A comparison of Polysorbates and Alternative Surfactants for Interfacial Stress Protection and Mitigation of Fatty Acid Particle Formation in the Presence of an Esterase

Abstract

The hydrolysis of polysorbate surfactants in large molecule drug product formulations caused by residual host cell proteins presents numerous stability concerns for pharmaceuticals. The fatty acids (FA) released by polysorbate hydrolysis can nucleate into particulates or challenge the conformational stability of the proteinaceous active pharmaceutical ingredient (API). The loss of intact polysorbate may also leave the Drug Product (DP) vulnerable to interfacial stresses. Polysorbate 20 and 80 are available in several different quality grades (Multi-compendial, Super Refined, Pure Lauric Acid (PLA)/Pure Oleic Acid (POA)). All variations of polysorbate as well as three alternative surfactants: Brij L23, Brij O20 and Poloxamer 188 were compared for their ability to protect against air-water interfacial stresses as well as their risk for developing particulates when in the presence of lipoprotein lipase (LPL) (Pseudomonas).

Results show a meaningful difference in the timing and morphology of FA particle formation depending on the type of polysorbate used. All grades of polysorbate, while susceptible to hydrolysis, still offered sufficient protection to interfacial stresses, even when hydrolyzed to concentrations as low as 0.005 % (w/v). Alternative surfactants that lack an ester bond were resistant to lipase degradation and showed good protection against shaking stress.

Introduction

Large molecule drug products utilize surfactants to prevent adsorption and to protect the protein against interfacial stresses such as the air-water interface or the primary container contact area-water interfaces.¹ Polysorbate (PS) 20 and 80 are the most widely utilized surfactants in large molecule formulations, with Poloxamer 188 being used in some medications.² Brij L23 and Brij O20 are common surfactants in household consumer products but are currently not approved for pharmaceutical use.

Polysorbates are amphipathic, nonionic surfactants characterized as ethoxylated sorbitan esterified with fatty acids. While the primary fatty acids esterified to PS20 and 80 are lauric acid and oleic acid respectively, polysorbates are heterogeneous mixtures where the identity of the esterified fatty acid and arrangement ethylene oxides around the sorbitan can vary between grades and batches.³ PS20 and 80 are available in several grades: Multi-compendial (MC), Super Refined (SR), Pure Lauric Acid (PLA) and Pure Oleic Acid (POA). A representative distribution of fatty acids for each of these PS grades is provided in Table 1.

Distributions of fatty acids esters for each polysorbate grade are examples and based on the specific certificate of analysis of the used batch in this study.
MC PS20 and MC PS80 are the most commonly used grades in pharmaceuticals. SR grades of PS are similar to MC in terms of fatty acid composition but have fewer process related impurities such as primary and secondary oxidation products and unesterified fatty acids. SR PS20 also contains a higher percentage of higher-order esters which gives it a lower Critical Micelle Concentration (CMC).4-6 PLA and POA PS have minimal process related impurities and have largely uniform incorporations of lauric acid (PS20) and oleic acid (PS80) as their hydrophobic components. Previous research has shown that PLA PS20 is more sensitive to oxidation than the comparable POA PS80.7 Currently PLA PS20 is a non-compendial grade of PS.

The distribution of fatty acids in PS is significant because populations of residual host cell proteins, carried through monoclonal antibody (mAb) purification, can present enough enzymatic activity to hydrolyze polysorbate. This results in the release of free fatty acids (FFAs) into the DP formulation. FFAs have limited solubility and can nucleate into meaningful populations of visible and subvisible particles. Fatty acid degradants also have the potential to impact the DP stability by interacting directly with the mAb.8-15 Several factors impact how the products of PS hydrolysis develop into particulates: the population of fatty acids, the rate at which they enter solution, the pH and temperature of the formulation, and the identity and concentration of intact surfactant and other impurities such as e.g., glass leachables. Together each of these factors play a role in the rate and morphology of the fatty acid particles formation.16-20

Alternative surfactants devoid of ester bonds are an effective means of avoiding surfactant degradation by host cell esterases and lipases. Polyethoxylated fatty alcohol (PFA) surfactants contain hydrophobic and hydrophilic subunits joined by an ether linkage that allows this surfactant class to be impervious to host cell impurities that would otherwise degrade the standard PS surfactants. Two notable examples of PFAs, Laureth-23 and Oleth-20, commercially known as Brij L23 and Brij O20, respectively, contain the corresponding fatty alcohol subunits to the fatty acids nominally found in PS20 and PS80. This structure enables them to perform the protective functions required in large molecule formulations yet remain resistant to enzymatic degradation. Poloxamer 188 is another alternative surfactant that lacks an ester bond. It is a triblock copolymer featuring a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene.21,22 Fig. 1 shows the structure and physical properties of the surfactants under evaluation.23,24

In this study a performance comparison for fatty acid particle formation caused by lipoprotein lipase (LPL) PS hydrolysis and interfacial stress protection during stability storage and shaking was conducted. Formulations were prepared using different grades of PS20/80 as well as Brij L23,-O20, and P188. LPL (Pseudomonas) was used to provide a controlled rate of PS hydrolysis.

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Materials

BrijL23, BrijO20, SRPS80 ,POAPS80, SRPS20, and PLAPS2 were purchased from Croda;MCPS20 and MCPS80 were purchased fromJ .T.Baker; Sorbitol and L-Histidinemonohydrochloridemonohydrate were purchased from Merck; L-Histidine was purchased from Ajino-moto; P188 (Poloxamer 188, Kolliphor P 188) was from Thermo; LPL Lipoprotein Lipasepseudomonas (>1200units/mg) was from Sigma. Themonoclonal antibody(mAb) used in this study was produced and purified at Janssen, Malvern,PA. The water used in all studies was from a Millipore Milli-Qwatersys-tem with an average conductivity of 1 8.2Mohm. The 25 R glassvialswerepurchasedfromSchott. The20mmrubberstopperswereacquiredfromWest.

A comparison of Polysorbates and Alternative Surfactants for Interfacial Stress Protection and Mitigation of Fatty Acid Particle Formation in the Presence of an Esterase, Roy, Ian et al., Journal of Pharmaceutical Sciences, Volume 113, Issue 9, 2688 – 2698

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