Applying UHPLC-HRAM MS/MS Method to Assess Host Cell Protein Clearance during the Purification Process Development of Therapeutic mAbs.

Host cell proteins (HCPs) are one of the process-related impurities that need to be well characterized and controlled throughout biomanufacturing processes to assure the quality, safety, and efficacy of monoclonal antibodies (mAbs) and other protein-based biopharmaceuticals. Although ELISA remains the gold standard method for quantification of total HCPs, it lacks the specificity and coverage to identify and quantify individual HCPs. As a complementary method to ELISA, the LC-MS/MS method has emerged as a powerful tool to identify and profile individual HCPs during the downstream purification process. In this study, we developed a sensitive, robust, and reproducible analytical flow ultra-high-pressure LC (UHPLC)-high-resolution accurate mass (HRAM) data-dependent MS/MS method for HCP identification and monitoring using an Orbitrap Ascend BioPharma Tribrid mass spectrometer. As a case study, the developed method was applied to an in-house trastuzumab product to assess HCP clearance efficiency of the newly introduced POROS™ Caprylate Mixed-Mode Cation Exchange Chromatography resin (POROS Caprylate mixed-mode resin) by monitoring individual HCP changes between the trastuzumab sample collected from the Protein A pool (purified by Protein A chromatography) and polish pool (purified by Protein A first and then further purified by POROS Caprylate mixed-mode resin). The new method successfully identified the total number of individual HCPs in both samples and quantified the abundance changes in the remaining HCPs in the polish purification sample.

Mitigating In-Column Artificial Modifications in High-Temperature LC-MS for Bottom-Up Proteomics and Quality Control of Protein Biopharmaceuticals.

Elevating the column temperature is an effective strategy for improving the chromatographic separation of peptides. However, high temperatures induce artificial modifications that compromise the quality of the peptide analysis. Here, we present a novel high-temperature LC-MS method that retains the benefits of a high column temperature while significantly reducing peptide modification and degradation during reversed-phase liquid chromatography. Our approach leverages a short inline trap column maintained at a near-ambient temperature installed upstream of a separation column. The retentivity and dimensions of the trap column were optimized to shorten the residence time of peptides in the heated separation column without compromising the separation performance. This easy-to-implement approach increased peak capacity by 1.4-fold within a 110 min peptide mapping of trastuzumab and provided 10% more peptide identifications in exploratory LC-MS proteomic analyses compared with analyses conducted at 30 °C while maintaining the extent of modifications close to the background level. In the peptide mapping of biopharmaceuticals, where in-column modifications can falsely elevate the levels of some critical quality attributes, the method reduced temperature-related artifacts by 66% for N-terminal pyroGlu and 63% for oxidized Met compared to direct injection at 60 °C, thus improving reliability in quality control of protein drugs. Our findings represent a promising advancement in LC-MS methodology, providing researchers and industry professionals with a valuable tool for improving the chromatographic separation of peptides while significantly reducing the unwanted modifications.

Characterization of a Monoclonal Antibody by Native and Denaturing Top-Down Mass Spectrometry.

Established in recent years as an important approach to unraveling the heterogeneity of intact monoclonal antibodies, native mass spectrometry has been rarely utilized for sequencing these complex biomolecules via tandem mass spectrometry. Typically, top-down mass spectrometry has been performed starting from highly charged precursor ions obtained via electrospray ionization under denaturing conditions (i.e., in the presence of organic solvents and acidic pH). Here we systematically benchmark four distinct ion dissociation methods─namely, higher-energy collisional dissociation, electron transfer dissociation, electron transfer dissociation/higher-energy collisional dissociation, and 213 nm ultraviolet photodissociation─in their capability to characterize a therapeutic monoclonal antibody, trastuzumab, starting from denatured and native-like precursor ions. Interestingly, native top-down mass spectrometry results in higher sequence coverage than the experiments carried out under denaturing conditions, with the exception of ultraviolet photodissociation. Globally, electron transfer dissociation followed by collision-based activation of product ions generates the largest number of backbone cleavages in disulfide protected regions, including the complementarity determining regions, regardless of electrospray ionization conditions. Overall, these findings suggest that native mass spectrometry can certainly be used for the gas-phase sequencing of whole monoclonal antibodies, although the dissociation of denatured precursor ions still returns a few backbone cleavages not identified in native experiments. Finally, a comparison of the fragmentation maps obtained under denaturing and native conditions strongly points toward disulfide bonds as the primary reason behind the largely overlapping dissociation patterns.

Analysis of therapeutic monoclonal antibodies by imaged capillary isoelectric focusing (icIEF).

Imaged capillary isoelectric focusing (icIEF) is a preferred analytical method for determining isoelectric points (pIs) and charge heterogeneity profiles in biotherapeutic proteins. In this study, we optimized the icIEF method for an in-house IgG1κ monoclonal antibody (mAb-1) and assessed its reproducibility, robustness, and autosampler stability. The optimized method was used to evaluate batch-to-batch consistency in pIs for multiple lots of mAb-1 and determine the relative percentages of charge variants. We also tested the method's performance using multiple lots of another IgG1 mAb, commercially available as Herceptin (trastuzumab). Additionally, we designed and assessed native and denaturing platform icIEF methods for 11 other marketed mAbs, with pIs ranging from 6.0 (eculizumab) to 9.22 (tocilizumab).

Comparative analysis of Herceptin N-Linked glycosylation by HILIC-FLD and LC-MS/MS methods.

Monoclonal antibodies like Herceptin play a pivotal role in modern therapeutics, with their glycosylation patterns significantly influencing their bioactivity. To characterize the N-glycan profile and their relative abundance in Herceptin, we employed two analytical methods: hydrophilic interaction chromatography with fluorescence detection (HILIC-FLD) for released glycans and liquid chromatography tandem mass spectrometry (LC-MS/MS) for glycopeptides. Our analysis included 21 European Union (EU)-Herceptin lots and 14 United States (US)-Herceptin lots. HILIC-FLD detected 25 glycan species, including positional isomers, revealing comparable chromatographic profiles for both EU and US lots. On the other hand, LC-MS/MS identified 26 glycoforms within the glycopeptide EEQYNSTYR. Both methods showed that a subset of glycans dominated the total abundance. Notably, EU-Herceptin lots with an expiration date of October 2022 exhibited increased levels of afucosylated and high mannose N-glycans. Our statistical comparisons showed that the difference in quantitative results between HILIC-FLD and LC-MS/MS is significant, indicating that the absolute quantitative values depend on the choice of the analytical method. However, despite these differences, both methods demonstrated a strong correlation in relative glycan proportions. This study contributes to the comprehensive analysis of Herceptin's glycosylation, offering insights into the influence of analytical methods on glycan quantification and providing valuable information for the biopharmaceutical industry.

Design of a Ratiometric Plasmonic Biosensor for Herceptin Detection in HER2-Positive Breast Cancer.

Breast cancer is the most common cause of cancer death in women; therefore, its early detection and treatment are crucial. To achieve this goal, we designed an optical sensor based on direct interaction of trastuzumab [Herceptin (HER)], a monoclonal antibody used to treat HER2-positive breast cancer, with plasmonic nanoparticles. Surface-modified gold nanoparticles (AuNPs) have gained considerable attention in biosensing techniques over the last years, which actuated these nanoparticles to the heart of various biosensing notions. We have exploited the localized surface plasmon resonance (LSPR) of gold nanoparticles to determine HER in human serum. AuNPs were decorated with negatively charged citrate ions, yielding enhanced direct-surface interaction with HER antibodies. The AuNPs are mixed with silver nanoparticles (AgNPs) in an optimized ratio to increase selectivity and sensitivity further. AuNPs detect the HER antibodies using LSPR, whereas AgNPs help monitor interferences' effect on the sensing media. The three effective factors in HER sensing, including the nanoparticle ratio, temperature, and pH were optimized via response surface methodology (RSM) based on the central composite design (CCD). The sensor's response toward HER was achieved in the linear range of 0.5 × 10-7 to 40 × 10-7 M with the detection limit of 3.7 × 10-9 M and relative standard deviation (RSD) less than 5%. The selectivity of the LSPR sensor was assessed by monitoring its response toward HER in the presence of other biological molecules with similar physicochemical properties. Rapid response time (less than 1 min), selectivity, and the simplicity of the developed LSPR-based sensor are the key advantages of the developed sensor.

Coupling of Trastuzumab chromatographic profiling with machine learning tools: A complementary approach for biosimilarity and stability assessment.

Biosimilar products present a growing opportunity to improve the global healthcare systems. The amount of accepted variability during the comparative assessments of biosimilar products introduces a significant challenge for both the biosimilar developers and the regulatory authorities. The aim of this study was to explore unsupervised machine learning tools as a mathematical aid for the interpretation and visualization of such comparability under control and stress conditions using data extracted from high throughput analytical techniques. For this purpose, a head-to-head analysis of the physicochemical characteristics of three Trastuzumab (TTZ) approved biosimilars and the originator product (Herceptin®) was performed. The studied quality attributes included the primary structure and identity by peptide mapping (PM) with reversed-phase chromatography-UV detection, size and charge profiles by stability-indicating size exclusion and cation exchange chromatography. Stress conditions involved pH and thermal stress. Principal component analysis (PCA) and two of the widely used cluster analysis tools, namely, K-means and Density-based Spatial Clustering of Applications with Noise (DBSCAN), were explored for clustering and feature representation of varied analytical datasets. It has been shown that the clustering patterns delineated by the used algorithms changed based on the included chromatographic profiles. The applied data analysis tools were found effective in revealing patterns of similarity and variability between i) intact and stressed as well as ii) originator and biosimilar samples.

Quantification of HER2 in COS7 cells using quantum weak measurement.

A Mach-Zehnder interferometer system based on weak measurement was set up to determinate the concentration variation of molecule by measuring the phase difference change between the two optical paths. The spectrum of the light was recorded to monitor the concentration of trastuzumab (Herceptin), which is a humanised monoclonal antibody, targeted to human epidermal growth factor receptor 2 (HER2). The trastuzumab targeting to HER2 was real-time detected and continuously monitored, the HER2 numbers of COS7 cells on a coverslip was determined at pico-molar level. Our weak measurement enabled method proposes an alternative approach for the concentration detection of molecules, providing a promising functional tool for the quantification of HER2 in cancer cells, possibly promoting fields such as the diagnosis and treatment of cancer.

Collecting antibodies and large molecule biomarkers in mouse interstitial brain fluid: a comparison of microdialysis and cerebral open flow microperfusion.

The determination of concentrations of large therapeutic molecules, like monoclonal antibodies (mAbs), in the interstitial brain fluid (ISF) is one of the cornerstones for the translation from preclinical species to humans of treatments for neurodegenerative diseases. Microdialysis (MD) and cerebral open flow microperfusion (cOFM) are the only currently available methods for extracting ISF, and their use and characterization for the collection of large molecules in rodents have barely started. For the first time, we compared both methods at a technical and performance level for measuring ISF concentrations of a non-target-binding mAb, trastuzumab, in awake and freely moving mice. Without correction of the data for recovery, concentrations of samples are over 10-fold higher through cOFM compared to MD. The overall similar pharmacokinetic profile and ISF exposure between MD (corrected for recovery) and cOFM indicate an underestimation of the absolute concentrations calculated with in vitro recovery. In vivo recovery (zero-flow rate method) revealed an increased extraction of trastuzumab at low flow rates and a 6-fold higher absolute concentration at steady state than initially calculated with the in vitro recovery. Technical optimizations have significantly increased the performance of both systems, resulting in the possibility of sampling up to 12 mice simultaneously. Moreover, strict aseptic conditions have played an important role in improving data quality. The standardization of these complex methods makes the unraveling of ISF concentrations attainable for various diseases and modalities, starting in this study with mAbs, but extending further in the future to RNA therapeutics, antibody-drug conjugates, and even cell therapies.

Characterization of Glycosylated Proteins at Subunit Level by HILIC/MS.

Hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry (MS) is considered as the reference analytical technique for glycans profiling, especially for the characterization of glycosylated protein therapeutics such as monoclonal antibodies (mAbs) and mAbs-related products. Although HILIC/MS is mainly known to profile enzymatically released and fluorescently labeled N-glycans, the recent commercialization of new widepore HILIC amide bonded stationary phases packed with sub-2 µm particles has allowed for remarkable separations also at the subunit level. Here, we describe a simple protocol to perform the mAb glycans profiling at subunit level by HILIC/MS.

Platform Methods to Characterize the Charge Heterogeneity of Three Common Protein Therapeutics by Imaged Capillary Isoelectric Focusing.

Imaged capillary isoelectric focusing (icIEF) is a gold standard method for characterizing the charge heterogeneity of protein therapeutics. A broad range of protein therapeutics such as monoclonal antibodies, antibody-drug conjugates (ADCs), and fusion proteins are routinely analyzed by icIEF due to its high resolution and high reproducibility. Platform methods, which can be applied without modification to the analysis of different protein therapeutics, save valuable time and resources in method development and quality control. Here, we provide platform methods for icIEF analysis of three classes of protein therapeutics, a biosimilar to the monoclonal antibody trastuzumab, recombinant human erythropoietin (rhEPO), and a fusion protein. The details of sample preparation and separation conditions for each molecule are described in this chapter.

Optimisation of the use of sliding window deconvolution for comprehensive characterisation of trastuzumab and adalimumab charge variants by native high resolution mass spectrometry.

The biopharmaceutical industry continues to develop mAb-based biotherapeutics in increasing numbers. Due to their complexity, there are several critical quality attributes (CQAs) that need to be measured and controlled to guarantee product safety and efficacy. Charge variant analysis is a widely used method to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs) and, together with a bottom-up peptide centred approach, acts as a key analytical platform to fulfil regulatory requirements. Native MS measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact proteins such as mAbs. The resulting native mass spectrum of a mAb is characterized by a narrower charge-state envelope that simplifies the spectra and also condenses the ion signals into fewer peaks, increasing the signal-to-noise ratio. Algorithmic spectral deconvolution is needed for routine accurate and rapid molecular weight determination, and consequently, multiple deconvolution algorithms have evolved over the past decade. Here, we demonstrate the utility of the sliding window algorithm as a robust and powerful deconvolution tool for comprehensive characterisation of charge variant analysis data for mAbs. Optimum performance is evaluated by studying the impact of critical software parameters on detection, identification and relative quantitation of protein isoforms. By combining molecular mass and retention time information, it was possible to identify multiple modifications on adalimumab and trastuzumab, both IgG1 mAbs, including lysine truncation, deamidation and succinimide formation, along with the N-glycan distribution of each of the identified charge variants. Sliding window deconvolution also provides a key benefit of low abundant variant detection in a single analysis and the ability to detect co-eluting components with different relative abundances. The studied mAbs demonstrate the algoritms applicability for efficient data processing of both simple and complex mAbs analysed using pH gradient cation exchange chromatography coupled to native mass spectrometry.

Using differential scanning calorimetry for the development of non-reduced capillary electrophoresis sodium dodecyl sulfate methods for monoclonal antibodies.

Analysis of non-reduced and reduced monoclonal antibodies (mAbs) by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is routinely used to detect product size variants and process-related impurities. Levels of high molecular weight (HMW) forms obtained from this method usually trend comparably to those obtained by orthogonal methods such as size-exclusion ultra-high performance liquid chromatography (SE-UHPLC). However, in the presented case study, comparison of CE-SDS data for three IgG1 mAbs (trastuzumab, mAb1, and mAb2) showed a discrepancy between amounts of observed HMW forms in mAb2 compared with its native forms determined by SE-UHPLC (~17% vs. ~0.5%, respectively). SDS chemical denaturation, as measured by differential scanning calorimetry, demonstrated that the high thermal stability of mAb2 caused an unidentified HMW peak observed by non-reduced (NR)-CE-SDS, which was the result of improper denaturing, resulting in a partially folded species. More so, this strategy enabled the rapid identification of optimal SDS concentration and temperature conditions needed for suitable denaturation for mAb2. This case study presents an alternative option for quick optimization of NR-CE-SDS methods when characterizing mAbs or other thermally stable proteins. Also, this strategy can be used to understand basic biophysical mechanisms of protein unfolding and investigate the higher-order structure imparted by specific sequences and understand how these sequences might affect the results of an analytical method such as CE-SDS.

Aptamers as Versatile Molecular Tools for Antibody Production Monitoring and Quality Control.

Antibody drugs have been used to treat many diseases, and to date, this has been the most rapidly growing drug class. However, the lack of suitable methods for real-time and high-throughput monitoring of antibody production and quality control has been a hindrance to the further advancement of antibody drugs or biosimilars. Therefore, we herein report a versatile tool for one-step fluorescence monitoring of antibody production by using aptamer probes selected through the in vitro SELEX method. In this case, DNA aptamers were selected against the humanized IgG1 antibody drug trastuzumab with high specificity and affinity with a Kd value of aptamer CH1S-3 of 10.3 nM. More importantly, the obtained aptamers were able to distinguish native from heat-treated, whereas antibodies failed this test. On the basis of the advantages of rapid detection for aptamers, we designed aptamer molecular beacons for direct and sensitive detection of trastuzumab in complex samples. Unlike traditional antibody-based ELISA, the signal was observed directly upon interaction with the target without the need for time-consuming binding and multiple washing steps. To further highlight biomedical applications, the use of aptamers as potential tools for quality control and traceless purification of antibody drugs was also demonstrated. Thus, aptamers are shown to be promising alternatives for antibody production monitoring, quality control, and purification, providing technical support to accelerate antibody drug development.

An application of Nano Differential Scanning Fluorimetry for Higher Order Structure assessment between mAb originator and biosimilars: Trastuzumab and Rituximab as case studies.

Differential scanning fluorimetry (DSF) or thermal shift has emerged in recent years as a high-throughput screening method in biotherapeutic formulation studies. The present article reports on a fast-track assessment platform for rapid investigation of therapeutic proteins such as monoclonal antibodies (mAb) with minimal sample concentration, volume, and preparation. The proposed nanoDSF platform has been demonstrated for rapid assessment of two commercial IgG 1 drug products (DP), trastuzumab and rituximab, and their biosimilars with respect to their conformational and colloidal stability. Domain specific differences for each of the IgGs have been elucidated with respect to onset of domain unfolding (Tonset) and melting temperatures. These thermal unfolding and transition midpoint (Tm) measurements are based on the intrinsic aromatic amino acid residue fluorescence of proteins. Moreover, to understand the possibility of nanoDSF as a predictive tool, data from nanoDSF has been correlated with accelerated stability studies. Melting temperatures across brands were found to be highly comparable to the rate of heating, thereby exhibiting a significant domain specific effect on melting temperatures for both trastuzumab and rituximab. Conservation of higher order structure (HOS) through reversible unfolding was also examined and both the mAbs were found to regain tertiary structure up till the first transition midpoint. No clear correlation was found between formation of higher molecular weight species (HMWS) and unfolding parameters (Tonset and Tagg) for accelerated stability studies. Finally, a discussion on the need for fast predictive assessment of conformation and colloidal stability as well as a comparison of advantages and limitations of the technique with routine/classical tools such as circular dichroism spectrophotometry and differential scanning calorimetry has been presented.

Detection of doxorubicin, cisplatin and therapeutic antibodies in formalin-fixed paraffin-embedded human cancer cells.

A major limitation in the pharmacological treatment of clinically detectable primary cancers and their metastases is their limited accessibility to anti-cancer drugs (cytostatics, inhibitory antibodies, small-molecule inhibitors) critically impairing therapeutic efficacies. Investigations on the tissue distribution of such drugs are rare and have only been based on fresh frozen material or methanol-fixed cell culture cells so far. In this paper, we expand the detection of cisplatin-induced DNA adducts and anthracyclines as well as therapeutic antibodies to routinely prepared formalin-fixed, paraffin-embedded sections (FFPE). Using pre-treated cell lines prepared as FFPE samples comparable to tissues from routine analysis, we demonstrate that our method allows for the detection of chemotherapeutics (anthracyclines by autofluorescence, cisplatin by immune detection of DNA adducts) as well as therapeutic antibodies. This methodology thus allows for analyzing archival FFPE tissues, as demonstrated here for the detection of cisplatin, doxorubicin and trastuzumab in FFPE sections of tumor xenografts from drug-treated mice. Analyzing human tumor samples, this will lead to new insights into the tissue penetration of drugs.

Conformation assessment of therapeutic monoclonal antibodies by SEC-MS: Unravelling analytical biases for application to quality control.

Immunogenicity related to the degradation of therapeutic monoclonal antibodies (mAbs) remains a major concern for their therapeutic efficacy and safety. Therefore, an analytical method allowing characterization and detection of mAbs degradation is mandatory. In this study, a simultaneous coupling of size exclusion chromatography (SEC) to native mass spectrometry (MS) and fluorescence detection (FLD) is proposed to detect degraded therapeutic mAbs and biases of structural changes (e.g. dimerization, denaturation) that may occur during native MS. A comprehensive study on infliximab behaviors have been performed under different mobile phase conditions (e.g. composition, pH, organic solvent, etc.) and MS parameters (e.g. gas temperatures, CID energies, etc.). Experimental conditions avoiding artificial denaturation and/ or dimerization have been defined. We have also demonstrated that under the developed conditions infliximab affinity towards its biological target TNFα is preserved. In addition, using this method dimers, denatured monomers and fragments could be detected in trastuzmab samples stressed by a long-term storage. These results were confirmed by using SEC coupled to ion mobility mass spectrometry as an orthogonal method for the detection of denatured monomer.

In-depth analysis of monoclonal antibodies using microfluidic capillary electrophoresis and native mass spectrometry.

Charge variant profiling of therapeutic proteins is required by the International Council for Harmonisation guidelines and is traditionally performed by capillary electrophoresis or ion exchange chromatography. Recently, improvements in the hyphenation of capillary electrophoresis with mass spectrometry and the introduction of mass spectrometry compatible background electrolytes has allowed the implementation of native mass spectrometric determination of the charge variant profile obtained from the electrophoretic separation. The low flow operation of the microfluidic electrophoretic platform significantly boosts mass spectrometric sensitivity and increases the dynamic range, even when using sample amounts as low as 1 ng in capillary. In the current study, rituximab, trastuzumab and bevacizumab drug products were analysed using the ZipChip microfluidic CE-ESI-MS platform that facilitated confident identification of proteoforms with an average mass accuracy of <15 ppm. Up to 52 proteoforms were identified for trastuzumab drug product, while rituximab sample revealed the presence of fragments and sialylated N-glycans. Overall, the CE-ESI-MS platform proved to be a fast and robust tool for therapeutic protein charge variant profiling and facilitated efficient coupling with native mass spectrometry for the generation of highly informative characterisation data.

Improving the throughput of immunoaffinity purification and enzymatic digestion of therapeutic proteins using membrane-immobilized reagent technology.

Continued interest in protein therapeutics has motivated the development of improved bioanalytical tools to support development programs. LC-MS offers specificity, sensitivity, and multiplexing capabilities without the need for target-specific reagents, making it a valuable alternative to ligand binding assays. Immunoaffinity purification (IP) and enzymatic digestion are critical, yet extensive and time-consuming components of the "gold standard" bottom-up approach to LC-MS-based protein quantitation. In the present work, commercially available technology, based on membrane-immobilized reagents in spin column and plate format, is applied to reduce IP and digestion times from hours to minutes. For a standard monoclonal antibody, the lower limit of quantitation was 0.1 ng µL-1 compared to 0.05 ng µL-1 for the standard method. A pharmacokinetics (PK) study dosing Herceptin in rat was analyzed by both the membrane and the standard method with a total sample processing time of 4 h and 20 h, respectively. The calculated concentrations at each time point agreed within 8% between both methods, and PK values including area under the curve (AUC), half-life (T1/2), mean residence time (MRT), clearance (CL), and volume of distribution (Vdss) agreed within 6% underscoring the utility of the membrane methodology for quantitative bioanalysis workflows.

Demonstrating Analytical Similarity of Trastuzumab Biosimilar HLX02 to Herceptin® with a Panel of Sensitive and Orthogonal Methods Including a Novel FcγRIIIa Affinity Chromatography Technology.

BACKGROUND: A biosimilar needs to demonstrate its similarity to the originator reference product (RP) in terms of structural and functional properties as well as nonclinical and clinical outcomes. OBJECTIVES: The aim was to assess the analytical similarity between the trastuzumab biosimilar HLX02 and Europe-sourced Herceptin® (EU-Herceptin®) and China-sourced Herceptin® (CN-Herceptin®) following a quality-by-design (QbD) quality study and tier-based quality attribute evaluation. METHODS: A panel of highly sensitive and orthogonal methods, including a novel Fc gamma receptor IIIa (FcγRIIIa) affinity chromatography technique that enables quantitative comparison of glycan effects on effector function, was developed for the assessment. To ensure the full product variability was captured, ten batches of HLX02 were compared with 39 RP batches with expiry dates from August 2017 to March 2021. RESULTS: The extensive three-way similarity assessment demonstrated that HLX02 is highly similar to the RPs. Furthermore, the %afucose, %galactose, and FcγRIIIa affinity of the RPs were observed to first decrease and then return to the original level in relation to their expiry dates, and the RP batches can be subgrouped by their FcγRIIIa affinity chromatograms. HLX02 is demonstrated to be more similar to the RPs of the high FcγRIIIa affinity group. CONCLUSION: Besides having an overall high analytical similarity to both EU-Herceptin® and CN-Herceptin®, HLX02 is more similar to Herceptin® with high FcγRIIIa affinity, a result that demonstrates the power of the novel FcγRIIIa affinity chromatography technology in biosimilarity evaluation.