Expression of recombinant anti‐colorectal cancer large single‐chain monoclonal antibody in insect cells

Colorectal cancer is the third most commonly diagnosed cancer in the world. Monoclonal antibody (mAb) CO17‐1A recognizes the tumor‐associated antigen GA733‐2, a cell surface glycoprotein highly expressed in colorectal carcinoma cells which is applicable for preventing and curing colorectal cancer. In this study, we tried to produce a new recombinant anti‐colorectal cancer large single chain (lsc) mAb based on mAb CO17‐1A in the baculovirus‐insect cell protein expression system. Two kinds of recombinant lsc mAbs were generated where variable light chain (VL) and heavy chain (HC) of mAb CO17‐1A were fused together by an interchain linker. The only difference between the two mAbs is based on fusion of an ER retention signal (KDEL) at its C‐terminus of HC. Polymerase chain reaction analysis verified the presence of both recombinant genes in the bacmid for generating viral expression vectors in insect cells. Western blot confirmed the expression of lsc mAbs in baculovirus‐infected insect cells. Cell enzyme linked immunosorbent assay (ELISA) showed that the mAbs from cell lysates bound to SW480 and SW620 human colorectal cancer cells. These results indicate that the baculovirus insect expression system can produce anti‐colorectal lsc mAb recognizing human colorectal cancer cells.     

Plant-derived mouse IgG monoclonal antibody fused to KDEL endoplasmic reticulum-retention signal is N-glycosylated homogeneously throughout the plant with mostly high-mannose-type N-glycans

Plants are potential hosts for the expression of recombinant glycoproteins intended for therapeutic purposes. However, N-glycans of mammalian glycoproteins produced in transgenic plants differ from their natural counterparts. The use of the endoplasmic reticulum (ER)-retention signal has been proposed to restrict glycosylation of plantibodies to only high-mannose-type N-glycans. Furthermore, little is known about the influence of plant development and growth conditions on N-linked glycosylation. Here, we report a detailed N-glycosylation profiling study of CB.Hep1, a mouse IgG2b monoclonal antibody (mAb) against hepatitis B surface antigen (HBsAg) currently expressed in tobacco plants (Nicotiana tabacum L.). The KDEL ER-retention signal was fused to the C-terminal of both light and heavy chains. The structures of the N-linked glycans of this mAb produced in transgenic tobacco plants at various growth stages were analysed by high-performance liquid chromatography (HPLC) profiling techniques and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and compared with those of murine origin. The high-mannose-type oligosaccharides accounted for more than 80% of the total N-glycans, with Man7GlcNAc2 being the most abundant species. Some complex N-glycans bearing xylose and small amounts of oligosaccharides with both xylose and fucose were identified. No appreciable differences were detected when comparing glycosylation at different leaf ages, e.g. from seedling leaves up to 8 weeks old and top or basal leaves of mature plants, or between leaves, stems and whole plants. A strict retention of glycoproteins to ER by the use of the tetrapeptide KDEL was not sufficient, even though the majority of the resulting N-glycosylation was of the high-mannose type. It is highly likely to be dependent on other factors, which are most probably protein specific.     

Expression of recombinant anti‐breast cancer immunotherapeutic monoclonal antibody in baculovirus–insect cell system

The anti‐breast cancer monoclonal antibody (mAb) BR55 was expressed in the baculovirus–insect cell expression system, which is advantageous because of its high production capacity, cell culture flexibility and glycosylation capability. The baculovirus–insect cell expression system was successfully established for production of mAb BR55 and mAb BR55 fused with the KDEL (Lys–Asp–Glu–Leu) endoplasmic reticulum (ER) retention signal (mAb BR55K). The heavy chain (HC) and light chain (LC) genes of mAb BR55 were cloned under the control of the polyhedrin (P_PH) and P₁₀ promoters, respectively, in the pFastBacDual vector. The antibody gene‐expression cassettes carrying both the HC and LC genes were transferred into a bacmid in Escherichia coli (DH10Bac). The bacmid carrying the expression cassettes was transfected into Sf9 insect cells to generate baculovirus expressing mAb BR55 and BR55K. Western blot analysis confirmed the expression of mAb BR55 and BR55K in baculovirus‐infected insect cells. Cell direct enzyme linked immunosorbent assay (ELISA) showed that both mAbs from insect cell lysates or cell culture medium bound to MCF‐7 human breast cancer cells. Both mAb BR55 and BR55K were successfully purified using a Protein A affinity column. Collectively, these results suggest that the anti‐breast cancer mAb BR55 can be expressed, properly assembled and purified from the baculovirus expression system, which can serve as an alternative system for antibody production.     

Glycomodification and characterization of anti-colorectal cancer immunotherapeutic monoclonal antibodies in transgenic tobacco

Anti-colorectal cancer mAb CO17-1A (IgG_2a) recognizes the antigen GA733, which is highly expressed on the surface membrane of human colorectal carcinoma cells. In this study, a transgenic tobacco system for the production of mAb CO17-1A was developed. The mAb construct included a KDEL sequence, an endoplasmic reticulum (ER) retention signal attached to the C-terminus of the heavy chain, to target accumulation of mAb into ER. An immunoblot showed significantly enhanced levels of expression of the plant-derived mAbK (mAb^PK) CO17-1A compared to mAb^P CO17-1A mAb without the KDEL sequence. An ELISA assay using human colorectal carcinoma cells confirmed that expression of mAb^PK was also significantly higher than that of mAb^P. Glycosylation analysis revealed that mAb^P had plant-specific glycans; whereas, mAb^PK primarily had oligomannose glycans. FACS showed that the Fc domains of both mAb^PK and mammalian-derived mAb (mAb^M) had similar binding activity to the FcγRI receptor (CD64). However, the Fc domains of the mAb^P had slightly lower binding activity to the Fc_γRI receptor than both mAb^PK and mAb^M. The antibody-dependent cell cytotoxicity of mAb^PK, against human colorectal cancer cells, was as efficient as mAb^M; whereas mAb^P was very low. These results suggest that KDEL localized and accumulated mAb^P in the ER and eventually enhanced the expression of mAb^P with oligomannose glycan and similar anti-cancer biological activity to the parental mAb^M.     

In vitro and in vivo efficacy of anti‐chikungunya virus monoclonal antibodies produced in wild‐type and glycoengineered Nicotiana benthamiana plants

Chikungunya virus (CHIKV) is a mosquito‐transmitted alphavirus, and its infection can cause long‐term debilitating arthritis in humans. Currently, there are no licensed vaccines or therapeutics for human use to combat CHIKV infections. In this study, we explored the feasibility of using an anti‐CHIKV monoclonal antibody (mAb) produced in wild‐type (WT) and glycoengineered (?XFT) Nicotiana benthamiana plants in treating CHIKV infection in a mouse model. CHIKV mAb was efficiently expressed and assembled in plant leaves and enriched to homogeneity by a simple purification scheme. While mAb produced in ?XFT carried a single N‐glycan species at the Fc domain, namely GnGn structures, WT produced mAb exhibited a mixture of N‐glycans including the typical plant GnGnXF₃ glycans, accompanied by incompletely processed and oligomannosidic structures. Both WT and ?XFT plant‐produced mAbs demonstrated potent in vitro neutralization activity against CHIKV. Notably, both mAb glycoforms showed in vivo efficacy in a mouse model, with a slight increased efficacy by the ?XFT‐produced mAbs. This is the first report of the efficacy of plant‐produced mAbs against CHIKV, which demonstrates the ability of using plants as an effective platform for production of functionally active CHIKV mAbs and implies optimization of in vivo activity by controlling Fc glycosylation.     

In vitro wound healing: Inhibition activity of insect‐derived mAb CO17‐1A in human colorectal cancer cell migration

The monoclonal antibody (mAb) CO17‐1A specifically binds to the tumor‐associated cell surface glycoprotein GA733 in colorectal cancer cells. Thus, mAb CO17‐1A has the potential to act as an immune therapeutic protein against colorectal cancer. Recently, it was shown that the baculovirus insect cell expression system produces anti‐colorectal cancer mAb CO17‐1A. In this study, the colorectal cancer antibody mAb CO17‐1A fused to the endoplasmic reticulum (ER) retention signal sequence (KDEL), and the (mAb CO17‐1AK) was expressed in Spodoptera frugiperda Sf9 insect cells. The yield, cell cytotoxicity, and in vitro anti‐tumor activity of mAb CO17‐1AK were verified. Western blotting was performed to confirm that both heavy and light chains of mAb CO17‐1A were expressed in Sf9 insect cells. The insect‐derived mAb (mAb^I) CO17‐1A was purified using a protein G affinity column. An in vitro wound healing assay was conducted to determine the inhibition activity of mAb CO17‐1A during tumor cell migration, showing that mAb^I CO17‐1AK was effective as mammalian‐derived mAb CO17‐1A (mAb^M CO17‐1A). These results suggest that the insect cell expression system can produce and properly assemble mAbs that inhibit tumor cell migration.     

Production of monoclonal antibodies with a controlled N‐glycosylation pattern in seeds of Arabidopsis thaliana

Seed‐specific expression is an appealing alternative technology for the production of recombinant proteins in transgenic plants. Whereas attractive yields of recombinant proteins have been achieved by this method, little attention has been paid to the intracellular deposition and the quality of such products. Here, we demonstrate a comparative study of two antiviral monoclonal antibodies (mAbs) (HA78 against Hepatitis A virus; 2G12 against HIV) expressed in seeds of Arabidopsis wild‐type (wt) plants and glycosylation mutants lacking plant specific N‐glycan residues. We demonstrate that 2G12 is produced with complex N‐glycans at great uniformity in the wt as well as in the glycosylation mutant, carrying a single dominant glycosylation species, GnGnXF and GnGn, respectively. HA78 in contrast, contains additionally to complex N‐glycans significant amounts of oligo‐mannosidic structures, which are typical for endoplasmic reticulum (ER)‐retained proteins. A detailed subcellular localization study demonstrated the deposition of both antibodies virtually exclusively in the extracellular space, illustrating their efficient secretion. In addition, although a KDEL‐tagged version of 2G12 exhibited an ER‐typical N‐glycosylation pattern, it was surprisingly detected in protein storage vacuoles. The different antibody variants showed different levels of degradation with hardly any degradation products detectable for HA78 carrying GnGnXF glycans. Finally, we demonstrate functional integrity of the HA78 and 2G12 glycoforms using viral inhibition assays. Our data therefore demonstrate the usability of transgenic seeds for the generation of mAbs with a controlled N‐glycosylation pattern, thus expanding the possibilities for the production of optimally glycosylated proteins with enhanced biological activities for the use as human therapeutics.     

Dynamics of immature mAb glycoform secretion during CHO cell culture: An integrated modelling framework

Ensuring consistent glycosylation‐associated quality of therapeutic monoclonal antibodies (mAbs) has become a priority in pharmaceutical bioprocessing given that the distribution and composition of the carbohydrates (glycans) bound to these molecules determines their therapeutic efficacy and immunogenicity. However, the interaction between bioprocess conditions, cellular metabolism and the intracellular process of glycosylation remains to be fully understood. To gain further insight into these interactions, we present a novel integrated modelling platform that links dynamic variations in mAb glycosylation with cellular secretory capacity. Two alternative mechanistic representations of how mAb specific productivity (q_p) influences glycosylation are compared. In the first, mAb glycosylation is modulated by the linear velocity with which secretory cargo traverses the Golgi apparatus. In the second, glycosylation is influenced by variations in Golgi volume. Within our modelling framework, both mechanisms accurately reproduce experimentally‐observed dynamic changes in mAb glycosylation. In addition, an optimisation‐based strategy has been developed to estimate the concentration of glycosylation enzymes required to minimise mAb glycoform variability. Our results suggest that the availability of glycosylation machinery relative to cellular secretory capacity may play a crucial role in mAb glycosylation. In the future, the modelling framework presented here may aid in selecting and engineering cell lines that ensure consistent mAb glycosylatio.     

Expression of a recombinant chimeric protein of human colorectal cancer antigen GA733-2 and Fc fragment of antibody using a replicating vector based on Beet curly top virus in infiltrated Nicotiana benthamiana leaves

We describe expression and characterization of recombinant human colorectal cancer antigen GA733-2 fused to Fc fragment of antibody (GA733-2-Fc) using a replicating vector based on Beet curly top virus in infiltrated Nicotiana benthamiana leaves. Recombinant GA733-2-Fc/KDEL with a molecular mass of ~68?kDa was transiently expressed. The level of expression of GA733-2-Fc with ER retention signal KDEL (GA733-2-Fc/KDEL) in the expression vector system was 0.96% of total soluble proteins. Recombinant GA733-2-Fc/KDEL was purified using an affinity chromatography. Mice immunized with recombinant GA733-2-Fc/KDEL mounted a strong GA733-2-Fc/KDEL-specific serum antibody response. Vaccination of plant-derived recombinant GA733-2-Fc/KDEL regressed tumor volumes in BALB/c mice. The population of activated-T and NK-T cells increased notably in lymph node, spleen, and tumor-infiltrating lymphocytes derived from the tumor-regressed mice. Taken together, recombinant GA733-2-Fc/KDEL expressed in plants can be used as an effective experimental immunogen for research in cancer vaccine development.     

Production of a tumour‐targeting antibody with a human‐compatible glycosylation profile in N. benthamiana hairy root cultures

Hairy root (HR) cultures derived from Agrobacterium rhizogenes transformation of plant tissues are an advantageous biotechnological manufacturing platform due to the accumulation of recombinant proteins in an otherwise largely protein free culture medium. In this context, HRs descending from transgenic Nicotiana tabacum plants were successfully used for the production of several functional mAbs with plant‐type glycans. Here, we expressed the tumor‐targeting monoclonal antibody mAb H10 in HRs obtained either by infecting a transgenic N. tabacum line expressing H10 with A. rhizogenes or a glyco‐engineered N. benthamiana line (ΔXTFT) with recombinant A. rhizogenes carrying mAb H10 heavy and light chain cDNAs. Selected HR clones derived from both plants accumulated mAb H10 in the culture medium with similar yields (2–3 mg/L). N‐glycosylation profiles of antibodies purified from HR supernatant revealed the presence of plant‐typical complex structures for N. tabacum‐derived mAb H10 and of GnGn structures lacking xylose and fucose for the ΔXTFT‐derived counterpart. Both antibody glyco‐formats exhibited comparable antigen binding activities. Collectively, these data demonstrate that the co‐infection of ΔXTFT Nicotiana benthamiana with recombinant A. rhizogenes is an efficient procedure for the generation of stable HR cultures expressing the tumor‐targeting mAb H10 with a human‐compatible glycosylation profile, thus representing an important step towards the exploitation of root cultures for the production of ‘next generation’ human therapeutic antibodies.     

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     

A novel plant-made monoclonal antibody enhances the synergetic potency of an antibody cocktail against the SARS-CoV-2 Omicron variant

This study describes a novel, neutralizing monoclonal antibody (mAb), 11D7, discovered by mouse immunization and hybridoma generation, against the parental Wuhan-Hu-1 RBD of SARS-CoV-2. We further developed this mAb into a chimeric human IgG and recombinantly expressed it in plants to produce a mAb with human-like, highly homogenous N-linked glycans that has potential to impart greater potency and safety as a therapeutic. The epitope of 11D7 was mapped by competitive binding with well-characterized mAbs, suggesting that it is a Class 4 RBD-binding mAb that binds to the RBD outside the ACE2 binding site. Of note, 11D7 maintains recognition against the B.1.1.529 (Omicron) RBD, as well neutralizing activity. We also provide evidence that this novel mAb may be useful in providing additional synergy to established antibody cocktails, such as Evusheld™ containing the antibodies tixagevimab and cilgavimab, against the Omicron variant. Taken together, 11D7 is a unique mAb that neutralizes SARS-CoV-2 through a mechanism that is not typical among developed therapeutic mAbs and by being produced in ΔXFT Nicotiana benthamiana plants, highlights the potential of plants to be an economic and safety-friendly alternative platform for generating mAbs to address the evolving SARS-CoV-2 crisis.     

Expression,glycosylation and function of recombinant anti‐colorectal cancer mAb CO17‐1A in SfSWT4 insect cells

Colorectal cancer is a commonly diagnosed cancer in the world. Monoclonal antibody (mAb) CO17‐1A recognizes the tumor‐associated antigen GA733, a cell surface glycoprotein highly expressed in colorectal carcinoma cell, which is considered to be applicable for diagnosis and therapeutic treatment against colorectal cancer. In addition antibodies are glycoproteins, efficiently recognize and eliminate specific pathogenic and disease antigens. We have currently established baculovirus insect cell expression system to produce anti‐colorectal cancer mAb CO17‐1A. In this study, mAb CO17‐1A was expressed in the transgenic insect cell line SfSWT4, in which glycosylation processing pathway has been humanized. Immunoblot confirmed that mAb CO17‐1A properly expressed in SfSWT4 insect cells. mAb CO17‐1A was purified using protein G affinity column. In addition, MALDI‐TOF verified that the mAb CO17‐1A fused to KDEL, endoplasmic reticulum (ER) retention signal (mAb CO17‐1AK) had high mannose type of glycan structure. Migration assay showed that insect cell‐derived mAb CO17‐1AK (mAb^I CO17‐1AK) with high mannose type of glycan structure was effective as mammalian‐derived mAb CO17‐1A (mAb^M CO17‐1A) in inhibition of metastasis. Kinetic analysis of antigen‐antibody interaction using Surface Plasmon Resonance (SPR) confirmed that mAb^I CO17‐1AK is efficient to interact with antigen GA733 as mAb^M CO17‐1A. These results suggest that the insect cell expression system with the SfSWT4 possibly can be used as a useful alternative way to produce full‐size mAb for cancer immunotherapy.     

A novel approach for the simultaneous quantification of a therapeutic monoclonal antibody in serum produced from two distinct host cell lines

Therapeutic monoclonal antibodies (mAbs) possess a high degree of heterogeneity associated with the cell expression system employed in manufacturing, most notably glycosylation. Traditional immunoassay formats used to quantify therapeutic mAbs are unable to discriminate between different glycosylation patterns that may exist on the same protein amino acid sequence. Mass spectrometry provides a technique to distinguish specific glycosylation patterns of the therapeutic antibody within the same sample, thereby allowing for simultaneous quantification of the same mAb with different glycosylation patterns. Here we demonstrate a two-step approach to successfully differentiate and quantify serum mixtures of a recombinant therapeutic mAb produced in two different host cell lines (CHO vs. Sp2/0) with distinct glycosylation profiles. Glycosylation analysis of the therapeutic mAb, CNTO 328 (siltuximab), was accomplished through sample pretreatment consisting of immunoaffinity purification (IAP) and enrichment, followed by liquid chromatography (LC) and mass spectrometry (MS). LC-MS analysis was used to determine the percentage of CNTO 328 in the sample derived from either cell line based on the N-linked G1F oligosaccharide on the mAb. The relative amount of G1F derived from each cell line was compared with ratios of CNTO 328 reference standards prepared in buffer. Glycoform ratios were converted to concentrations using an immunoassay measuring total CNTO 328 that does not distinguish between the different glycoforms. Validation of the IAP/LC-MS method included intra-run and inter-run variability, method sensitivity and freeze-thaw stability. The method was accurate (%bias range = -7.30–13.68%) and reproducible (%CV range = 1.49–10.81%) with a LOQ of 2.5 μg/mL.     

Purification of anti‐colorectal cancer monoclonal antibody CO17‐1A from insect cell culture using a French press and sonication

Monoclonal antibody (mAb) CO17‐1A binds to GA733, which is a tumor‐associated glycoprotein antigen highly expressed on the colorectal cancer cell surface. Thus, mAb CO17‐1A is considered a useful biomolecule for diagnosis and treatment against colorectal cancer. Previously, we established a baculovirus–insect cell expression system for the production of mAb CO17‐1A. In order to use mAb CO17‐1A as a diagnostic and therapeutic tool, however, the antibody must be properly purified from the insect cells. In this study, our aim was to investigate effective purification processes of mAb CO17‐1A expressed in Spodoptera frugiperda (Sf9) insect cells, using a French press and sonication for cell disruption. SDS‐PAGE confirmed that both mAb CO17‐1A and mAb CO17‐1A fused to the KDEL endoplasmic reticulum (ER) retention signal (mAb CO17‐1AK) were expressed clearly in Sf9 insect cells. Western blot analysis showed that detection levels of mAb CO17‐1A and CO17‐1AK were higher when the insect cells were disrupted two times by the French press and then sonicated, compared to only one French press disruption plus sonication. Optical microscopy confirmed that insect cells treated with both the French press and sonication were properly disrupted. Analysis of gene sequence information on mAb CO17‐1A verified that a signal peptide is present but a transmembrane protein does not exist. These results suggest that cell disruption by the French press twice and sonication once is an effective method for improving purification efficiency.     

Recombinant anti-hCG antibodies retained in the endoplasmic reticulum of transformed plants lack core-xylose and core-alpha(1,3)-fucose residues

Plant-based expression systems are attractive for the large-scale production of pharmaceutical proteins. However, glycoproteins require particular attention as inherent differences in the N-glycosylation pathways of plants and mammals result in the production of glycoproteins bearing core-xylose and core-alpha(1,3)-fucose glyco-epitopes. For treatments requiring large quantities of repeatedly administered glycoproteins, the immunological properties of these non-mammalian glycans are a concern. Recombinant glycoproteins could be retained within the endoplasmic reticulum (ER) to prevent such glycan modifications occurring in the late Golgi compartment. Therefore, we analysed cPIPP, a mouse/human chimeric IgG1 antibody binding to the beta-subunit of human chorionic gonadotropin (hCG), fused to a C-terminal KDEL sequence, to investigate the efficiency of ER retrieval and the consequences in terms of N-glycosylation. The KDEL-tagged cPIPP antibody was expressed in transgenic tobacco plants or Agrobacterium-infiltrated tobacco and winter cherry leaves. N-Glycan analysis showed that the resulting plantibodies contained only high-mannose (Man)-type Man-6 to Man-9 oligosaccharides. In contrast, the cPIPP antibody lacking the KDEL sequence was found to carry complex N-glycans containing core-xylose and core-alpha(1,3)-fucose, thereby demonstrating the secretion competence of the antibody. Furthermore, fusion of KDEL to the diabody derivative of PIPP, which contains an N-glycosylation site within the heavy chain variable domain, also resulted in a molecule lacking complex glycans. The complete absence of xylose and fucose residues clearly shows that the KDEL-mediated ER retrieval of cPIPP or its diabody derivative is efficient in preventing the formation of non-mammalian complex oligosaccharides.     

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.     

Impact on N-Glycosylation profile of monoclonal anti-D antibodies as a way to control their immunoregulatory and cytotoxic properties

Prophylaxis of hemolytic disease of newborns is based on the ability of polyclonal anti-D antibodies for sup-pressing maternal immune response against D-positive fetal red blood cells. The immunosuppressive effect of anti-D antibody is mediated by interaction between its Fc-fragment and low-affinity IgG Fc-receptor (FcγR) on the immune cell. No clinically effective monoclonal anti-D antibody (mAb) that can replace polyclonal anti-D immunoglobulin has been developed yet. The goals of this study were comparison of structural and functional properties of human anti-D polyclonal and monoclonal Abs and assessment of the possibility to manipulate the effector properties of the mAb. N-Glycosylation and particularly the content of nonfucosylated glycans are crucial for affinity of mAb to FcγRIIIA, which plays the key role in the clearance of sensitized cells. We studied and compared glycoprofiles and FcγRIIIA-mediated hemolytic ability of human polyclonal antibodies and anti-D mAbs produced by human B-cell lines, human-rodent heterohybridomas, and a human non-lymphoid cell line PER.C6. Replacement of producing cell line and use of glycosylation modulators can convert an inert mAb into an active one. Nevertheless, rodent cell lines, as well as human non-lymphoid cells, distort natural glycosylation of human IgG and could lead to the loss of immunosuppressive properties. All of the anti-D mAbs secreted by human B-cell lines have a glycoprofile close to human serum IgG. Hence, the constant ratio of IgG glycoforms in human serum is predetermined by glycosylation at the level of the individual antibody-producing cell. The anti-D fraction of polyclonal anti-D immunoglobulin compared to the total human IgG contains more nonfucosylated glycans. Thus, only human trans-formed B-cells are an appropriate source for efficient anti-D mAbs that can imitate the action of polyclonal anti-D IgG.