Glass vial breakage during freezing and lyophilization processes poses a significant concern, as it can lead to product loss and potential safety hazards. In aqueous formulation buffers containing amorphous excipients, vial breakage has been correlated with the strains associated to the plugging-off of the frozen product from the inner vial surface during freezing. This study aimed to systematically identify parameters influencing this strain and to propose risk-mitigating options. The effects of formulation composition, vial type, and freezing process were investigated. The strain was assessed using strain gauge measurements and simulated via finite element analysis. Our findings show that the formulation composition and vial type had the greatest influence on the generated strains. Thermal contraction of the frozen formulation, and thus strain on the vial, occurred only below Tg'. The presence of a monoclonal antibody in trehalose-based formulation buffer led to an increased strain, with higher strain for higher protein concentrations. The strain was independent of the solid content of the formulation within the range probed in this study. The most effective way to mitigate the strain was to promote an earlier plugging-off, e.g. by modifying the vial surface using a hydrophobic coating (water contact angle > 120°). Bake-on siliconized vials showed the most substantial strain reduction, with a fivefold decrease compared to uncoated vials. In contrast, variation in freezing rate and freezing protocol had only marginal impact on the strain formed, as well as vial wall thickness or material. For formulations containing trehalose and mannitol at mass ratios of 2:1 and 1:2, the incorporation of an annealing step was beneficial due to a decreased plugging-off strain. Overall, this study provides valuable insights into the factors affecting the strain on vials during freezing and offers practical measures to minimize this strain and the associated risk of vial breakage, thereby enhancing the safety of frozen drug products.

Polysorbate degradation by host cell-derived hydrolases presents a critical challenge in biopharmaceutical formulations. It can lead to fatty acid release, particle formation and reduced product stability. Mass spectrometry-based host cell protein (HCP) analysis is widely used for HCP identification, but detection becomes challenging in formulations where monoclonal antibodies are present in large excess. In such cases, hydrolases can remain undetected, despite being enzymatically active at trace levels.In this study, we demonstrate that individual CHO-derived hydrolases generate distinct polysorbate degradation fingerprints that can be detected by reverse phase ultra performance liquid chromatography coupled to mass spectrometry (RP-UPLC-MS) and classified using supervised machine learning. Models were trained on single time point fingerprints comprising approximately 50 measurements for five hydrolases (CES1F, CES2C, LPLA2, PPT1 and PAF-AH). Evaluated algorithms included Logistic Regression, Random Forest, Gradient Boosting, Support Vector Classifier, AdaBoost, and Artificial Neural Networks. Seven out of eight models achieved 100 % accuracy on the test set, confirming that enzyme-specific information is preserved in single measurements in the presence of individual enzymes, independent of enzyme concentration or degradation time.External validation using an independently prepared hydrolase spike sample confirmed the robustness of the models. Prediction confidence was high at early degradation stages and decreased at late stages, as enzyme-specific degradation fingerprints became more similar. This work presents an activity-based classification framework for the functional identification of polysorbate degrading hydrolases. The approach supports downstream monitoring and risk-based mitigation strategies by identifying the enzymes that drive polysorbate hydrolysis under formulation conditions.

Although therapeutic proteins are susceptible to visible light-induced photooxidation, the underlying mechanisms remain unclear because amino acid residues do not directly absorb light above 400 nm. To evaluate the role of light source characteristics, three monoclonal antibodies formulated in water were exposed to spectrally distinct fluorescent and light-emitting diode (LED) sources, eliminating excipient-related effects. Following exposure to visible light (400-800 nm), degradation was assessed by size-exclusion, Protein A, and ion-exchange chromatography, together with mass spectrometry. The LED spectrum displayed a pronounced emission at 451 nm and a broad band between 480 and 800 nm, whereas the fluorescent lamp exhibited prominent emissions at 436, 545, and 612 nm, with additional blue-light contributions overlapping antibody absorption. Both light sources induced dose- and antibody-dependent photodegradation, characterized by increased high molecular weight species, Fc-oxidation, acidic and basic charge variants, and methionine and tryptophan oxidation, without detectable fragmentation. Monomer content decreased by 1-4 %, Fc-oxidized variants increased by 5-20 %, and the ion-exchange main peak decreased by 2-8 %. LED-induced degradation was less pronounced than that induced by fluorescent light, reflecting reduced short-wavelength emission. These findings support photosensitizer-mediated oxidation at 400-450 nm and highlight the importance of controlling light exposure during bioprocessing.

Polysorbates are commonly used in biotherapeutic drug formulations, but their stability over the course of the product's shelf life is a matter of concern. An industry-wide survey involving 15 biopharmaceutical companies found that 23 biotherapeutic drug products (DPs) in clinical development exhibited significant reductions in polysorbate (PS) content during long-term storage at 2-8 °C. In all cases, this decline did not impact critical quality attributes (CQAs), except for the formation of fatty acid (FA)-related sub-visible particles (SVP) in 7 DPs and FA-visible particles (VP) in 1 DP. Particle formation predominantly resulted from enzymatic or uncharacterized degradation mechanisms, not oxidative pathways. Corrective measures, such as optimization of downstream purification or reformulation, were undertaken only when SVP levels exceeded acceptable thresholds. For PS20 and PS80, the levels of FAs generated were estimated and translated into predicted SVP levels based on theoretical assumptions. Additionally, the current understanding of PS degradation in biopharmaceuticals, based on the latest literature, is summarized, with consideration of safety and immunogenicity aspects related to the primary PS degradation products. Overall, PS degradation is considered manageable and not problematic under practical conditions. Enzymatic hydrolysis of PS is generally deemed acceptable, provided that all CQAs are maintained within specified limits. If FA-related particles are formed it is recommended that the PS degradation pathway is well characterized, and an appropriate control strategy be implemented.

The importance of biologics has increased over the last few decades. Since proteins in liquid formulations tend to form protein particles in response to external stress factors, surfactants are added to prevent this. Currently, polysorbates (PSs) 20 and 80 are mainly used for this purpose. However, alternatives are being investigated, as PSs have stability issues, primarily due to oxidative or enzymatic degradation. The most frequently discussed alternative, which is already used in commercial biologics, is poloxamer 188 (P188). However, this triblock copolymer, which consists of a polyoxypropylene block flanked by polyoxyethylene units, may also degrade through oxidative processes. Surprisingly, there are no direct comparative stability studies for PSs and P188. Therefore, the present study compared the oxidative degradation profiles of PS20 and PS80 with those of P188 in various relevant buffer systems. To this end, the formulations were subjected to stress in the form of 50 ppb (approximately 0.9 µM) of Fe2+ and light in the visible range, in line with their exposure during pharmaceutical production. Samples that received neither an iron spike nor light served as controls. The solutions contained 0.4mg·mL-1 PS20, PS80, or P188, respectively. The surfactants were formulated in either a 25 mM acetate, citrate, or histidine buffer solution (all pH 5.5) or in pure water. Significant differences were observed between these buffers, with greater effects at higher temperatures. The stability of the surfactant depended greatly on the buffer and the applied stress. All surfactants exhibited high rates of oxidative degradation when formulated in acetate buffer or pure water. PSs demonstrated the highest overall stability in citrate buffer, while P188 remained most stable in histidine, as well as in citrate buffer. However, both PSs exhibited instability in the presence of iron and light in histidine buffer, a phenomenon not observed in P188.

Frozen storage or lyophilization of biopharmaceuticals is often used to stabilize drug products. In most cases, the drug is formulated in an aqueous formulation in glass vials. During freezing, physicochemical changes of the formulation, such as excipient crystallization and differences in the coefficients of thermal expansion between glass and the aqueous phase can induce mechanical strain in the vial wall and ultimately lead to glass vial breakage. To systematically characterize strain formation during freezing and its spatial distribution in the vial walls, we used strain gauge measurements and finite element analysis (FEA) simulations. Strain gauge rosette measurements revealed that the direction of principal strains at the vial walls during strain peaks varied with excipients present in the aqueous phase and sensor position. These findings emphasize that correct alignment of linear strain gauges with the principal strain direction is crucial; misalignment can significantly diminish the measured signal and may lead to incorrect conclusions regarding strain distribution and magnitude. Using linear strain gauges, the influence of the gauge position and the vial size was subsequently investigated using a trehalose-based formulation buffer. The measured strain strongly depended on the position of the strain gauge with respect to the fill level, as strain was not evenly distributed throughout the vial wall. Highest strains occurred just below the fill level of the formulation. This was comparable across different vial sizes. Significantly higher strains were observed depending on the freezing method, with shelf cooling producing greater strain than air circulation cooling due to product temperature gradients along the vertical axis of the vial. While locally elevated strains are generated in colder regions, plug detachment is delayed by warmer regions that mechanically constrain the frozen plug. Also, the chamber pressure inside the lyophilizer affected the strain signal. FEA simulations confirmed that the regions of highest strain can be found just below and at the fill level. With increasing fill heights, additional areas of high strain in the lower third of the vial develop which is attributed to a constraining effect of the vial bottom to radial deformation. Overall, the combination of strain gauge measurements and finite element analysis allows systematic identification of parameters governing vial strain formation during freezing, enabling identification of critical parameters relevant to vial breakage during freezing.

Background/Objectives: Surfactants are commonly used to protect proteins from denaturation and particle formation, thereby ensuring the long-term stability of biopharmaceuticals. Polysorbates (PS) 20 and 80 are the most widely used surfactants in the pharmaceutical industry. However, alternative excipients such as poloxamers are currently under investigation. In this study, mixed micelles (MMs) composed of phospholipids (PL) and polysorbate 20 (PS20) were explored as a novel stabilisation strategy, aiming to reduce the PS content in protein formulations by partial substitution with PL. Despite their favourable properties, including thermodynamic stability and small particle size, MMs have seen limited application, and no reports exist on their use for stabilising antibody solutions. Results: In a first step, PS20/PL ratios were identified, which are advantageous to form stable MM solutions, followed by an optimization of the formulation process by introducing a second heating step using the direct dispersion method. Successful MM formation was confirmed via transmission and dynamic light scattering analyses at total surfactant concentrations of up to 20 mg·mL-1 and 50 mg·mL-1, with PL contents of 50% and up to 40%, respectively. These surfactant concentrations of up to 20 mg·mL-1 and 50 mg·mL-1 are substantially higher than the surfactant concentrations that are typically used in final biopharmaceutical formulations (0.01-2 mg·mL-1). Consequently, the mixed micellar system enables operation even at concentrations substantially above practical formulation limits. In the ensuing study, the stabilizing potential of the PL/PS20 micellar system was appraised through agitation studies. Methods: In these studies, bovine serum albumin was employed as a model protein, while a monoclonal antibody was used as a candidate therapeutic molecule. Stability was assessed through visual inspection, turbidity measurements, particle analysis, and size-exclusion chromatography. Conclusions: A protective effect comparable to that of PS20 alone was observed for both model proteins, demonstrating for the first time that MMs can effectively stabilise biologics.

Polysorbate 20 (PS20) is one of the most commonly used non-ionic surfactants in cosmetics, pharmaceuticals and food products. Considered as biocompatible and non-irritating, it is further valued for its solubilising and protein stabilising properties. PS20 is manufactured through a multi-stage reaction of sorbitol with various fatty acids and ethylene oxide, resulting in a complex mixture of components with different molecular weights and polarity. Since variations in the distribution of these components can influence its performance, such as the emulsifying or solubilising efficiency, a detailed understanding of the PS20 composition is of importance. Herein we introduce a combined approach of reversed-phase chromatography with mass detection and automated stochastic modelling that enables the quantitative characterisation of PS20 at the component level. With two straightforward sample preparations and two methods for an ultra-high performance liquid chromatography (UPLC) system coupled to a single quadrupole mass (QDa) detector, this technique ensures efficient data acquisition. Seven PS20 products of different manufacturers, age and qualities were studied using the presented approach. Molar contents and weight percentages were calculated for each of the more than 27'700 components of the PS20, which were fully characterised by i) the substance class (i.e. sorbitan, isosorbide or polyoxyethylene (POE)), ii) the number of esters, iii) the fatty acid combination and iv) the number of OE units. The obtained results allowed not only an accurate prediction of bulk parameters, such as hydroxyl and saponification values, but also a detailed product comparison.

Surfactants like poloxamer 188 are commonly used to stabilise antibody and other protein drug products. This study investigated the impact of poloxamer 188 on the stability of an antibody model protein in an aqueous buffered solution in comparison to mixtures composed of both polyethylene glycol and polypropylene glycol block polymers. The separate consideration of the block polymer units is important to understand the stabilising characteristics of poloxamer 188 and its degradation products. Therefore, interfacial stress was applied using a 48-hour shaking test to evaluate the stabilisation properties of the surfactants for an antibody. The effect of shaking was investigated by visual inspection, nephelometric turbidity measurement, size exclusion chromatography, and sub-visible particle formationvia backgrounded membrane imaging. Surface tension measurements revealed the impact of polyethylene glycol and polypropylene glycol on the air-water interface and showed that high surface activity is not the only decisive factor for protein stabilisation. It was determined that all excipients have the capacity to protect the antibody from protein particle formation to a certain extent. Polypropylene glycol performed better than polyethylene glycol due to a higher tendency to interact with the airwater interface. Surprisingly, mixtures of polyethylene glycol and polypropylene glycol, similar to their molecular weight ratio in poloxamer 188, led to comparable stabilisation properties to those of the triblock polymer poloxamer. This study indicates different stabilisation mechanisms for polyethylene glycol and polypropylene glycol combined in the poloxamer 188 molecule.

Although polysorbate 20 (PS20) and 80 (PS80) are regarded as the gold standard for stabilising biologics against colloidal destabilisation and particle formation, concerns have been raised due to polysorbate hydrolysis. The released hydrophobic free fatty acids are poorly soluble in water, and formation of fatty acid (FA) particles may occur after the solubility limits are exceeded. It is therefore essential to understand and estimate the solubility limits of fatty acids in biotherapeutic formulations. To date, solubility limits of individual fatty acids were studied and predicted. However, when considering polysorbate hydrolysis, a mixture of fatty acids is invariably released, which has the potential to affect the solubility limits of individual fatty acid components. In this study, we investigated the solubility limits of individual fatty acids and fatty acid mixtures in the presence of PS20. The determination of the solubility limit of free fatty acids (FFAs) in solution was achieved through the utilisation of visual inspection, turbidity measurements, and liquid chromatography-mass spectrometry (LC-MS). In addition, a design of experiments (DoE) approach was used to statistically evaluate how the solubility limits of FAs are affected by (i) degree of hydrolysis of PS20, (ii) PS20 concentration, (iii) pH, (iv) buffer system, (v) presence of tonicifier, (vi) protein and (vii) protein concentration. The resulting data were used to develop a model to estimate the solubility of fatty acids in complex formulation conditions. A comprehensive understanding of how formulation conditions affect the solubility limits of fatty acid blends is crucial for effective mitigation of particle formation caused by polysorbate degradation.

Polysorbates (PSs) are key excipients for the colloidal stability of biopharmaceuticals with unique properties. A comprehensive understanding of the physicochemical properties of these multicomponent products is essential to address potential stability issues without compromising their functionality. Here, we demonstrate that polysorbate 80 HP (PS80) shows an anomalous clouding, i.e., a thermotropic liquid-liquid phase separation behavior, which cannot adequately be explained by the conventional interpretation assuming a pseudobinary system. In a binary two-phase system of surfactant and buffer, an increase in the total surfactant concentration increases the fraction of the surfactant-rich phase in the respective proportion (lever rule). PS80 within about 7 K of the lower critical solution temperature fails to comply with this; concentrations and compositions of the coexisting phases change with the total concentration. This renders the phases more alike and, at some point, eliminates phase separation. This significant deviation from the pseudobinary phase behavior can be resolved by conceptually dividing the numerous chemical species in PS80 into two independent pseudocomponents, PS80-I and -II. Ternary phase diagrams derived from this approach successfully explain the observed anomalous behavior. RP-UPLC-MS analysis indicated a concentration-dependent redistribution of the nonesterified components (NECs), suggesting, along with other evidence, that NECs are key constituents of component II. Specifically, free polyethylene glycol (PEG) and/or PEG-sorbitans seem to function as intrinsic cosurfactant(s) within PS80, modulating its wetting and clouding properties. The latter is important for interaction, association, and phase separation properties in biologics.

Given that the amphiphilicity of polysorbates represents a key factor in the protection of proteins from particle formation, the loss of this property through degradative processes is a significant concern. Therefore, the present study sought to identify the factors that contribute to the oxidative cleavage of the polysorbate (PS) molecule and to ascertain the preferred sites of degradation. In order to gain insight into the radical susceptibility of the individual polysorbate segments and their accessibility to water, conceptual density functional theory calculations and molecular dynamics simulations were performed. The behavior of monoesters and diesters was examined in both monomer form and within the context of micelles. The theoretical results were corroborated by experimental findings, wherein polysorbate 20 was subjected to 50 ppb Fe2+ and 100,000 lx·h of visible light, and subsequently stored at 25 °C/60% r.h. or 40 °C/75% r.h. for a period of 3 months. Molecular dynamics simulations demonstrated that unesterified polyoxyethylene(POE) chains within a polysorbate 20 molecule exhibited the greatest water accessibility, indicating their heightened susceptibility to oxidation. Nevertheless, the oxidative cleavage of esterified polyoxyethylene chains of a polysorbate 20 molecule is highly detrimental to the protective effect on protein particle formation. This occurs presumably at the oxyethylene (OE) units in the vicinity of the sorbitan ring, leaving a nonamphiphilic molecule in the worst case. Consequently, the critical degradation sites were identified, resulting in the formation of degradation products that indicate a loss of amphiphilicity in PS.

Polysorbate 20 (PS20) and polysorbate 80 (PS80) are essential surfactants used to stabilize biopharmaceutical products, yet their highly heterogeneous mixtures and susceptibility to oxidation and enzymatic hydrolysis complicate routine analysis. We developed a hierarchical generative model that reconstructs entire liquid chromatography-mass spectrometry (LC-MS) measurements to automatically interpret complex polysorbate datasets. By embedding domain knowledge of base structures, oxyethylene chain lengths, fatty acid esterification, and isotope patterns, the model resolves individual subspecies and provides molecular-level composition. Applied to PS20 and PS80, the approach distinguishes oxidative from hydrolytic degradation and yields pathway-specific fingerprints. Model outputs agree closely with manual integration while delivering greater depth and automation. This transforms polysorbate analysis from labor-intensive peak-by-peak workflows into an objective, comprehensive characterization tool suited for quality control, batch selection and degradation monitoring throughout development and manufacturing.

The viscosity of high-concentration protein solutions is a critical parameter in biopharmaceutical formulation development. Conventionally, the viscosity is measured and optimized in labor-intensive experimental workflows that require a lot of material. While predicting the viscosity with atomistic molecular dynamics (MD) simulations is feasible, they are computationally prohibitively expensive due to the large system sizes and the long simulation times involved. Coarse-grained MD (CG-MD) simulations significantly reduce computational demands, but evaluating their accuracy and predictive power requires rigorous validation. Here, we assess the capability of the Martini 3 CG force field to predict the viscosity of high-concentration antibody solutions. We show that a refined Martini 3 force field, with optimized protein-protein interactions, can predict the elevated viscosities observed in concentrated solutions of F(ab')2 fragments of the therapeutic monoclonal antibody (mAb) omalizumab. Furthermore, we show that our previously developed Martini 3-exc model for arginine excipients successfully captures the trend of lowering viscosity, as observed in our rheology experiments. These findings open the way to physics-based computational prediction of the properties of dense biopharmaceutical solutions via large-scale MD simulations.

Polysorbate degradation in biopharmaceutical formulations can impact the stability and efficacy of therapeutic proteins. This degradation is predominantly caused by specific residual host cell hydrolases present at sub-ppm concentrations. Their low abundance, combined with the lack of sensitive, enzyme specific detection methods, is a major analytical challenge. This study presents a novel approach using reverse-phase ultra-performance liquid chromatography coupled with mass spectrometry (RP-UPLC-MS) to systematically analyse the specific polysorbate degradation patterns of hydrolases expressed by Chinese hamster ovary (CHO) cells. Our findings reveal distinct degradation fingerprints of five CHO-derived hydrolases, highlighting their unique preferences for different polysorbate species based on ester linkages (e.g. monoester or multiester), hydrophilic head groups and FA chain length. This study is the first of its kind to provide such detailed insights into the enzymatic cleavage patterns of polysorbates using enzymes directly derived from CHO cells. Our results underscore the limitations of indirect polysorbate quantification assays, such as the fluorescence micelle assay (FMA) and 4-methylumbelliferone (MU4)-based hydrolytic activity assays, in accurately reflecting the activity of specific hydrolases. For instance, the FMA tends to overestimate the contribution of certain polysorbate species due to its reliance on micelle formation, while the MU4 assay's predictive reliability is limited by its use of surrogate substrates. In contrast, RP-UPLC-MS enables the precise identification of individual polysorbate species and their degradation products, providing a direct measure of hydrolase activity. The detailed enzymatic fingerprints obtained in this study pave the way for future advancements in enzyme classification and the potential application of computational tools for automated hydrolase identification.

Polysorbates, in particular polysorbate (PS) 20 and 80, are the most commonly used surfactants for stabilising biotherapeutics produced by biotechnological processes. PSs are derived from ethoxylated sorbitan (a derivative of sorbitol) esterified with fatty acids of varying chain length and degree of saturation. In the past, these surfactants have been reported to have specific liabilities. Chemical (oxidations and hydrolyses) and enzymatic degradations have been reported to affect the stability of PS in drug products. Specifically, the presence of trace amounts (sub-ppm) of certain host cell proteins (HCPs) can induce enzymatic PS degradation, which can lead to the release of free fatty acids during storage over time. Enzymatic polysorbate degradation may impair the functionality of the surfactant in stabilising therapeutic proteins, leading to the formation of visible and/or sub-visible particles in biopharmaceutical drug products. This review summarises the enzymes currently known to be involved in the degradation of polysorbate in mammalian biotechnological processes for therapeutic proteins. In recent years, advanced analytical methods have been developed to qualify and quantify the PS-degrading enzymes. Most of these assays are based on mass spectrometry with a preceding HCP enrichment approach. Efforts were made to measure the enzyme activity and correlate it with observed PS degradation. The impact on drug product quality attributes, including fatty acid solubility and phase separation, up to the formation of visible particles, and the potential induction of protein and protein/fatty acid mixed particles as well as the sensitivity of specific PS quality towards enzymatic degradation, was considered. Various drug substance (DS) mitigation strategies related to the occurrence of PS degrading enzymes are discussed as amongst them the generation of stable HCP knockout cell lines, which are also carefully analysed. The underlying opinion article reflects the undergoing discussions related to PS degrading enzymes and focusses on (i) impact on drug product, (ii) analytics for identification/quantification (characterisation) of the PS degrading enzymes, (iii) enzyme activity (iv) currently identified enzymes, and (v) potential mitigation strategies to avoid enzymatic PS degradation during DS manufacturing.

The frozen storage of biopharmaceuticals brings new challenges to the primary packaging material. Due to an increasing demand and the downsides of standard type I glass vials, such as vial breakage, novel vial types for special applications of parenteral drug products have been introduced to the market in the past years. Mechanical stresses due to dimensional changes experienced during freezing and thawing could change the material properties, hence affecting the interaction with the drug product stored in the vial or functionality such as overall integrity. Therefore, we studied the suitability of different vial qualities related to the thermally induced mechanical stresses experienced during frozen drug product preparation and storage. First, the possible failure modes for each vial type were identified. The interaction between vial surface and drug product were investigated considering surface hydrophobicity, surface free energy and surface roughness as well as microscopically visible changes analyzed by confocal laser scanning microscopy. Differences in surface hydrophobicity, roughness and surface free energy between the vial types did not impact the performance upon freeze-thaw stress and did not change with the stress. Screening the vial content for particles originating from the container using light and electron scanning microscopy combined with energy-dispersive X-ray spectroscopy showed only rare cases of particles in coated glass vials. Under extreme stress conditions, including a drop-test in the frozen state, a low number of particles was also detected in coated polymer vials. No quality issues regarding the functionality were observed upon container closure integrity testing, while the oxygen permeability was slightly increased for uncoated and especially coated polymer vials. Overall, the results show that several vial types are appropriate for the frozen storage of drug products and selection should be based on the formulation and other product requirements.

Understanding the structure and self-organisation of monoclonal antibodies (mAbs) at the air–water interface is crucial for the stability and efficacy of protein drug formulations. This paper investigates the competitive adsorption of mAb and two amphiphilic polymers, poloxamer 188 (P188) and polysorbate 20 (PS20), commonly used to stabilise mAb formulations. Our objective was twofold: to ascertain whether the surfactants in question are capable of preventing mAb adsorption; and to determine whether it is possible to desorb mAb molecules from the air–water interface by surfactant addition. Langmuir film balance measurements and drop shape tensiometry were used to obtain surface pressure and surface tension data. Infrared Reflection–Absorption Spectroscopy (IRRAS) provided information on the surface composition, including the amount of adsorbed molecules. The state adopted by P188 is contingent upon its surface concentration, which determines the self-assembled phases it adopts. We show that the phase state of P188 has a considerable influence on mAb adsorption. The presence of P188 in the brush phase (≥ 0.3 mg/L) consistently inhibits mAb adsorption, but addition of P188 subsequent to the formation of the mAb film does not result in mAb desorption. However, addition of PS20 results in the desorption of freshly-formed interfacial mAb layers of up to two hours’ age, whereas an aged mAb layer of 17 h was unable to be desorbed by PS20. Thus there is a time-dependent reorganisation of mAb at the air–water interface, increasing resistance to desorption, which we discuss in the context of potential intermolecular interactions within the interfacial film.

The high doses of drugs required for biotherapeutics, such as monoclonal antibodies (mAbs), and the small volumes that can be administered to patients by subcutaneous injections pose challenges due to high-concentration formulations. The addition of excipients, such as arginine and glutamate, to high-concentration protein formulations can increase solubility and reduce the tendency of protein particle formation. Molecular dynamics (MD) simulations can provide microscopic insights into the mode of action of excipients in mAb formulations but require large system sizes and long time scales that are currently beyond reach at the fully atomistic level. Computationally efficient coarse-grained models such as the Martini 3 force field can tackle this challenge but require careful parametrization, testing, and validation. This study extends the popular Martini 3 force field toward realistic protein-excipient interactions of arginine and glutamate excipients, using the Fab domains of the therapeutic mAbs trastuzumab and omalizumab as model systems. A novel all-atom to coarse-grained mapping of the amino acid excipients is introduced, which explicitly captures the zwitterionic character of the backbone. The Fab-excipient interactions of arginine and glutamate are characterized concerning molecular contacts with the Fabs at the single-residue level. The Martini 3 simulations are compared with results from all-atom simulations as a reference. Our findings reveal an overestimation of Fab-excipient contacts with the default interaction parameters of Martini 3, suggesting a too strong attraction between protein residues and excipients. Therefore, we reparametrized the protein-excipient interaction parameters in Martini 3 against all-atom simulations. The excipient interactions obtained with the new Martini 3 mapping and Lennard-Jones (LJ) interaction parameters, coined Martini 3-exc, agree closely with the all-atom reference data. This work presents an improved parameter set for mAb-arginine and mAb-glutamate interactions in the Martini 3 coarse-grained force field, a key step toward large-scale coarse-grained MD simulations of high-concentration mAb formulations and the stabilizing effects of excipients.