monitoring (QCM-D). The obtained data shows important differences between the surfactants as well as a clear influence of the type of interface considered on the observed behavior. In order to link these physicochemical results to the performance of the chosen surfactants in the stabilization of antibodies, we performed another series of tests to quantify protein aggregation (i.e., the formation of (sub)visible particles in formulations under stress) as well as the release of oil from siliconized vials. In addition, the stability of the surfactants against enzymatic degradation was investigated. It is demonstrated that HS15 can compete with the widely used polysorbates in terms of interfacial activity and protein stabilization, while offering higher robustness against degradation by a lipase and an esterase. On the other hand, P188 shows poor interfacial activity but can still suppress the aggregation of at least some proteins, indicating that different mechanisms of stabilization are at play. Our findings and the broad spectrum of tests described in this work are instructive toward a better understanding of protein stabilization in distinct primary packaging systems through surfactants in aqueous formulations.

Introduction

Degradation of therapeutic antibodies on their way from initial production to final administration is a common challenge in the pharmaceutical industry.1 Uncontrolled antibody aggregation and/or adsorption onto different interfaces may lead to reduced activity or enhanced immunogenicity, which can pose severe risks to the patient. Therefore, most formulations contain surfactants to stabilize the drug molecules against aggregation and suppress their adsorption to interfaces. Today, polysorbates such as polyoxyethylene 20 sorbitan monolaurate (PS20) or monooleate (PS80) represent the most frequently used class of nonionic surfactants for the stabilization of antibody-containing products.2−5 However, also other types of amphiphilic structures are already applied in such formulations, most notably Poloxamer 188 (P188), a triblock copolymer consisting of a ∼1800 Da poly(propylene oxide) (PPO) middle block flanked by two poly(ethylene oxide) (PEO) chains, with a typical total PEO content of approximately80%.3,6,7 Novel excipients that are still under development include polyether-modified N-acyl amino acids, such as Nmyristoyl phenylalanine-N-polyetheramine diamide (FM1000).8−11 In a very recent study, a comprehensive comparison of both established and potential new surfactants for biopharmaceuticals led to the conclusion that analogues of α-tocopherol poly(ethylene glycol) succinate may serve as promising new structures for antibody stabilization.10 Furthermore, hydroxypropylated cyclodextrins,12,13 polyethoxylated fatty ethers (Brij 35 or 58),10,11,14−17 as well as highly purified polysorbates17 were proposed as alternatives to the conventional multicompendial polysorbates. Finally, a
more hydrophobic version of P188 was found to be beneficial for formulations enclosed in siliconized primary packaging material.18 Further information on the development of alternative surfactants for biopharmaceutical formulations can be found in several recent review articles.3,19,20

Chemical stability is an important factor to consider when deciding which surfactant to use in the formulation of a therapeutic antibody. For example, after downstream processing, some residual host cell proteins (HCPs), such as lipases or esterases, may cleave ester bonds present in surfactants like the polysorbates.21,22 Another critical pathway for surfactant degradation is oxidation, which can be triggered by impurities in excipients or the conditions prevailing during raw material storage and handling; for example, metal ions originating from the manufacturing process of the drug substance may catalyze oxidation.23 Although sufficient residual antibody stabilization was reported after partial polysorbate oxidation below a certain threshold,24 the susceptibility of excipients to chemical reactions in pharmaceutical products still raises serious patient safety and quality concerns.25 According to a recent industry survey,26 both hydrolytic and oxidative degradation were
reported for polysorbates by a comparable number of participating companies, with enzymatic degradation being declared as the primary cause of hydrolysis. The hydrolysis of polysorbates yields free fatty acids and PEGylated sorbitan or isosorbides, which are not amphiphilic and thus cannot protect biologicals from interfacial stresses.20 Depending on the degree of hydrolysis, the fraction of intact polysorbate species may become critical, and a loss of drug stabilization can occur, for example when performing shaking studies at the end of the shelf life of a product. In addition, released free fatty acids may result in particle formation.27,28 Compared to polysorbates, P188 should generally provide higher chemical stability due to the lack of labile ester bonds. Moreover, poloxamers were shown to contain significantly lower levels of hydroperoxides and aldehydes in neat form than other pegylated surfactants such as PS20 or PS80.29,30 However, the situation appears to become more complex in aqueous systems under pharmaceutically relevant conditions. For example, histidine, a common
buffer, may promote chemical degradation of P188 in the presence of metal ions31 and/or hydroxyl radicals32 with a strong dependence of the observed detrimental effect on the solution environment, including parameters such as temperature, poloxamer concentration, and type of oxidizing species.25,26 This highlights that comprehensive analyses are needed to assess the true safety profile of excipients for use in drug formulations, as the degradation products will also be parenterally administered and should not cause any adverse effects.33,34 Although the formulation space has become more homogeneous over the years,5 the continuous development of novel surfactants (including safety assessment) is still required to ensure drug efficacy and excipient chemical stability in all relevant environments.

As mentioned, the main purpose of surfactants in biological drug products is to maintain the intended functionality of the active pharmaceutical ingredient (API) by preventing uncontrolled aggregation and potential denaturation. In principle, protein aggregation may occur in the bulk solution.32 While surfactants may suppress these processes, the composition of antibody formulations is typically chosen to optimize the conformational, chemical, and colloidal stability of the bulk protein and thus achieve maximum shelf life independent of any added surface-active excipients.35,36 The actual need for amphiphilic stabilizers in such systems derives from the fact that protein aggregation can be dramatically enhanced at surfaces and interfaces,37 where local conditions may induce structural rearrangements and conformational changes that potentially result in attractive protein−protein interactions. One prominent interface to consider here is that between the (hydrophilic) aqueous formulation and the surrounding (hydrophobic) gas phase (usually air). Such liquid/gas interfaces are omnipresent throughout the whole lifecycle of a therapeutic antibody, for example in the form of air bubbles
caused by agitation during processing or transport. Their detrimental impact on protein aggregation as well as the potential mitigating influence of surfactants have been demonstrated in numerous previous studies.38−41 Similar aggregation-triggering effects can occur on solid/liquid interfaces, for example when antibodies encounter technical surfaces like steel during production42 or interact with the materials used in primary packaging devices such as prefilled syringes (PFS).43 Although PFS units typically consist of glass or, to a lesser extent, plastics like cyclic olefin polymers (COPs), the main solid surfaces of concern in the context of protein aggregation are silicones like polydimethylsiloxane (PDMS) due to their inherent hydrophobicity. The presence of silicone-water interfaces in PFS originates from the common use of PDMS for lubrication to reduce the force required for drug administration. In addition to the PDMS(-like) surfaces exposed at the stoppers and syringe barrel walls, limited interfacial stability of siloxane species may result in certain degrees of leaching into the formulation, yielding (sub)visible solid particles and/or liquid oil droplets with high interfacial area for interaction with the therapeutic proteins.44,45 In principle, adsorption on the formed solid−liquid or liquid−
liquid interfaces can lead to a decrease in the concentration of active drug species; this, however, is only significant in the case of formulations with low API contents. The main concern in terms of drug efficacy is that antibodies may undergo denaturation upon adsorption, followed by aggregation and particle formation,37,46−50 potentially in combination with effects caused by agitation and air/liquid interfaces51−53 The challenging task for surfactants thus is to compete with, or rather outcompete, the (intrinsically surface-active) proteins in their affinity to bind to the various types of interfaces that evolve in time and space during drug production, processing, transport, storage and administration, ideally by faster and/or more efficient coverage of the relevant (hydrophobic) interfaces and their subsequent “passivation” against interactions
with the APIs.37 Successful reduction (or even complete elimination) of interfacial protein adhesion has
been reported for both polysorbates and poloxamers under a broad range of conditions.37,39,40,42,46,49,50 For example, Kannan et al. used quartz-crystal microbalance (QCM) measurements to demonstrate the lower affinity of two monoclonal antibodies to adsorb on PDMS in the presence of PS20 and P188, whereby the polysorbate showed stronger surface passivation and reduced antibody aggregation more efficiently.47 Grapentin et al. observed mixed particles of PDMS and proteins in formulations containing P188 and attributed their formation to the lower interfacial activity of the poloxamer compared to polysorbates.54 In a study by Lu et al.,49 sum frequency generation (SFG) spectroscopy revealed that PS80 adsorption on the interface between water and silicone oil may still allow for antibody adhesion at low surfactant dosages, but prevented the surface-bound proteins from undesired denaturation. Apart from protecting APIs against detrimental interactions at interfaces, surfactants also influence the actual interfacial area available for such interactions, most notably by promoting the release of silicone droplets in agitated systems and modulating their coalescence behavior. For instance, the relatively low water/oil interfacial tension provided by a more surface-active excipient like PS20 can cause higher numbers of dispersed oil droplets than with P188, which may lead to more pronounced losses of API by
monomeric antibody adsorption.47

In the light of the growing body of evidence for the key role of liquid/gas, liquid/liquid, and liquid/solid interfaces in the potential degradation of therapeutic antibody formulations, we have applied a complementary suite of experimental techniques to probe the behavior of different surfactants at several of the identified relevant interfaces − alone and in competition with antibodies. For this purpose, we chose PS20, PS80, and P188 as the three dominant excipients in the market today. In addition, two other nonionic surfactants with approval for use in parenteral applications55 were studied − namely Kolliphor HS 15 (polyoxyethylene 15 hydroxystearate), which is a mixture comprising mono- and diesters of PEG with 12-hydroxy stearic acid and residual unfunctionalized PEG56 and Kolliphor ELP (polyoxyethylene 35 castor oil), which contains PEG ricinoleate as main ingredient, accompanied by fatty acid esters of PEG, unfunctionalized PEG and ethoxylated glycerol.57 The chemical structures of the surfactants investigated in this work are depicted in Figure S1 of the Supporting Information (SI). The same set of surfactants was independently investigated with respect to their ability to prevent antibody aggregation under different pharmaceutically relevant conditions. Finally, the susceptibility of the surfactants
toward enzymatic degradation was assessed in order to establish a holistic picture of the advantages and potential disadvantages of a representative set of excipients in terms of chemical stability, interfacial activity, and stabilization performance.

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Materials

Acetic acid (≥99.8%), ammonia (25% solution in water; LC-MS grade), sodium acetate (anhydrous; USP), dibasic sodium phosphate (anhydrous; Ph. Eur., BP, JP, USP), L-histidine (≥99.8%), and sodium chloride (Ph. Eur., BP, USP) were used as received from Merck (Darmstadt, Germany). Monobasic sodium phosphate (anhydrous; USP), formic acid (≥99%), and D-(+)-maltose monohydrate (USP/NF) were purchased from Sigma-Aldrich (Taufkirchen, Germany), VWR (Darmstadt, Germany), and Glentham Life Sciences (Corsham, UK), respectively. The solvents methanol, acetonitrile, and 2-propanol were obtained from Fisher Scientific (Schwerte, Germany) in LC-MS grade. Ultrapure water with a resistivity of 18.2 MΩ·cm was taken from a Merck Millipore purification system (Burlington, USA). The surfactants Kolliphor PS 20 (PS20), Kolliphor PS 80 (PS80), Kolliphor P 188 Bio (P188), Kolliphor HS 15 (HS15) and Kolliphor ELP (ELP) were supplied by BASF SE (Ludwigshafen, Germany) in commercial pharmaceutical quality. Peroxide contents were determined for PS20, PS80 and HS15 and found to be 0.8, 2, and <1 mequiv/kg. The enzymes used in the present work were lipase from Aspergillus oryzae (AOL; lyophilized white powder, ∼50 U/mg), porcine liver esterase (PLE; lyophilized slightly beige powder, ≥50 U/mg), and type II porcine pancreas lipase (PPL; lyophilized cake, 30−90 U/mg), as provided by Sigma-Aldrich (Taufkirchen, Germany).

As model proteins, polyclonal bovine immunoglobulin (IgG; ≥95%) from MP Biochemicals (Irvine, USA), monoclonal panitumumab (Vectibix; liquid formulation containing 20 mg/mL protein, 80 mM sodium acetate, and 100 mM sodium chloride at pH 5.6−6.0), and the fusion protein abatacept (Orencia; lyophilized powder containing 250 mg protein, 17.2 mg monobasic sodium phosphate, 500 mg maltose, and 14.6 mg sodium chloride with a typical pH of 7.2−7.8) were investigated. For the preparation of siliconized surfaces, a surfactant-stabilized 35% emulsion of dimethicone NF (Liveo 366 from DuPont (Wilmington, USA)) was employed, while experiments with liquid silicone oil were performed using the AK 5 grade from Wacker (Burghausen, Germany).

Physicochemical Comparison of Kolliphor HS 15, ELP, and Conventional Surfactants for Antibody Stabilization in Biopharmaceutical Formulations, Nadine Löw, Coralie Schneider, Maksymilian Marek Zegota, Adam Grabarek, Eleonora Corradini, Georg Schuster, Meike Maria Roskamp, Felicitas Guth, Andrea Hawe, and Matthias Kellermeier
Molecular Pharmaceutics 2025 22 (8), 4890-4908, DOI: 10.1021/acs.molpharmaceut.5c00519

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