Conversion of a CHO cell culture process from perfusion to fed-batch technology without altering product quality

During the development of a new drug product, it is a common strategy to develop a first-generation process with the aim to rapidly produce material for pre-clinical and early stage clinical trials. At a later stage of the development, a second-generation process is then introduced with the aim to supply late-stage clinical trials as well as market needs. This work was aimed at comparing the performance of two different CHO cell culture processes (perfusion and fed-batch) used for the production of a therapeutically active recombinant glycoprotein at industrial pilot-scale. The first-generation process was based on the Fibra-Cel packed-bed perfusion technology. It appeared during the development of the candidate drug that high therapeutic doses were required (>100mg per dose), and that future market demand would exceed 100 kg per year. This exceeded by far the production capacity of the first-generation process, and triggered a change of technology from a packed-bed perfusion process with limited scale-up capabilities to a fed-batch process with scale-up potential to typical bioreactor sizes of 15m(3) or more. The productivity per bioreactor unit volume (in product m(-3)year(-1)) of the fed-batch process was about 70% of the level reached with the first-generation perfusion process. However, since the packed-bed perfusion system was limited in scale (0.6m(3) maximum) compared to the volumes reached in suspension cultures (15m(3)), the fed-batch was selected as second-generation process. In fact, the overall process performance (in product year(-1)) was about 18-fold higher for the fed-batch compared to the perfusion mode. Data from perfusion and fed-batch harvests samples indicated that comparable product quality (relative abundance of monomers dimers and aggregates; N-glycan sialylation level; isoforms distribution) was obtained in both processes. To further confirm this observation, purification to homogeneity of the harvest material from both processes, followed by a complementary set of studies (e.g. full physico-chemical characterization, assessment of in vitro and in vivo bioactivity, comparative pharmacokinetics and pharmacodynamics studies in relevant species, etc.) would be required. Finally, this illustrates the need to fix the production process early during the development of a new drug product in order to minimize process conversion efforts and to shorten product development time lines.     

Analytical comparability study of recombinant monoclonal antibody therapeutics

Process changes are inevitable in the life cycle of recombinant monoclonal antibody therapeutics. Products made using pre- and post-change processes are required to be comparable as demonstrated by comparability studies to qualify for continuous development and commercial supply. Establishment of comparability is a systematic process of gathering and evaluating data based on scientific understanding and clinical experience of the relationship between product quality attributes and their impact on safety and efficacy. This review summarizes the current understanding of various modifications of recombinant monoclonal antibodies. It further outlines the critical steps in designing and executing successful comparability studies to support process changes at different stages of a product's lifecycle.     

Robust factor selection in early cell culture process development for the production of a biosimilar monoclonal antibody

This work presents a multivariate methodology combining principal component analysis, the Mahalanobis distance and decision trees for the selection of process factors and their levels in early process development of generic molecules. It is applied to a high throughput study testing more than 200 conditions for the production of a biosimilar monoclonal antibody at microliter scale. The methodology provides the most important selection criteria for the process design in order to improve product quality towards the quality attributes of the originator molecule. Robustness of the selections is ensured by cross‐validation of each analysis step. The concluded selections are then successfully validated with an external data set. Finally, the results are compared to those obtained with a widely used software revealing similarities and clear advantages of the presented methodology. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:181–191, 2017     

A systematic approach to the validation of process control parameters for monoclonal antibody production in fed-batch culture of a murine myeloma

A systematic approach to the validation of control ranges of control parameters for a cell culture process producing a monoclonal antibody is described. Specifically, the structure and functional activity of a monoclonal IgG1 antibody produced at the outer limits of numerical ranges of fed-batch culture control parameters such as pH and temperature were examined, with the aim of providing assurance that antibody produced under varying culture conditions was of consistent quality based on a carefully defined set of specifications. An experimental design was created using a half-fractional factorial design for fed-batch culture incorporating half of the thirty two possible combinations of five selected control parameters at high and low levels. Statistical analysis of all data gathered from the study allowed an assessment of the effects of the process control parameters at either high or low outer limits on fed-batch culture response variables such as growth rate and specific antibody productivity. Measured values for the responses of growth rate and specific antibody productivity throughout this study ranged from 0.22-0.44 d(-1) and 6.4-32 microg monoclonal antibody/10(6) cells/d respectively. Analytical characterisation of monoclonal antibody purified from each fed-batch culture considered the purity, structure and biological activity of the glycoprotein. All antibody preparations were identical to each other and to the current antibody reference standard or control. Glycosylation analysis of certain samples from the study demonstrated that the distribution of glycoforms of the antibody was not affected by the varying process control conditions of the fed-batch cultures.     

Waiving in vivo studies for monoclonal antibody biosimilar development: National and global challenges

Biosimilars are biological medicinal products that contain a version of the active substance of an already authorised original biological medicinal product (the innovator or reference product). The first approved biosimilar medicines were small proteins, and more recently biosimilar versions of innovator monoclonal antibody (mAb) drugs have entered development as patents on these more complex proteins expire. In September 2013, the first biosimilar mAb, infliximab, was authorised in Europe. In March 2015, the first biosimilar (Zarxio?, filgrastim-sndz, Sandoz) was approved by the US Food and Drug Administration; however, to date no mAb biosimilars have been approved in the US. There are currently major differences between how biosimilars are regulated in different parts of the world, leading to substantial variability in the amount of in vivo nonclinical toxicity testing required to support clinical development and marketing of biosimilars. There are approximately 30 national and international guidelines on biosimilar development and this number is growing. The European Union's guidance describes an approach that enables biosimilars to enter clinical trials based on robust in vitro data alone; in contrast, the World Health Organization's guidance is interpreted globally to mean in vivo toxicity studies are mandatory.

We reviewed our own experience working in the global regulatory environment, surveyed current practice, determined drivers for nonclinical in vivo studies with biosimilar mAbs and shared data on practice and study design for 25 marketed and as yet unmarketed biosimilar mAbs that have been in development in the past 5y. These data showed a variety of nonclinical in vivo approaches, and also demonstrated the practical challenges faced in obtaining regulatory approval for clinical trials based on in vitro data alone. The majority of reasons for carrying out nonclinical in vivo studies were not based on scientific rationale, and therefore the authors have made recommendations for a data-driven approach to the toxicological assessment of mAb biosimilars that minimises unnecessary use of animals and can be used across all regions of the world.     

Enhanced expression of a biosimilar monoclonal antibody with a novel NS0 platform

The precise product quality and lower cost of goods demands of the growing biosimilars industry are driving biomanufacturing innovation. Biosimilar cell lines that produce complex glycoproteins such as monoclonal antibodies must be both highly productive and express a product with critical quality attributes closely matching those of the innovator reference. In this work, a biomanufacturing platform is described that harnesses the commercially‐established NS0 host cell in new ways to create stable, highly productive cell lines with characteristics meeting the current demands. A cholesterol metabolic selection marker and implementation strategy that can be generically applied are shown to yield high expressing cell lines as well as eliminate the need for cholesterol addition, which has been a significant barrier in both stainless steel reactors as well as in single‐use plastic systems. Additionally, for the first time, a multiplex selection strategy was implemented that served to increase NS0 cell line specific productivity >10‐fold and volumetric yields >6‐fold. The best overall performing cell line had a Qp of 28.5 picograms per cell per day and was rapidly adapted to a lean production medium. Yields in l ‐glutamine fed‐batch shaker cultures exceeded 500 mg/L. An initial screening of four feeding strategies resulted in a final 13‐day yield of over 1.4 g/L in small shaker culture. Overall, this work shows both the strategy to develop biosimilar cell lines and the commercial potential of a novel expression system highly suited for the manufacture of biosimilars of reference biologics currently produced in murine cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:455–462, 2018     

Impact of cell culture on recombinant monoclonal antibody product heterogeneity

Recombinant monoclonal antibodies are commonly expressed in mammalian cell culture and purified by several steps of filtration and chromatography. The resulting high purity bulk drug substance still contains product variants differing in properties such as charge and size. Posttranslational modifications and degradations occurring during cell culture are the major sources of heterogeneity in bulk drug substance of recombinant monoclonal antibodies. The focus of the current review is the impact of cell culture conditions on the types and levels of various modifications and degradations of recombinant monoclonal antibodies. Understanding the relationship between cell culture and product variants can help to make consistently safe and efficacious products. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1103–1112, 2016     

Effect of ambient light on monoclonal antibody product quality during small‐scale mammalian cell culture process in clear glass bioreactors

During a small‐scale cell culture process producing a monoclonal antibody, a larger than expected difference was observed in the charge variants profile of the harvested cell culture fluid (HCCF) between the 2 L and larger scales (e.g., 400 L and 12 kL). Small‐scale studies performed at the 2 L scale consistently showed an increase in acidic species when compared with the material made at larger scale. Since the 2 L bioreactors were made of clear transparent glass while the larger scale reactors are made of stainless steel, the effect of ambient laboratory light on cell culture process in 2 L bioreactors as well as handling the HCCF was carefully evaluated. Photoreactions in the 2 L glass bioreactors including light mediated increase in acidic variants in HCCF and formulation buffers were identified and carefully analyzed. While the acidic variants comprised of a mixture of sialylated, reduced disulfide, crosslinked (nonreducible), glycated, and deamidated forms, an increase in the nonreducible forms, deamidation and Met oxidation was predominantly observed under light stress. The monoclonal antibody produced in glass bioreactors that were protected from light behaved similar to the one produced in the larger scale. Our data clearly indicate that care should be taken when glass bioreactors are used in cell culture studies during monoclonal antibody production. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:562–570, 2014     

Fed-batch bioreactor process scale-up from 3-L to 2,500-L scale for monoclonal antibody production from cell culture

This case study focuses on the scale-up of a Sp2/0 mouse myeloma cell line based fed-batch bioreactor process, from the initial 3-L bench scale to the 2,500-L scale. A stepwise scale-up strategy that involved several intermediate steps in increasing the bioreactor volume was adopted to minimize the risks associated with scale-up processes. Careful selection of several available mixing models from literature, and appropriately applying the calculated results to our settings, resulted in successful scale-up of agitation speed for the large bioreactors. Consideration was also given to scale-up of the nutrient feeding, inoculation, and the set-points of operational parameters such as temperature, pH, dissolved oxygen, dissolved carbon dioxide, and aeration in an integrated manner. It has been demonstrated through the qualitative and the quantitative side-by-side comparison of bioreactor performance as well as through a panel of biochemical characterization tests that the comparability of the process and the product was well controlled and maintained during the process scale-up. The 2,500-L process is currently in use for the routine clinical production of Epratuzumab in support of two global Phase III clinical trials in patients with lupus. Today, the 2,500 L, fed-batch production process for Epratuzumab has met all scheduled batch releases, and the quality of the antibody is consistent and reproducible, meeting all specifications, thus confirming the robustness of the process.     

Recovery and purification process development for monoclonal antibody production

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography, and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation, and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs.     

Recovery and purification process development for monoclonal antibody production

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs.Key words: monoclonal antibody, recovery, purification, chromatography, membrane, filtration, platform process     

Defining process design space for monoclonal antibody cell culture

The concept of design space has been taking root as a foundation of in‐process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non‐key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product‐related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified. Biotechnol. Bioeng. 2010;106: 894–905. © 2010 Wiley Periodicals, Inc.     

Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies

This study shows that state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) can be used for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for antibody products. A candidate biosimilar IgG1 monoclonal antibody (mAb) was compared in detail to a commercially available innovator product. Intact protein mass, primary sequence, PTMs, and the micro-differences between the two mAbs were identified and quantified simultaneously. Although very similar in terms of sequences and modifications, a mass difference observed by LC-MS intact mass measurements indicated that they were not identical. Peptide mapping, performed with data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode (LC-MS^E), located the mass difference between the biosimilar and the innovator to a two amino acid residue variance in the heavy chain sequences. The peptide mapping technique was also used to comprehensively catalogue and compare the differences in PTMs of the biosimilar and innovator mAbs. Comprehensive glycosylation profiling confirmed that the proportion of individual glycans was different between the biosimilar and the innovator, although the number and identity of glycans were the same. These results demonstrate that the combination of accurate intact mass measurement, released glycan profiling, and LC-MS^E peptide mapping provides a set of routine tools that can be used to comprehensively compare a candidate biosimilar and an innovator mAb.     

Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies

This study shows that state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) can be used for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for antibody products. A candidate biosimilar IgG1 monoclonal antibody (mAb) was compared in detail to a commercially available innovator product. Intact protein mass, primary sequence, PTMs and the micro-differences between the two mAbs were identified and quantified simultaneously. Although very similar in terms of sequences and modifications, a mass difference observed by LC-MS intact mass measurements indicated that they were not identical. Peptide mapping, performed with data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode (LC-MS^E), located the mass difference between the biosimilar and the innovator to a two amino acid residue variance in the heavy chain sequences. The peptide mapping technique was also used to comprehensively catalogue and compare the differences in PTMs of the biosimilar and innovator mAbs. Comprehensive glycosylation profiling confirmed that the proportion of individual glycans was different between the biosimilar and the innovator, although the number and identity of glycans were the same. These results demonstrate that the combination of accurate intact mass measurement, released glycan profiling and LC-MS^E peptide mapping provides a set of routine tools that can be used to comprehensively compare a candidate biosimilar and an innovator mAb.Key words: biosimilar mAb, innovator mAb, molecular similarity, sequence variants, posttranslational modifications, N-linked glycosylation, chemical degradations, micro-heterogeneities, characterization, intact protein mass measurement, peptide mapping, glycan profiling, LC-MS, LC-fluorescence, MALDI MS     

Dynamic analysis of GS-NS0 cells producing a recombinant monoclonal antibody during fed-batch culture

In this study we have analyzed the dynamic covariation of the mammalian cell proteome with respect to functional phenotype during fed-batch culture of NS0 murine myeloma cells producing a recombinant IgG(4) monoclonal antibody. GS-NS0 cells were cultured in duplicate 10 L bioreactors (36.5 degrees C, 15% DOT, pH 7.0) for 335 h and supplemented with a continuous feed stream after 120 h. Cell-specific growth rate declined continuously after 72 h of culture. Cell-specific recombinant monoclonal antibody production rate (qP) varied sixfold through culture. Whilst qP correlated with relative recombinant heavy chain mRNA abundance up to 216 h, qP subsequently declined, independent of recombinant heavy chain or light chain mRNA abundance. GS-NS0 cultures were sampled at 48 h intervals between 24 and 264 h of culture for proteomic analyses. Total protein abundance and nascent polypeptide synthesis was determined by 2D PAGE of unlabeled proteins visualized by SYPRO Ruby and autoradiography of (35)S-labeled polypeptides, respectively. Covariation of nascent polypeptide synthesis and abundance with biomass-specific cell growth, glucose and glutamate consumption, lactate and Mab production rates were then examined using two partial least squares regression models. Most changes in polypeptide synthesis or abundance for proteins previously identified by mass spectrometry were positively correlated with biomass-specific growth rate. We conclude that the substantial transitions in cell physiology and qP that occur during culture utilize a relatively constant complement of the most abundant host cell machines that vary primarily with respect to induced changes in cell growth rate.     

Analysis of monoclonal antibody product heterogeneity resulting from alternate cleavage sites of signal peptide

Signal peptides used in biosynthesis of proteins are cleaved at a very specific site by signal peptidase during posttranslational translocation of cytoplasmic proteins across the membrane. In some cases, however, there can be cleavage at nonspecific sites, giving rise to heterogeneity in the mature protein, which manifests itself as either elongation or truncation of the N terminus of the mature protein. When used as biopharmaceutical therapeutics, such heterogeneities may be a cause for concern, depending on the nature of the heterogeneity. This article describes the determination of such heterogeneity by peptide mapping in both the heavy chain and the light chain (LC) of a Chinese hamster ovary (CHO) cell-expressed monoclonal antibody (mAb). The peptide map method described here was capable of detecting the extended N-terminal peptides at levels as low as 1% relative to the peak area of the intact N-terminal peptide. The LC of a mAb product was truncated at its N termini by two amino acid residues at approximately 3-4% levels, resulting from alternate signal peptide cleavage. This article describes the quantitation of this truncation by liquid chromatography-mass spectrometry (LC-MS) peptide mapping. Also described is analysis and characterization of LC truncation by reduced and denatured capillary electrophoresis in sodium dodecyl sulfate (CE-SDS). The truncated mAb, which was devoid of the two N-terminal amino acids, was engineered and shown to migrate as the “pre-LC” peak in reduced CE-SDS assay. The amount of the pre-LC peak recovered from the CE-SDS assay was shown to correlate with the amount of truncated peptide observed from the reduced and alkylated peptide map of the engineered mAb.     

天然产物莪术醇单克隆抗体的制备

为制备小分子化合物莪术醇的单克隆抗体,先将莪术醇(curcumol)与载体蛋白牛血清蛋白(BSA)偶联形成完全抗原,用基质辅助激光解吸飞行时间质谱法(MALDI-TOF-MS)鉴定莪术醇人工抗原的偶联率,然后采用杂交瘤技术获得杂交瘤株,并对其进行小鼠腹水的制备与纯化.结果表明:莪术醇半抗原与载体的偶联比为19.6,单克...     

An innovative approach for the characterization of the isoforms of a monoclonal antibody product

Protein biopharmaceuticals, such as monoclonal antibodies (mAbs) are widely used for the prevention and treatment of various diseases. The complex and lengthy upstream and downstream production methods of the antibodies make them susceptible to physical and chemical modifications. Several IgG1 immunoglobulins are used as medical agents for the treatment of colon, breast, and head and neck cancers, and at least four to eight isoforms exist in the products. The regulatory agencies understand the complex nature of the antibody molecules and allow the manufactures to set their own specifications for lot release, provided the safety and efficacy of the products are established in animal models prior to clinical trials. During the manufacture of a mAb product, we observed lot-to-lot variability in the isoform content and, although the variability is within the set specifications for lot release, made attempts to gain mechanistic insight by isolating and characterizing the individual isoforms. Matrix-assisted laser desorption/ionization (MALDI) and liquid chromatography (LC)/mass spectrometry (MS)/MS analyses of the isolated isoforms indicate that this variability is caused by sialic acid content, as well as truncation of C-terminal lysine of the individual isoforms. Sialidase and carboxypeptidase treatment of the product confirm the observations made by MALDI and LC/MS/MS.     

An innovative approach for the characterization of the isoforms of a monoclonal antibody product

Protein biopharmaceuticals, such as monoclonal antibodies (mAbs) are widely used for the prevention and treatment of various diseases. The complex and lengthy upstream and downstream production methods of the antibodies make them susceptible to physical and chemical modifications. Several IgG1 immunoglobulins are used as medical agents for the treatment of colon, breast and head and neck cancers, and at least four to eight isoforms exist in the products. The regulatory agencies understand the complex nature of the antibody molecules and allow the manufactures to set their own specifications for lot release, provided the safety and efficacy of the products are established in animal models prior to clinical trials. During the manufacture of a mAb product, we observed lot-to-lot variability in the isoform content and, although the variability is within the set specifications for lot release, made attempts to gain mechanistic insight by isolating and characterizing the individual isoforms. Matrix-assisted laser desorption/ionization (MALDI) and liquid chromatography (LC)/mass spectrometry (MS)/MS analyses of the isolated isoforms indicate that this variability is caused by sialic acid content, as well as truncation of C-terminal lysine of the individual isoforms. Sialidase and carboxypeptidase treatment of the product confirm the observations made by MALDI and LC/MS/MS.Key words: IgG1, isoforms, charge heterogeneity, monoclonal antibody, glycosylation, silaic acidMonoclonal antibodies (mAbs) are used as medical agents to treat a variety of diseases including cancer, cardiovascular diseases and blood disorders.^1–3 Although a few IgG2 (e.g., panitumumab, denosumab) and IgG4 antibody molecules are in the market, most of the approved products are IgG1 molecules. IgG1 antibodies are glycoproteins with a conserved N-glycosylation site at Asn 297. Glycosylation influences the biological functions, such as antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) of the antibodies. The oligosaccharides present in the IgG1 molecules are heterogeneous due to the presence of various sugar residues, including sialic acid, galactose, N-acetylglucasmine and fucose residues. Molecular alterations in antibodies can take place at every stage of manufacturing: upstream and downstream processing, formulation and storage. These alterations can take place enzymatically or non-enzymatically and may produce charge or size heterogeneity. Deamidation, proteolytic fragmentation, oxidation, disulfide bond shuffling and glycosylation are the most common modifications that occur during the production of protein therapeutics.^4–7 These modifications can reduce the biological activity and may induce immunogenicity in patients. Hence, the regulatory agencies require a comprehensive characterization of the structural integrity, purity and stability of the protein therapeutics.⁸To date, eight chimeric, humanized and human IgG1 mAbs have been approved in the United States, Europe, as well as other countries, for the treatment of several types of cancers.^9–12 One such molecule produced at ImClone has two N-glycosylation sites and at least six to eight isoforms with isoelectric points (pIs) between 7.9–8.9 are present in this product. Although techniques such as ion exchange chromatography (IEX) and capillary isoelectic focusing (IEF) are available for the separation and characterization of charge varients,^13,14 we were not successful in separating the individual isoforms with these techniques from the IgG1 product used in this investigation. The peaks from IEX showed the presence of multiple bands on IEF. Hence, an alternative approach was used to isolate each isoform of this IgG1 product, and we demonstrated the involvement of sialic acid and C-terminal lysine as the root causes for lot-to-lot variation observed during the production of this molecule. The method is fast and very effective in separating isoforms with a difference in the pI values < 0.1.     

Analytical similarity assessment of rituximab biosimilar CT-P10 to reference medicinal product

CT-P10 (Truxima?) was recently approved as the world's first rituximab biosimilar product in the European Union (EU) and South Korea. To demonstrate biosimilarity of CT-P10 with the reference medicinal product (RMP), extensive 3-way similarity assessment has been conducted between CT-P10, EU-Rituximab and US-Rituximab, focusing on the physicochemical and biological quality attributes. A multitude of state-of-the-art analyses revealed that CT-P10 has identical primary and higher order structures compared to the original product. Purity/impurity profiles of CT-P10 measured by the levels of aggregates, fragments, non-glycosylated form and process-related impurities were also found to be comparable with those of RMPs. In terms of the post-translational modification, CT-P10 contains slightly less N-terminal pyro-glutamate variant, which has been known not to affect product efficacy or safety. Oligosaccharide profiling has revealed that, although CT-P10 contains the same conserved glycan species and relative proportion with the RMPs, the content of total afucosylated glycan in CT-P10 was slightly higher than in EU- or US-Rituximab. Nevertheless, the effect of the observed level of afucosylation in CT-P10 drug product on Fc receptor binding affinity or antibody-dependent cell-mediated cytotoxicity was found to be negligible based on the spiking study with highly afucosylated sample. Arrays of biological assays representative of known and putative mechanisms of action for rituximab have shown that biological activities of CT-P10 are within the quality range of RMPs. Recent results of clinical studies have further confirmed that the CT-P10 exhibits equivalent clinical efficacy and safety profiles compared to EU- and US-Rituximab. The current 3-way similarity assessment together with clinical study results confidently demonstrate that CT-P10 is highly similar with EU- and US-Rituximab in terms of physicochemical properties, biological activities, efficacy, and safety for its final approval as a biosimilar product.