Surfactant Protection Efficacy at Surfaces Varies with the Nature of Hydrophobic Materials.

OBJECTIVE: Monoclonal antibodies are in contact with many different materials throughout their life cycle from production to patient administration. Plastic surfaces are commonly found in single use bags, syringes, perfusion bags and tubing and their hydrophobic nature makes them particularly prone for adsorption of therapeutic proteins. The addition of surfactants in therapeutic formulations aims at minimizing surface and interface adsorption of the active molecules. However, their protection efficacy related to the nature of the plastic material is still poorly investigated. METHODS: We use real-time surface-sensitive techniques and immunosorbent assays, to quantify surfactant and monoclonal antibody adsorption on hydrophobic model surfaces and different plastic polymers to analyse the effect of material surface properties on the level of surfactant protection. RESULTS: We show that Polysorbate 80 protects monoclonal antibodies significantly better from adsorption on a polystyrene surface than on a hexadecane self-assembled monolayer, used as a model surface with similar hydrophobicity. This enhanced protective effect on polystyrene is observed for different antibodies and also other surfactants, and its extent depends on the surfactant concentration for a given antibody concentration. A comparative adsorption study allows ranking different in-use plastics and highlights the dependence of Polysorbate 80 protection efficacy on the nature of the plastic material. CONCLUSION: This study demonstrates that, beyond hydrophobicity, the nature of plastic polymer surfaces affects surfactant adsorption and thereby impacts their protection efficacy in therapeutic antibody formulations.

Clogging of microfluidic constrictions by monoclonal antibody aggregates: role of aggregate shape and deformability.

The formation of aggregates in solutions of monoclonal antibodies is difficult to prevent. Even if the occurrence of large aggregates is rare, their existence can lead to partial or total clogging of constrictions in injection devices, with drastic effects on drug delivery. Little is known on the origin and characteristics of such clogging events. Here we investigate a microfluidic model system to gain fundamental understanding of the clogging of constrictions by monoclonal antibody aggregates. Highly concentrated solutions of monoclonal antibodies were used to create protein aggregates (larger than 50 microns) using mechanical or heat stress. We show that clogging occurs when aggregates reach the size of the constriction and that clogs can in some cases be released by increasing the applied pressure. This indicates the important role of protein aggregate deformability. We perform systematic experiments for different relative aggregate sizes and applied pressures, and measure the resulting flow-rate. This allows us to present first in situ estimates of an effective Young's modulus. Despite their different shapes and densities, we can predict the number of clogging events for a given constriction size from the aggregate size distribution measured by Flow Imaging Microscopy (MFI). In addition our device can detect the occurrence of very rare big aggregates often overlooked by other detection methods.

Optimised hyperbolic microchannels for the mechanical characterisation of bio-particles.

The transport of bio-particles in viscous flows exhibits a rich variety of dynamical behaviour, such as morphological transitions, complex orientation dynamics or deformations. Characterising such complex behaviour under well controlled flows is key to understanding the microscopic mechanical properties of biological particles as well as the rheological properties of their suspensions. While generating regions of simple shear flow in microfluidic devices is relatively straightforward, generating straining flows in which the strain rate is maintained constant for a sufficiently long time to observe the objects' morphologic evolution is far from trivial. In this work, we propose an innovative approach based on optimised design of microfluidic converging-diverging channels coupled with a microscope-based tracking method to characterise the dynamic behaviour of individual bio-particles under homogeneous straining flow. The tracking algorithm, combining a motorised stage and a microscopy imaging system controlled by external signals, allows us to follow individual bio-particles transported over long-distances with high-quality images. We demonstrate experimentally the ability of the numerically optimised microchannels to provide linear velocity streamwise gradients along the centreline of the device, allowing for extended consecutive regions of homogeneous elongation and compression. We selected three test cases (DNA, actin filaments and protein aggregates) to highlight the ability of our approach for investigating dynamics of objects with a wide range of sizes, characteristics and behaviours of relevance in the biological world.

Nanoparticulate Impurities in Pharmaceutical-Grade Sugars and their Interference with Light Scattering-Based Analysis of Protein Formulations.

PURPOSE: In the present study we investigated the root-cause of an interference signal (100-200 nm) of sugar-containing solutions in dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) and its consequences for the analysis of particles in biopharmaceutical drug products. METHODS: Different sugars as well as sucrose of various purity grades, suppliers and lots were analyzed by DLS and NTA before and (only for sucrose) after treatment by ultrafiltration and diafiltration. Furthermore, Fourier transform infrared (FTIR) microscopy, scanning electron microscopy coupled energy-dispersive X-ray spectroscopy (SEM-EDX), and fluorescence spectroscopy were employed. RESULTS: The intensity of the interference signal differed between sugar types, sucrose of various purity grades, suppliers, and batches of the same supplier. The interference signal could be successfully eliminated from a sucrose solution by ultrafiltration (0.02 µm pore size). Nanoparticles, apparently composed of dextrans, ash components and aromatic colorants that were not completely removed during the sugar refinement process, were found responsible for the interference and were successfully purified from sucrose solutions. CONCLUSIONS: The interference signal of sugar-containing solutions in DLS and NTA is due to the presence of nanoparticulate impurities. The nanoparticles present in sucrose were identified as agglomerates of various impurities originating from raw materials.

Small amounts of sub-visible aggregates enhance the immunogenic potential of monoclonal antibody therapeutics.

PURPOSE: Determine the effect of minute quantities of sub-visible aggregates on the in vitro immunogenicity of clinically relevant protein therapeutics. METHODS: Monoclonal chimeric (rituximab) and humanized (trastuzumab) antibodies were subjected to fine-tuned stress conditions to achieve low levels (<3% of total protein) of sub-visible aggregates. The effect of stimulating human dendritic cells (DC) and CD4(+) T cells with the aggregates was measured in vitro using cytokine secretion, proliferation and confocal microscopy. RESULTS: Due to its intrinsic high clinical immunogenicity, aggregation of rituximab had minimal effects on DC activation and T cell responses compared to monomeric rituximab. However, in the case of trastuzumab (low clinical immunogenicity) small quantities of aggregates led to potent CD4(+) T cell proliferation as a result of strong cytokine and co-stimulatory signals derived from DC. Consistent with this, confocal studies showed that stir-stressed rituximab was rapidly internalised and associated with late endosomes of DC. CONCLUSIONS: These data link minute amounts of aggregates with activation of the innate immune response, involving DC, resulting in T cell activation. Thus, when protein therapeutics with little or no clinical immunogenicity, such as trastuzumab, contain minute amounts of sub-visible aggregates, they are associated with significantly increased potential risk of clinical immunogenicity.

In vivo fluorescence imaging of IgG1 aggregates after subcutaneous and intravenous injection in mice.

PURPOSE: To monitor the biodistribution of IgG1 aggregates upon subcutaneous (SC) and intravenous (IV) administration in mice and measure their propensity to stimulate an early immune response. METHODS: A human mAb (IgG1) was fluorescently labeled, aggregated by agitation stress and injected in SKH1 mice through SC and IV routes. The biodistribution of monomeric and aggregated formulations was monitored over 47 days by fluorescence imaging and the early immune response was measured by quantifying the level of relevant cytokines in serum using a Bio-plex assay. RESULTS: The aggregates remained at the SC injection site for a longer time than monomers but after entry into the systemic circulation disappeared faster than monomers. Upon IV administration, both monomers and aggregates spread rapidly throughout the circulation, and a strong accumulation in the liver was observed for both species. Subsequent removal from the circulation was faster for aggregates than monomers. No accumulation in lymph nodes was observed after SC or IV administration. Administration of monomers and aggregates induced similar cytokine levels, but SC injection resulted in higher cytokine levels than IV administration. CONCLUSION: These results show differences in biodistribution and residence time between IgG1 aggregates and monomers. The long residence time of aggregates at the SC injection site, in conjunction with elevated cytokine levels, may contribute to an enhanced immunogenicity risk of SC injected aggregates compared to that of monomers.

How spray drying processing and solution composition can affect the mAbs stability in reconstituted solutions for subcutaneous injections. Part II: Exploring each protein stabilizer effect.

Despite extensive research in spray drying of biopharmaceuticals, identifying the optimal formulation composition and process conditions to minimize the various stresses a biopharmaceutical undergoes during this drying process. The current study extends previous research on investigating how spray drying processing and solution composition can affect the stability of monoclonal antibodies (mAbs) in reconstituted solutions for subcutaneous injections. The decoupling process stresses on a model mAb (mAb-A) compared to the effect of coupled spray-drying stresses revealed that excipients and protein concentration had a more pronounced effect on stabilizing mAb-A against shear and thermal/dehydration stresses than spray drying operating conditions. These results prompted the continuation of the study, with the aim to investigate in greater depth the effect of mAb-A concentration in the formulation designated to spray-drying and then the effect of type and the concentration of individual excipients (sugars, amino acids and surfactants). The outcomes of this investigation suggest that a general increase in the concentration of excipients, particularly surfactants, correlates with a reduction in aggregation and turbidity observed in the reconstituted spray-dried mAb-A powders. These results, contribute to the identification of a suitable composition for a spray-dried mAb-A powder that ensures robust stability of the protein in reconstituted solutions intended for subcutaneous injection. This valuable insight has important implications for advancing the development of pharmaceutical formulations with enhanced stability and efficacy.

How spray drying processing and solution composition can affect the mAbs stability in reconstituted solutions for subcutaneous injections. Part I: Contribution of processing stresses against composition.

Spray drying is increasingly being applied to process biopharmaceuticals, particularly monoclonal antibodies (mAbs). However, due to their protein nature, mAbs are susceptible to degradation when subjected to various stresses during a drying process. Despite extensive research in this domain, identifying the appropriate formulation composition and spray drying conditions remains a complex challenge, requiring further studies to enhance the understanding on how process and formulation parameters impact mAb stability in reconstituted solutions. This research aims to explore spray drying as technique for producing pharmaceutical mAbs-based powders intended for reconstitution and subcutaneous injection. In the initial phase of this study, using a model mAb (mAb-A), the influence of dissociated and coupled process stresses on protein stability after solution reconstitution was investigated. The findings revealed a detrimental interplay of mechanical, interfacial, and thermal/dehydration stresses on mAb-A stability, notably characterized by an increase in protein aggregation. Subsequently, in a second phase, the study delved into the impact of spray drying processing conditions, the level of excipients, and protein concentration on mAb-A aggregation in reconstituted solutions. The obtained results highlighted the critical role of formulation composition as a parameter deserving further study, specifically concerning the selection of type and concentration of stabilizers to be added in the liquid mAb-A solution to be dried.

Native and Non-Native aggregation pathways of antibodies anticipated by cold-accelerated studies.

Assessment of cold stability is essential for manufacture and commercialization of biotherapeutics. Storage stability is often estimated by measuring accelerated rates at elevated temperature and using mathematical models (as the Arrhenius equation). Although, this strategy often leads to an underestimation of protein aggregation during storage. In this work, we measured the aggregation rates of two antibodies in a broad temperature range (from 60 °C to -25 °C), using an isochoric cooling method to prevent freezing of the formulations below 0 °C. Both antibodies evidenced increasing aggregation rates when approaching extreme temperatures, because of hot and cold denaturation. This behavior was modelled using Arrhenius and Gibbs-Helmholtz equations, which enabled to deconvolute the contribution of unfolding from the protein association kinetics. This approach made possible to model the aggregation rates at refrigeration temperature (5 °C) in a relatively short timeframe (1-2 weeks) and using standard characterization techniques (SEC-HPLC and DLS).

Detection and characterization of subvisible aggregates of monoclonal IgG in serum.

PURPOSE: To detect and characterize the aggregation of therapeutic monoclonal antibodies in undiluted biological fluids. METHODS: Fluorescently labeled subvisible IgG aggregates formed by applying either heat stress or by pH-shift were investigated immediately after addition to human serum, and after 24 h. Unstressed and stressed IgG formulations were analyzed by fluorescence single particle tracking, confocal laser scanning microscopy and flow cytometry. RESULTS: Unstressed formulations remained free from subvisible aggregates in serum, whereas heat-stressed and pH-shift stressed formulations showed dissimilar aggregation behaviors. The aggregation profile of the heat-stressed formulation diluted in serum remained practically the same as the one diluted in buffer, even after the 24 h incubation period. The pH-shift stressed formulation had strikingly smaller and more numerous subvisible aggregates immediately after dilution in serum compared to buffer. These aggregates became noticeably larger in both diluents after 24 h, but in serum they appeared to be formed by other types of constituents than the labeled protein itself. CONCLUSION: These results show that subvisible therapeutic protein aggregates may undergo changes in number, type and size distribution upon contact with human serum. This emphasizes the importance of analytical strategies for monitoring aggregation in undiluted biological fluids.

Mechanistic understanding of metal-catalyzed oxidation of polysorbate 80 and monoclonal antibody in biotherapeutic formulations.

Surfactants are commonly used in biotherapeutic formulations to prevent the formation of aggregates and protect proteins from denaturation. Among them polysorbates are the most widely used. However, they are known to be prone to degradation, mainly via enzymatic hydrolysis and oxidation. In this study, the impact of different conditions and factors on the oxidation of polysorbate 80 (PS80) and of a monoclonal antibody (mAb) was evaluated. In particular, the role of different formulation components (e.g., mAb concentration, pH, buffer, surfactant grade, chelators) was investigated in the presence of iron as transition metal contaminant. The results of our studies demonstrated that PS80 oxidation was accelerated even in the presence of iron levels as low as 20 ppb. In addition, the results showed that the oxidation of a specific solvent-exposed mAb methionine increased with PS80 oxidation, in particular under accelerated stress conditions and that the oxidation phenomenon was hindered in absence of iron or after addition of EDTA. Our results showed that PS80 "all oleate" (PS80-AO) was more sensitive to oxidative degradation than PS80 "multi-compendial" (PS80-MC). Contrary to acetate and citrate buffers, the results showed that the kinetics of PS80 oxidation was pH-dependent in presence of histidine buffer. It was also demonstrated that, when increasing its concentration, the mAb exhibited a protective effect against metal catalyzed PS80 and methionine oxidation. Our systematic studies on the role of the formulation components and potential contaminants (i.e., iron) demonstrated the complexity of the oxidative mechanism and the importance of different competitive systems, including pro-oxidant factors (e.g., iron, pH, PS80 quality) and antioxidant factors (e.g., protein concentration, EDTA, citrate) that may occur in biologic formulations containing PS80.

A Systematic Approach to Evaluating Closed System Drug-Transfer Devices During Drug Product Development.

The use of Closed System Drug-Transfer Devices (CSTDs) has increased significantly in recent years due to NIOSH and USP recommendations to use them during preparation of hazardous drugs. Mechanistic and material differences between CSTDs and traditional in-use components warrant an assessment of their impact on product quality and dosing accuracy. Using a combination of prevalent CSTDs with biologic molecules, we performed an extensive assessment of the effect of using CSTDs for dose preparation and observed no negative impact on product quality attributes. Additionally, we found that the CSTD hold-up volume is 2 to 4-fold higher than conventional in-use components and exhibited a strong dependence on the CSTD brand used. We also found that the CSTD brand and dosing volume have a major influence on dosing accuracy with suboptimal protein recovery at very low dosing volumes. We identified entrapment of product in the CSTD spike as the root cause for this sub-optimal recovery and found that flushing the CSTD spike with a brand-new syringe and not the dosing syringe aided in complete protein recovery. Taken together we present a systematic approach to evaluate the risks and impact of CSTD to drug product quality, dose preparation, and dosing accuracy.

Stress Factors in Protein Drug Product Manufacturing and Their Impact on Product Quality.

Injectable protein-based medicinal products (drug products, or DPs) must be produced by using sterile manufacturing processes to ensure product safety. In DP manufacturing the protein drug substance, in a suitable final formulation, is combined with the desired primary packaging (e.g., syringe, cartridge, or vial) that guarantees product integrity and enables transportation, storage, handling and clinical administration. The protein DP is exposed to several stress conditions during each of the unit operations in DP manufacturing, some of which can be detrimental to product quality. For example, particles, aggregates and chemically-modified proteins can form during manufacturing, and excessive amounts of these undesired variants might cause an impact on potency or immunogenicity. Therefore, DP manufacturing process development should include identification of critical quality attributes (CQAs) and comprehensive risk assessment of potential protein modifications in process steps, and the relevant steps must be characterized and controlled. In this commentary article we focus on the major unit operations in protein DP manufacturing, and critically evaluate each process step for stress factors involved and their potential effects on DP CQAs. Moreover, we discuss the current industry trends for risk mitigation, process control including analytical monitoring, and recommendations for formulation and process development studies, including scaled-down runs.

Fluorescence single particle tracking for the characterization of submicron protein aggregates in biological fluids and complex formulations.

PURPOSE: To evaluate the potential of fluorescence single particle tracking (fSPT) for the characterization of submicron protein aggregates in human serum, plasma and formulations containing human serum albumin (HSA). METHODS: A monoclonal IgG was covalently labeled with a fluorescent dye and cross-linked with glutaraldehyde. IgG aggregates and fluorescent beads of 0.1 µm (control) were diluted in buffer, serum and plasma, and their size distributions were analyzed by fSPT and nanoparticle tracking analysis (NTA). In a separate experiment, IgG and HSA, fluorescently labeled with different dyes, were mixed and subjected to heat stress. The stressed sample was analyzed by fSPT using a dual color mode and by NTA. RESULTS: The accuracy and precision of fSPT proved to be comparable to NTA. fSPT was able to successfully measure all the samples in buffer, serum and plasma. The average size of the cross-linked protein aggregates showed a slight increase in biological fluids. Moreover, fSPT analysis showed that a significant proportion of the aggregates formed by subjecting an IgG/HSA mixture to heat stress were composed of both proteins. CONCLUSION: fSPT is a powerful technique for the characterization of submicron protein aggregates in biological fluids and complex formulations.

Adsorption rate constants of therapeutic proteins and surfactants for material surfaces.

Adsorption of therapeutic proteins to material surfaces can be a pivotal issue in drug development, especially for low concentration products. Surfactants are used to limit adsorption losses. For each formulation component, surface adsorption is the result of a combination of its diffusion and surface adsorption rates. The latter are difficult to measure accurately because a depletion layer forms rapidly in the bulk solution above a bare surface, slowing down adsorption. Adapting flow conditions and local surface chemistry, we are able to minimize depletion limitations and measure apparent adsorption rate constants of three monoclonal antibodies, other proteins and surfactants with a hydrophobic surface. We show that surface adsorption rates scale with the molecular mass of the molecule, with polysorbates therefore showing thousand times slower rates than antibodies. Moreover, we observed that the desorption dynamic of polysorbates from a given hydrophobic surface depends on surface coverage, whereas this is not the case for Poloxamer 188. These novel contributions to surface adsorption dynamics enable a new perspective on the evaluation of drug surface compatibility and can, together with diffusion rates, be used to predict the protective potential of surfactants in given conditions.

Fluorescent molecular rotors as dyes to characterize polysorbate-containing IgG formulations.

PURPOSE: The aim was to evaluate fluorescent molecular rotors (DCVJ and CCVJ), which are mainly sensitive to viscosity, for the characterization of polysorbate-containing IgG formulations and compare them to the polarity-sensitive dyes ANS, Bis-ANS and Nile Red. METHODS: IgG formulations with polysorbate 20 or 80 were stressed below the aggregation temperature and analyzed by steady-state and time-resolved fluorescence and by HP-SEC with UV and fluorescent dye detection (Bis-ANS and CCVJ). Furthermore, commercial protein preparations of therapeutic proteins (Enbrel 50 mg, Humira 40 mg and MabThera 100 mg) were aggregated accordingly and analyzed with CCVJ fluorescence and HP-SEC. RESULTS: Contrarily to (Bis-)ANS and Nile Red, the molecular rotors DCVJ and CCVJ showed low background fluorescence in polysorbate-containing buffers. Time-resolved fluorescence experiments confirmed the steady-state fluorescence data. Both DCVJ and CCVJ showed enhanced fluorescence intensity for aggregated IgG formulations and were suitable for the characterization of polysorbate-containing IgG formulations in steady-state fluorescence and HP-SEC with dye detection (CCVJ). CCVJ was capable of detecting thermally induced aggregation in the commercial polysorbate-containing products Enbrel 50 mg, Humira 40 mg and MabThera 100 mg. CONCLUSION: Fluorescent molecular rotors are suitable probes to detect aggregation in polysorbate-containing IgG formulations.

Critical evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates.

PURPOSE: To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic light scattering (DLS) and test its performance in characterizing drug delivery nanoparticles and protein aggregates. METHODS: Standard polystyrene beads of sizes ranging from 60 to 1,000 nm and physical mixtures thereof were analyzed with NTA and DLS. The influence of different ratios of particle populations was tested. Drug delivery nanoparticles and protein aggregates were analyzed by NTA and DLS. Live monitoring of heat-induced protein aggregation was performed with NTA. RESULTS: NTA was shown to accurately analyze the size distribution of monodisperse and polydisperse samples. Sample visualization and individual particle tracking are features that enable a thorough size distribution analysis. The presence of small amounts of large (1,000 nm) particles generally does not compromise the accuracy of NTA measurements, and a broad range of population ratios can easily be detected and accurately sized. NTA proved to be suitable to characterize drug delivery nanoparticles and protein aggregates, complementing DLS. Live monitoring of heat-induced protein aggregation provides information about aggregation kinetics and size of submicron aggregates. CONCLUSION: NTA is a powerful characterization technique that complements DLS and is particularly valuable for analyzing polydisperse nanosized particles and protein aggregates.

Mass spectrometric analysis of intact human monoclonal antibody aggregates fractionated by size-exclusion chromatography.

PURPOSE: The aim of this study was to develop a method to characterize intact soluble monoclonal IgG1 antibody (IgG) oligomers by mass spectrometry. METHODS: IgG aggregates (dimers, trimers, tetramers and high-molecular-weight oligomers) were created by subjecting an IgG formulation to several pH jumps. Protein oligomer fractions were isolated by high performance size exclusion chromatography (HP-SEC), dialyzed against ammonium acetate pH 6.0 (a mass spectrometry-compatible volatile buffer), and analyzed by native electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS). RESULTS: Monomeric and aggregated IgG fractions in the stressed IgG formulation were successfully isolated by HP-SEC. ESI-TOF MS analysis enabled us to determine the molecular weight of the monomeric IgG as well as the aggregates, including dimers, trimers and tetramers. HP-SEC separation and sample preparation proved to be necessary for good quality signal in ESI-TOF MS. Both the HP-SEC protocol and the ESI-TOF mass spectrometric technique were shown to leave the IgG oligomers largely intact. CONCLUSIONS: ESI-TOF MS is a useful tool complementary to HP-SEC to identify and characterize small oligomeric protein aggregates.

Stress Factors in mAb Drug Substance Production Processes: Critical Assessment of Impact on Product Quality and Control Strategy.

The success of biotherapeutic development heavily relies on establishing robust production platforms. During the manufacturing process, the protein is exposed to multiple stress conditions that can result in physical and chemical modifications. The modified proteins may raise safety and quality concerns depending on the nature of the modification. Therefore, the protein modifications potentially resulting from various process steps need to be characterized and controlled. This commentary brings together expertise and knowledge from biopharmaceutical scientists and discusses the various manufacturing process steps that could adversely impact the quality of drug substance (DS). The major process steps discussed here are commonly used in mAb production using mammalian cells. These include production cell culture, harvest, antibody capture by protein A, virus inactivation, polishing by ion-exchange chromatography, virus filtration, ultrafiltration-diafiltration, compounding followed by release testing, transportation and storage of final DS. Several of these process steps are relevant to protein DS production in general. The authors attempt to critically assess the level of risk in each of the DS processing steps, discuss strategies to control or mitigate protein modification in these steps, and recommend mitigation approaches including guidance on development studies that mimic the stress induced by the unit operations.

Stability of rituximab in freeze-dried formulations containing trehalose or melibiose under different relative humidity atmospheres.

The objective of the study was to compare the effectiveness of trehalose with that of melibiose in protecting a monoclonal antibody (rituximab) from aggregation, fragmentation, and secondary structure alterations during processing and subsequent storage. Because reducing disaccharides such as melibiose participate in Maillard reaction with proteins, especially in the presence of water, the lyophilizates were stored under different relative humidity (RH 5%, 11%, and 23%) atmospheres. Freeze drying was shown to cause clear alterations in rituximab secondary structure, an increase in noncovalent protein aggregation, and in some cases fragmentation. However, these changes were less pronounced in the formulation containing melibiose. Storing the lyophilizates under low RH (5%) proved to be most harmful to the stability of rituximab, intensifying secondary structure alterations and increasing protein aggregate content. Again, these changes were less aggravated in the formulation containing melibiose. Surprisingly, the concentration of aggregates larger than 1 µm decreased in some cases during storage at RH 11% and 23%. There was no indication that storage even under the highest RH (23%) would have caused significant amounts of Maillard reaction end products to be formed during 3 months of storage.