Evaluation of Proteolytic Digestion Efficiency in Hydrogen Exchange-Mass Spectrometry Experiments Using the Digestible Peptide Score.

In a hydrogen exchange-mass spectrometry (HX-MS) experiment, the enzymatic proteolysis of the deuterated protein is an essential step. Often the differences in the performance between different digestion protocols or between immobilized protease columns can be challenging to evaluate. To compare differences in the performance of immobilized protease columns, a new digestion efficiency metric known as digestible peptide scoring (DPS) was developed and is presented in this work. The measured response fraction of substance P peptide is used to assign a value between 0% and 100% based on the fraction of substance P digested by the enzyme, using angiotensin II as an undigested internal standard. In this work, the DPS approach was tested using multiple immobilized pepsin batches prepared using different protocols. The results demonstrate the repeatability of DPS values for batches prepared using the same conditions and the ability of the DPS evaluations to provide unique values when the immobilization conditions were altered. Protein digestions obtained with a higher scoring column were better than digestions obtained using a lower scoring column. The DPS evaluation is simple and quickly provides an unambiguous assessment which can be used to evaluate an immobilized enzyme column's suitability prior to performing an experiment, to track performance over a column's lifetime, to optimize protease immobilization protocols specifically for the quench conditions of a particular experiment, and to optimize the digestion conditions.

Understanding the Effects of Site-Specific Light Chain Conjugation on Antibody Structure Using Hydrogen Exchange-Mass Spectrometry (HX-MS).

Antibody drug conjugates (ADCs) represent one of the fastest growing classes of cancer therapeutics. Drug incorporation through site-specific conjugation in ADCs leads to uniform drug load and distribution. These site-specific modifications may have an impact on ADC quality attributes including protein higher order structure (HOS), which might impact safety and efficacy. In this study, we conducted a side-by-side comparison between the conjugated and unconjugated mAb. In the ADC, the linker-pyrrolobenzodiazepine was site specifically conjugated to an engineered unpaired C215 residue within the Fab domain of the light chain. Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) indicated a decrease in thermal stability for the CH2 transition of the ADC. Size exclusion chromatography (SEC) analysis showed that conjugation of the mAb resulted in earlier aggregation onset and increased aggregation propensity after 4 weeks at 40 °C. Differential hydrogen-exchange mass spectrometry (HX-MS) indicated that upon conjugation, light chain residues 150-155 and 197-204, close to the conjugation site, showed significantly faster HX kinetics, suggesting an increase in backbone flexibility within this region, while heavy chain residues 32-44 exhibited significantly slower kinetics, suggesting distal stabilization of the mAb backbone.

Single-domain antibodies reveal unique borrelicidal epitopes on the Lyme disease vaccine antigen, outer surface protein A (OspA).

Camelid-derived, single-domain antibodies (VHHs) have proven to be extremely powerful tools in defining the antigenic landscape of immunologically heterogeneous surface proteins. In this report, we generated a phage-displayed VHH library directed against the candidate Lyme disease vaccine antigen, outer surface protein A (OspA). Two alpacas were immunized with recombinant OspA serotype 1 from Borrelia burgdorferi sensu stricto strain B31, in combination with the canine vaccine RECOMBITEK Lyme containing lipidated OspA. The phage library was subjected to two rounds of affinity enrichment ("panning") against recombinant OspA, yielding 21 unique VHHs within two epitope bins, as determined through competition enzyme linked immunosorbent assays (ELISAs) with a panel of OspA-specific human monoclonal antibodies. Epitope refinement was conducted by hydrogen exchange-mass spectrometry. Six of the monovalent VHHs were expressed as human IgG1-Fc fusion proteins and shown to have functional properties associated with protective human monoclonal antibodies, including B. burgdorferi agglutination, outer membrane damage, and complement-dependent borreliacidal activity. The VHHs displayed unique reactivity profiles with the seven OspA serotypes associated with B. burgdorferi genospecies in the United States and Europe consistent with there being unique epitopes across OspA serotypes that should be considered when designing and evaluating multivalent Lyme disease vaccines.

Optimization of a Hydrogen Exchange-Mass Spectrometry Robotic Liquid Handler Using Tracers.

Hydrogen exchange-mass spectrometry (HX-MS) is a valuable analytical technique that can provide insight into protein interactions and structure. The deuterium labeling necessary to gain this insight is affected by many physical and chemical factors, making it challenging to achieve high reproducibility. Poor precision during dispensing, transfer, and mixing of solutions during the experiment contributes substantially to the overall variability. While the use of a robotic liquid handler can potentially improve precision, its operation must be optimized. We observed poor precision in data collected using a robotic liquid handler to perform HX-MS. In this work, we describe how we were able to improve that system's precision considerably based on tracking performance using caffeine, caffeine-d3, and caffeine-d9 as tracers for the sample, label, and quench to report on each operation of the liquid handling workflow. The insights gained about liquid handler performance and the three-tracer approach can aid in optimizing HX-MS workflow operations, whether performed manually or when using a liquid handling system. Additionally, these tracers can be incorporated as internal tracers during an experiment to report on the labeling and quench operations of each sample throughout the run and, if desired, be used to implement an uptake correction described previously.

Interaction between preservatives and a monoclonal antibody in support of multidose formulation development.

Multidose formulations have patient-centric advantages over single-dose formats. A major challenge in developing multidose formulations is the prevention of microbial growth that can potentially be introduced during multiple drawings. The incorporation of antimicrobial preservatives (APs) is a common approach to inhibit this microbial growth. Selection of the right preservative while maintaining drug product stability is often challenging. We explored the effects of three APs, 1.1 % (w/v) benzyl alcohol, 0.62 % (w/v) phenol, and 0.42 % (w/v) m-cresol, on a model immunoglobulin G1 monoclonal antibody, termed the "NIST mAb." As measured by hydrogen exchange-mass spectrometry (HX-MS) and differential scanning calorimetry, conformational stability was decreased in the presence of APs. Specifically, flexibility (faster HX) was significantly increased in the CH2 domain (HC 238-255) across all APs. The addition of phenol caused the greatest conformational destabilization, followed by m-cresol and benzyl alcohol. Storage stability studies conducted by subvisible particle (SVP) analysis at 40 °C over 4 weeks further revealed an increase in SVPs in the presence of phenol and m-cresol but not in the presence of benzyl alcohol. However, as monitored by size exclusion chromatography, there was neither a significant change in the monomeric content nor an accumulation of soluble aggregate in the presence of APs.

Structure of a transmission blocking antibody in complex with Outer surface protein A from the Lyme disease spirochete, Borreliella burgdorferi.

319-44 is a human monoclonal antibody capable of passively protecting mice against tick-mediated infection with Borreliella burgdorferi, the bacterial genospecies responsible for Lyme disease in North America. In vitro, 319-44 has complement-dependent borreliacidal activity and spirochete agglutinating properties. Here, we report the 2.2 Å-resolution crystal structure of 319-44 Fab fragments in complex with Outer surface protein A (OspA), the ~30 kDa lipoprotein that was the basis of the first-generation Lyme disease vaccine approved in the United States. The 319-44 epitope is focused on OspA ß-strands 19, 20, and 21, and the loops between ß-strands 16-17, 18-19, and 20-21. Contact with loop 20-21 explains competition with LA-2, the murine monoclonal antibody used to estimate serum borreliacidal activities in the first-generation Lyme disease vaccine clinical trials. A high-resolution B-cell epitope map of OspA will accelerate structure-based design of second generation OspA-based vaccines.

Structural Elucidation of a Protective B Cell Epitope on Outer Surface Protein C (OspC) of the Lyme Disease Spirochete, Borreliella burgdorferi.

Outer surface protein C (OspC) plays a pivotal role in mediating tick-to-host transmission and infectivity of the Lyme disease spirochete, Borreliella burgdorferi. OspC is a helical-rich homodimer that interacts with tick salivary proteins, as well as components of the mammalian immune system. Several decades ago, it was shown that the OspC-specific monoclonal antibody, B5, was able to passively protect mice from experimental tick-transmitted infection by B. burgdorferi strain B31. However, B5's epitope has never been elucidated, despite widespread interest in OspC as a possible Lyme disease vaccine antigen. Here, we report the crystal structure of B5 antigen-binding fragments (Fabs) in complex with recombinant OspC type A (OspCA). Each OspC monomer within the homodimer was bound by a single B5 Fab in a side-on orientation, with contact points along OspC's α-helix 1 and α-helix 6, as well as interactions with the loop between α-helices 5 and 6. In addition, B5's complementarity-determining region (CDR) H3 bridged the OspC-OspC' homodimer interface, revealing the quaternary nature of the protective epitope. To provide insight into the molecular basis of B5 serotype specificity, we solved the crystal structures of recombinant OspC types B and K and compared them to OspCA. This study represents the first structure of a protective B cell epitope on OspC and will aid in the rational design of OspC-based vaccines and therapeutics for Lyme disease. IMPORTANCE The spirochete Borreliella burgdorferi is a causative agent of Lyme disease, the most common tickborne disease in the United States. The spirochete is transmitted to humans during the course of a tick taking a bloodmeal. After B. burgdorferi is deposited into the skin of a human host, it replicates locally and spreads systemically, often resulting in clinical manifestations involving the central nervous system, joints, and/or heart. Antibodies directed against B. burgdorferi's outer surface protein C (OspC) are known to block tick-to-host transmission, as well as dissemination of the spirochete within a mammalian host. In this report, we reveal the first atomic structure of one such antibody in complex with OspC. Our results have implications for the design of a Lyme disease vaccine capable of interfering with multiple stages in B. burgdorferi infection.

High-Throughput Epitope Mapping by Hydrogen Exchange-Mass Spectrometry.

In this paper, we introduce a screening protocol for epitope mapping by hydrogen exchange mass spectrometry (HX-MS) that has higher throughput than a traditional HX-MS epitope mapping. In the screening protocol, three HX labeling times (20, 1000, and 86400 s) are each measured without replicates. The experimental protocol is anchored on a single epitope mapping experiment conducted using the traditional complete protocol (five HX times measured in triplicate) that is used to define HX times and define significance limits. Previously, we reported traditional epitope mapping results on the Borrelia burgdorferi outer surface protein A (OspA) antigen that are in excellent agreement with the X-ray crystallography results. Here, we show that the screening protocol and complete HX-MS identify identical epitopes of OspA but that the screening protocol has a 5-fold higher throughput.

Human B Cell Epitope Map of the Lyme Disease Vaccine Antigen, OspA.

The Lyme disease (LD) vaccine formerly approved for use in the United States consisted of recombinant outer surface protein A (OspA) from Borrelia burgdorferi sensu stricto (ss), the bacterial genospecies responsible for the vast majority of LD in North America. OspA is an ∼30 kDa lipoprotein made up of 21 antiparallel ß-strands and a C-terminal α-helix. In clinical trials, protection against LD following vaccination correlated with serum antibody titers against a single epitope near the C-terminus of OspA, as defined by the mouse monoclonal antibody (MAb), LA-2. However, the breadth of the human antibody response to OspA following vaccination remains undefined even as next-generation multivalent OspA-based vaccines are under development. In this report, we employed hydrogen exchange-mass spectrometry (HX-MS) to localize the epitopes recognized by a unique panel of OspA human MAbs, including four shown to passively protect mice against experimental B. burgdorferi infection and one isolated from a patient with antibiotic refractory Lyme arthritis. The epitopes grouped into three spatially distinct bins that, together, encompass more than half the surface-exposed area of OspA. The bins corresponded to OspA ß-strands 8-10 (bin 1), 11-13 (bin 2), and 16-20 plus the C-terminal α-helix (bin 3). Bin 3 was further divided into sub-bins relative to LA-2's epitope. MAbs with complement-dependent borreliacidal activity, as well as B. burgdorferi transmission-blocking activity in the mouse model were found within each bin. Therefore, the resulting B cell epitope map encompasses functionally important targets on OspA that likely contribute to immunity to B. burgdorferi.

Identification of Agitation-Induced Unfolding Events Causing Aggregation of Monoclonal Antibodies Using Hydrogen Exchange-Mass Spectrometry.

Due to significant safety tolerances on maximum levels of visible and sub-visible particles in parenterally dosed drug products like monoclonal antibodies (mAbs), particle formation rates must be determined during development and minimized. Agitation stress, encountered during transportation and manufacturing, increases particle formation rates in a protein and formulation dependent fashion in a phenomenon thought to be partially mediated by mAb adsorption to the continuously regenerating air-water interface that results from agitation. The goal of this study was to explore the structural dynamics of three mAbs with variable sensitivity to agitation to develop a mechanistic understanding of exactly what occurs at the air-water interface that leads to aggregation and particle formation. We observed preferential orientation at the interface and subsequent cooperative unfolding for the molecule which aggregates most extensively under agitation, and also that the magnitude of destabilization appears to scale with particle formation rates. We also show that polysorbate, a widely-used excipient in parenteral formulations to protect against particle formation, eliminates interface-induced destabilization. This study provides direct evidence that local unfolding events resulting from interface exposure precede particle formation and may play a causal role in the process.

Interaction of Aluminum-adjuvanted Recombinant P[4] Protein Antigen With Preservatives: Storage Stability and Backbone Flexibility Studies.

Eight antimicrobial preservatives used in parenteral multidose formulations (thimerosal, 2-phenoxy ethanol, phenol, benzyl alcohol, m-cresol, chlorobutanol, methyl paraben, propyl paraben) were examined for their effects on the storage stability (4 °C, 25 °C) of an Alhydrogel® (AH) adjuvanted formulation of the non-replicating rotavirus vaccine (NRRV) recombinant P[4] protein antigen. The stability of AH-adsorbed P[4] was monitored for antigen-antibody binding, conformational stability, and antigen-adjuvant interaction via competitive ELISA, DSC, and SDS-PAGE, respectively. There was an unexpected correlation between increasing storage stability of the AH-adsorbed P[4] and preservative hydrophobicity (log P) (e.g., the parabens and chlorobutanol were least destabilizing). We used hydrogen exchange-mass spectrometry (HX-MS) to better understand the destabilizing effects of temperature and preservative on backbone flexibility of AH-adsorbed P[4]. Thimerosal addition immediately increased the backbone flexibility across much of the AH-adsorbed P[4] protein backbone (except the N-terminal P2 region and residues G17-Y38), and further increase in P[4] backbone flexibility was observed after storage (4 °C, 4 weeks). HX-MS analysis of AH-adsorbed P[4] stored for 4 weeks at 25 °C revealed structural alterations in some regions of the epitope involved in P[4] specific mAb binding. These combined results are discussed in terms of a generalized workflow for multi-dose vaccine formulation development for recombinant protein antigens.

Rapid Assessment of Pepsin Column Activity for Reliable HDX-MS Studies.

Hydrogen/deuterium exchange with mass spectrometry (HDX-MS) is a widely used technique to probe protein structural dynamics, track conformational changes, and map protein-protein interactions. Most HDX-MS studies employ a bottom-up approach utilizing the acid active protease pepsin to digest the protein of interest, often utilizing immobilized protease in a column format. The extent of proteolytic cleavage will greatly influence data quality and presents a major source of variation in HDX-MS studies. Here, we present a simple cocktail of commonly available peptides that are substrates of pepsin and can serve as a rapid check of pepsin column activity. The peptide-based assay requires no system modifications and provides an immediate readout to check and benchmark pepsin activity across different HDX-MS platforms.

Statistical Equivalence Testing of Higher-Order Protein Structures with Differential Hydrogen Exchange-Mass Spectrometry (HX-MS).

Hydrogen exchange-mass spectrometry (HX-MS) is widely recognized for its potential utility for establishing the equivalence of the higher-order structures of proteins, particularly in comparability and similarity contexts. However, recent progress in the statistical analysis of HX-MS data has instead placed an emphasis on significance testing to identify regions of proteins where there are significant differences in HX between two or more protein states. In the cases involving assessment of similarity or equivalence of the higher-order structure of different protein samples (e.g., biosimilars), significance testing of HX-MS data is unsuitable. To meet this need, we have adapted the univariate two one-sided test (TOST) equivalence testing method for HX-MS data. Equivalence acceptance criteria were determined using maximum deviations from randomized resampling of truly equivalent samples to define hybrid equivalence criteria (maximum deviation of true equivalents, MDTE). Application of the TOST-MDTE test on differential HX-MS measurements of wild-type and mutated maltose-binding proteins demonstrates that the equivalence testing method was fit-for-purpose. Three infliximab biosimilars (Remsima, Renflexis, and Inflectra) were found to be equivalent to their Remicade reference product based on differential HX-MS measurements, while 5% deglycosylated NIST mAb was not statistically equivalent to the unmodified NIST mAb reference.

Recommendations for the Propagation of Uncertainty in Hydrogen Exchange-Mass Spectrometric Measurements.

Hydrogen exchange-mass spectrometry (HX-MS) is used widely to characterize higher-order protein structure and to locate changes in protein structure and dynamics that accompany, for example, ligand binding and protein-protein interactions. Quantitative differences in the amount of hydrogen exchange between two states (i.e., differential HX) are taken as evidence of significant differences in higher-order structure or dynamics. The quantitative measures range from simple mass differences at one HX labeling time to differences averaged across an HX time course with correction for deuterium recovery. This work applies the principles of uncertainty propagation to differential HX measurements to facilitate the identification of significant differences. Furthermore, it is shown that pooled estimates of experimental uncertainty result in a lower false positive rate than estimates of uncertainty based on individual standard deviations.

In Vivo Reoxidation Kinetics of Free Thiols in Multiple Domains of IgG1 Antibodies in Rats.

While free thiols in monoclonal antibodies (mAbs) have been extensively characterized by in vitro studies to probe its effect on antibody function and stability, their in vivo biotransformation has not been comprehensively studied. In this study, a panel of five recombinant IgG1 mAbs with elevated free thiols in the VH, VL, and CH2 domains were intravenously administered into Wistar rats. In vivo biotransformation of thirty-five free thiol sites in total (7 disulfide pairs in VL, CL, VH, CH1, HH, CH2, CH3 domains across the 5 mAbs) were monitored using a denaturing differential isotopic tagging procedure on immunopurified timepoints followed by LC-MS of tryptic digests. The free thiol levels in two VH domain and one CH2 domain disulfide sites decreased in vivo following first order kinetics. Free thiol levels of the remaining 32 sites were remarkably stable in vivo. Further analytical characterization highlighted a positive association between a free thiol's solvent accessibility and a free thiol's reoxidation propensity. The data and discussion presented here shed valuable insights into the in vivo fate of free thiols in several recombinant IgG1s and its implications for free thiols as a product quality attribute in therapeutic mAb products.

Protein A does not induce allosteric structural changes in an IgG1 antibody during binding.

Affinity chromatography is widely used for antibody purification in biopharmaceutical production. Although there is evidence suggesting that affinity chromatography might induce structural changes in antibodies, allosteric changes in structure have not been well-explored. Here, we used hydrogen exchange-mass spectrometry (HX-MS) to reveal conformational changes in the NIST mAb upon binding with a protein A (ProA) matrix. HX-MS measurements of NIST mAb bound to in-solution and resin forms of ProA revealed regions of the CH2 and CH3 domains with increased protection from HX upon ProA binding, consistent with the known ProA binding region. In-solution ProA experiments revealed regions in the Fab with increased HX uptake when the ProA:mAb molar ratio was increased to 2:1, suggesting an allosterically induced increase in backbone flexibility. Such effects were not observed with lower ProA concentration (1:1 molar ratio) or when ProA resin was used, suggesting some kind of change in binding mode. Since all pharmaceutical processes use ProA bound to resin, our results rule out reversible allosteric effects on the NIST mAb during interaction with resin ProA. However, irreversible effects cannot be ruled out since the NIST mAb was previously exposed to ProA during its original purification.

Mechanism of Thimerosal-Induced Structural Destabilization of a Recombinant Rotavirus P[4] Protein Antigen Formulated as a Multi-Dose Vaccine.

In a companion paper, a two-step developability assessment is presented to rapidly evaluate low-cost formulations (multi-dose, aluminum-adjuvanted) for new subunit vaccine candidates. As a case study, a non-replicating rotavirus (NRRV) recombinant protein antigen P[4] was found to be destabilized by the vaccine preservative thimerosal, and this effect was mitigated by modification of the free cysteine (C173S). In this work, the mechanism(s) of thimerosal-P[4] protein interactions, along with subsequent effects on the P[4] protein's structural integrity, are determined. Reversible complexation of ethylmercury, a thimerosal degradation byproduct, with the single cysteine residue of P[4] protein is demonstrated by intact protein mass analysis and biophysical studies. A working mechanism involving a reversible S-Hg coordinate bond is presented based on the literature. This reaction increased the local backbone flexibility of P[4] within the helical region surrounding the cysteine residue and then caused more global destabilization, both as detected by HX-MS. These effects correlate with changes in antibody-P[4] binding parameters and alterations in P[4] conformational stability due to C173S modification. Epitope mapping by HX-MS demonstrated involvement of the same cysteine-containing helical region of P[4] in antibody-antigen binding. Future formulation challenges to develop low-cost, multi-dose formulations for new recombinant protein vaccine candidates are discussed.

Characterization of Excipient Effects on Reversible Self-Association, Backbone Flexibility, and Solution Properties of an IgG1 Monoclonal Antibody at High Concentrations: Part 1.

Many challenges limit the formulation of antibodies as high-concentration liquid dosage forms including elevated solution viscosity, decreased physical stability, and in some cases, liquid-liquid phase separation. In this work, an IgG1 monoclonal antibody (mAb-J), which undergoes concentration-dependent reversible self-association (RSA), is characterized in the presence of 4 amino acids (Arg, Lys, Asp, Glu) and NaCl using biophysical techniques and hydrogen exchange-mass spectrometry. The 5 additives disrupt RSA, prevent phase separation, and reduce solution viscosity to varying extents. These excipients also cause decreased turbidity, reduced average hydrodynamic diameter, and increased relative solubility of mAb-J in solution. The RSA disrupting efficacy of the positively charged amino acids is greater than either negatively charged amino acids or NaCl. As measured by hydrogen exchange-mass spectrometry, anionic excipients induced more alterations of mAb-J backbone dynamics at pH 6.0, and weak Fab-Fab interactions likely remained with the addition of either cationic or anionic excipients at high protein concentrations. Along with a companion paper examining a different mAb with a different molecular mechanism of RSA, these results are discussed in the context of various excipient strategies to disrupt protein-protein interactions to formulate mAbs at high protein concentrations with good stability profiles and favorable pharmaceutical properties for subcutaneous administration.

Characterization of Excipient Effects on Reversible Self-Association, Backbone Flexibility, and Solution Properties of an IgG1 Monoclonal Antibody at High Concentrations: Part 2.

In this work, we continue to examine excipient effects on the reversible self-association (RSA) of 2 different IgG1 monoclonal antibodies (mAb-J and mAb-C). We characterize the RSA behavior of mAb-C which, similar to mAb-J (see Part 1), undergoes concentration-dependent RSA, but by a different molecular mechanism. Five additives that affect protein hydrophobic interactions to varying extents including a chaotropic salt (guanidine hydrochloride), a hydrophobic salt (trimethylphenylammonium iodide), an aromatic amino acid derivative (tryptophan amide hydrochloride), a kosmotropic salt (sodium sulfate, Na2SO4), and a less polar solvent (ethanol) were evaluated to determine their effects on the solution properties, molecular properties, and RSA of mAb-C at various protein concentrations. Four of the 5 additives examined demonstrated favorable effects on the pharmaceutical properties of high concentration mAb-C solutions (i.e., lower viscosity and weakened protein-protein interactions, PPIs) with a ranking order of guanidine hydrochloride > trimethylphenylammonium iodide > tryptophan amide hydrochloride > ethanol as measured by various biophysical techniques. Conversely, addition of Na2SO4 resulted in less desirable solution properties and enhanced PPIs. The effect of these 5 additives on mAb-C backbone dynamics were evaluated by hydrogen exchange-mass spectrometry (at high vs. low protein concentrations) to better understand their effects on the molecular sites of RSA in mAb-C.

Multifaceted assessment of rituximab biosimilarity: The impact of glycan microheterogeneity on Fc function.

Biosimilars are poised to reduce prices and increase patient access to expensive, but highly effective biologic products. However, questions still remain about the degree of similarity and scarcity of information on biosimilar products from outside of the US/EU in the public domain. Thus, as an independent entity, we performed a comparative analysis between the innovator, Rituxan® (manufactured by Genentech/Roche), and a Russian rituximab biosimilar, Acellbia® (manufactured by Biocad). We evaluated biosimilarity of these two products by a variety of state-of-the-art analytical mass spectrometry techniques, including tandem MS mapping, HX-MS, IM-MS, and intact MS. Both were found to be generally similar regarding primary and higher order structure, though differences were identified in terms of glycoform distribution levels of C-terminal Lys, N-terminal pyroGlu, charge variants and soluble aggregates. Notably, we confirmed that the biosimilar had a higher level of afucosylated glycans, resulting in a stronger FcγIIIa binding affinity and increased ADCC activity. Taken together, our work provides a comprehensive comparison of Rituxan® and Acellbia®.