Structural characterization of N-linked oligosaccharides on monoclonal antibody Nimotuzumab through process development.

Nimotuzumab (TheraCIM, CIMAher, h-R3, humanized anti-EGF-R antibody), monoclonal antibody (mAb) manufactured at the Center of Molecular Immunology (Havana, Cuba) is currently being tested in several clinical trials. Nimotuzumab has a single N-glycosylation site in the Fc-CH2 fragment but no N-glycosylation site in the Fab region. The current study reports the full characterization of the mAb N-glycosylation and the consistency observed in several production batches from a perfusion mode culturing system that lasted between 68 and 150 days. It confirms that the N-glycan structures of Nimotuzumab expressed in the NS0 murine myeloma cell line are of the murine type. They consist mainly of fucosylated G0, G1 and G2 oligosaccharides, which are normally found in the CH2 region of IgG. Other minor species found were high mannose and sialylated structures. A small portion of the glycans were sialylated (∼12%) and the only type of sialic acid detected was N-glycolyl-sialic acid, α2,6-linked to Gal. No Galα1-3Gal moieties were detected.

Physicochemical and biological characterization of 1E10 anti-idiotype vaccine.

BACKGROUND: 1E10 monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH)3, in several clinical trials for melanoma, breast, and lung cancer. During early clinical development this mAb was obtained in vivo from mice ascites fluid. Currently, the production process of 1E10 is being transferred from the in vivo to a bioreactor-based method. RESULTS: Here, we present a comprehensive molecular and immunological characterization of 1E10 produced by the two different production processes in order to determine the impact of the manufacturing process in vaccine performance. We observed differences in glycosylation pattern, charge heterogeneity and structural stability between in vivo-produced 1E10 and bioreactor-obtained 1E10. Interestingly, these modifications had no significant impact on the immune responses elicited in two different animal models. CONCLUSIONS: Changes in 1E10 primary structure like glycosylation; asparagine deamidation and oxidation affected 1E10 structural stability but did not affect the immune response elicited in mice and chickens when compared to 1E10 produced in mice.

Differential N-glycosylation of a monoclonal antibody expressed in tobacco leaves with and without endoplasmic reticulum retention signal apparently induces similar in vivo stability in mice.

Plant cells are able to perform most of the post-translational modifications that are required by recombinant proteins to achieve adequate bioactivity and pharmacokinetics. However, regarding N-glycosylation the processing of plant N-glycans in the Golgi apparatus displays major differences when compared with that of mammalian cells. These differences in N-glycosylation are expected to influence serum clearance rate of plant-derived monoclonal antibodies. The monoclonal antibody against the hepatitis B virus surface antigen expressed in Nicotiana tabacum leaves without KDEL endoplasmic reticulum (ER) retention signal (CB.Hep1(-)KDEL) and with a KDEL (Lys-Asp-Glu-Leu) fused to both IgG light and heavy chains (CB.Hep1(+)KDEL) were tested for in vivo stability in mice. Full characterization of N-glycosylation and aggregate formation in each monoclonal antibody batch was determined. The mouse counterpart (CB.Hep1) was used as control. Both (CB.Hep1(-)KDEL) and (CB.Hep1(+)KDEL) showed a faster initial clearance rate (first 24 h) compared with the analogous murine antibody while the terminal phase was similar in the three antibodies. Despite the differences between CB.Hep1(+)KDEL and CB.Hep1(-)KDEL N-glycans, the in vivo elimination in mice was indistinguishable from each other and higher than the murine monoclonal antibody. Molecular modelling confirmed that N-glycans linked to plantibodies were oriented away from the interdomain region, increasing the accessibility of the potential glycan epitopes by glycoprotein receptors that might be responsible for the difference in stability of these molecules.

The N-glycosylation of classical swine fever virus E2 glycoprotein extracellular domain expressed in the milk of goat.

Classical swine fever virus (CSFV) outer surface E2 glycoprotein represents an important target to induce protective immunization during infection but the influence of N-glycosylation pattern in antigenicity is yet unclear. In the present work, the N-glycosylation of the E2-CSFV extracellular domain expressed in goat milk was determined. Enzymatic N-glycans releasing, 2-aminobenzamide (2AB) labeling, weak anion-exchange and normal-phase HPLC combined with exoglycosidase digestions and mass spectrometry of 2AB-labeled and unlabeled N-glycans showed a heterogenic population of oligomannoside, hybrid and complex-type structures. The detection of two Man(8)GlcNAc(2) isomers indicates an alternative active pathway in addition to the classical endoplasmic reticulum processing. N-acetyl or N-glycolyl monosialylated species predominate over neutral complex-type N-glycans. Asn207 site-specific micro-heterogeneity of the E2 most relevant antigenic and virulence site was determined by HPLC-mass spectrometry of glycopeptides. The differences in N-glycosylation with respect to the native E2 may not disturb the main antigenic domains when expressed in goat milk.

Chemical and enzymatic N-glycan release comparison for N-glycan profiling of monoclonal antibodies expressed in plants.

Plants synthesize N-glycans containing the antigenic sugars alpha(1,3)-fucose and beta(1,2)-xylose. Therefore it is important to monitor these N-glycans in monoclonal antibodies produced in plants (plantibodies). We evaluated several techniques to characterize the N-glycosylation of a plantibody produced in tobacco plants with and without the KDEL tetrapeptide endoplasmic reticulum retention signal which should inhibit or drastically reduce the addition of alpha(1,3)-fucose and beta(1,2)-xylose. Ammonium hydroxide/carbonate-based chemical deglycosylation and PNGase A enzymatic release were investigated giving similar 2-aminobenzamide-labeled N-glycan HPLC profiles. The chemical release does not generate peptides which is convenient for MS analysis of unlabeled pool but its main drawback is that it induces degradation of alpha1,3-fucosylated N-glycan reducing terminal sugar. Three analytical methods for N-glycan characterization were evaluated: (i) MALDI-MS of glycopeptides from tryptic digestion; (ii) negative-ion ESI-MS/MS of released N-glycans; (iii) normal-phase HPLC of fluorescently labeled glycans in combination with exoglycosidase sequencing. The MS methods identified the major glycans, but the HPLC method was best for identification and relative quantitation of N-glycans. Negative-mode ESI-MS/MS permitted also the correct identification of the linkage position of the fucose residue linked to the inner core N-acteylglucosamine (GlcNAc) in complex N-glycans.

Production of plantibodies in Nicotiana plants.

Because of the wide use and high demand in medicine, monoclonal antibodies are among the main recombinant pharmaceuticals at present, although present limitations of the productive platforms for monoclonal antibodies are driving the improvement of the large-scale technologies and the development of alternative expression systems. This has drawn the attention on plants as expression system for monoclonal antibodies and related derivatives, owning the capacity of plants to properly express and process eukaryotic proteins with biological activity resembling that of the natural proteins. In this chapter, the procedures from the isolation of the monoclonal antibody genes to the biochemical and biological characterization of the plant-expressed monoclonal antibody are described.

N-glycosylation of plant recombinant pharmaceuticals.

N-glycosylation is a maturation event necessary for the correct function, efficiency, and stability of a high number of biopharmaceuticals. This chapter presented here proposes various methods to determine whether, how, and where a plant pharmaceutical is N-glycosylated. These methods rely on blot detection with glycan-specific probes, specific deglycosylation of glycoproteins followed by mass spectrometry, N-glycan profile analysis, and glycopeptide identification by LC-MS.

N-glycosylation pattern of E2 glycoprotein from classical swine fever virus.

The extracellular domain of E2 glycoprotein outer surface of the classical swine fever virus was expressed in epithelial kidney pig cells. The N-glycosylation determined by combination of Normal Phase-HPLC, Weak Anion Exchange-HPLC, exoglycosidase digestions and Mass Spectrometry revealed a complex mixture of neutral and monosialylated multiantennary N-glycans with variable number of alpha1-3-Gal-Gal antennae terminals. The most abundant neutral N-glycan has a composition of Hex(7)HexNAc(4)dHex(1), Negative ion ESI-MS/MS confirmed the presence of the alpha1-3-Gal-Gal motif on each arm of the fucosylated biantennary N-glycan. The most abundant monosialylated glycan was Hex(6)HexNAc(4)dHex(1)Neu(5)Ac(1), with the sialic acid linked to the terminal beta1-4-Gal-GlcNAc. Sialic acid on the antenna capping position was predominantly of the N-acetyl form.

Plant N-glycan profiling of minute amounts of material.

Development of convenient strategies for identification of plant N-glycan profiles has been driven by the emergence of plants as an expression system for therapeutic proteins. In this article, we reinvestigated qualitative and quantitative aspects of plant N-glycan profiling. The extraction of plant proteins through a phenol/ammonium acetate procedure followed by deglycosylation with peptide N-glycosidase A (PNGase A) and coupling to 2-aminobenzamide provides an oligosaccharide preparation containing reduced amounts of contaminants from plant cell wall polysaccharides. Such a preparation was also suitable for accurate qualitative and quantitative evaluation of the N-glycan content by mass spectrometry. Combining these approaches allows the profiling to be carried out from as low as 500 mg of fresh leaf material. We also demonstrated that collision-induced dissociation (CID) mass spectrometry in negative mode of N-glycans harboring alpha(1,3)- or alpha(1,6)-fucose residue on the proximal GlcNAc leads to specific fragmentation patterns, thereby allowing the discrimination of plant N-glycans from those arising from mammalian contamination.

Highly protective E2-CSFV vaccine candidate produced in the mammary gland of adenoviral transduced goats.

Classical swine fever virus is the etiological agent of the most economically important highly contagious disease of swine worldwide. E2 is the major envelope glycoprotein present as a homodimer on the outer surface of the virus and represents an important target for the induction of neutralizing immune response against the viral infection. The E2 extracellular domain was expressed in the milk of adenoviral transduced goats at the highest level about 1.2g/L. The recombinant glycoprotein was purified from clarified serum milk by a single metal chelate affinity chromatography step, as a homodimer of approximately 100kDa and purity over 98%. Glycosylation analysis showed the presence of oligomannoside, hybrid and complex type N-glycans, attached to the recombinant E2. The capacity of goat milk-derived E2 antigen to protect pigs from both classical swine fever clinical signs and viral infection was assessed in a vaccination and challenge trial. The immunized pigs became protected after challenge with 10(5) LD(50) of a highly pathogenic CSFV strain. In the context of veterinary vaccines, this expression system has the advantages that the recombinant antigen could be harvested in about 48h after adenoviral transduction with expression levels in the range of g/L. This approach may turn into a scalable expression system for the assessment and production of veterinary vaccines.

Rapid and sensitive anthrone-sulfuric acid assay in microplate format to quantify carbohydrate in biopharmaceutical products: method development and validation.

The need for an accurate, fast and reliable analysis of carbohydrate test is crucial for numerous biological processes. In that sense, anthrone-sulfuric acid assay is one of the most efficient quantification techniques successfully applied to carbohydrate determination. In this paper, a sensitive and accurate anthrone-sulfuric acid microplate assay was developed and validated for the quantitative estimation of yeast carbohydrates in the production of hepatitis B virus surface antigen, and the main component of the recombinant vaccine HEBERBIOVAC HB. A response surface methodology was applied to design and optimize the assay in order to maximize the differences on the expected effect and to minimize the number of experiments. The proposed method was linear over the concentration range from 10 to 120 microg/mL for glucose, with values for the coefficient of determination >0.99. Intra- and inter-assay variation coefficient ranged between 0.45-4.79% and 2.48-8.94%, respectively. The Student t-test used in the interference study, revealed good parallelism among curves (T(obs)< or =T(0.05)), which indicates the lack of interference in the working range. Yields obtained in accuracy test for two concentration levels varied between 90 and 105%, confirming the assay's reliability. In conclusion, the validated method, which has successfully been used for the process control monitoring of several samples generated from the production of hepatitis B vaccine, allows the quality and purity of the final product.

Analytical strategies to investigate plant N-glycan profiles in the context of plant-made pharmaceuticals.

Plants are attractive hosts for the production of recombinant proteins. However, their inability to process authentic human N-glycan structures imposes a major limitation on their use as expression systems for therapeutic products. Several strategies have emerged to engineer plant N-glycans into human-compatible molecules. In this context, fast and reliable analytical strategies for the identification of plant N-glycan profiles have been developed to define the N-glycosylation pathways of crops, to monitor the production of plant-made pharmaceuticals and to assess in planta remodelling strategies.

High-performance reverse phase chromatography with fluorescence detection assay for characterization and quantification of pneumococcal polysaccharides.

A methods using high-performance reverse phase (RP) chromatography with fluorescence detection, has been developed to determine the composition and identity of Streptococcus pneumoniae capsular polysaccharide used in formulating conjugate vaccine for prevention of pneumococcal infection. For the monosaccharide composition, the polysaccharides were subjected to hydrofluoric acid (HF) hydrolysis followed by trifluoroacetic acid (TFA). After acid hydrolysis, the released monosaccharides were re-N-acetylated and labeled with 2-aminobenzamide (2AB) by reductive amination reaction. High-performance RP chromatography was performed on C18 TSKODS 120T column. Nuclear magnetic resonance was used to confirm chemical structure and purity of pneumococcal capsular polysaccharides.

Enzyme-linked immunosorbent assay for quantitative determination of capsular polysaccharide production in Streptococcus pneumoniae clinical isolates.

A simple, specific, sensitive and reproducible ELISA has been developed to quantify the level of CPS (capsular polysaccharide) production in supernatants of Streptococcus pneumoniae cell cultures. CPSs from Strep. pneumoniae have been widely used as vaccine antigens. The quantification method is based on two type-23F serotype-specific polyclonal antibodies: IgG, purified from sera of mice immunized with a pneumococcal type-23F CPS conjugate, used in the coating step, and a serotype-specific rabbit serum as the second antibody. Solutions of purified type-23F CPS were used as standards. The relationship between A(492) and type-23F CPS concentration was linear over the range 1-310 ng/ml (r=0.989), with 1 ng/ml as the lower limit of sensitivity. The specificity of ELISA was assessed because purified type-19F CPS and cell-wall polysaccharide samples were not detected after their evaluation by the ELISA described in the present study. Repeatability and intermediate precision of the assay were good, the coefficients of variation being 3 and 10% respectively. This ELISA allowed selection of an appropriate vaccine strain, for a natural polysaccharide vaccine, among several 23F pneumococcal clinical isolates and constituted a valuable analytical tool for Strep. pneumoniae fermentation and CPS purification follow-up.

High expression level of recombinant human erythropoietin in the milk of non-transgenic goats.

The high degree of structural conservation of erythropoietin between species, make it, especially, difficult to produce this protein growth factor in the milk of transgenic animals. Here, we show that through the direct transduction of the mammary epithelium, it is possible to produce high levels of recombinant human erythropoietin in the milk of non-transgenic goats without causing harm to the animals. The efficiency of viral transduction was improved through a temporal disruption of tight-junctions with EGTA allowing for the expression of human erythropoietin at levels of up to 2g/L in milk. The human erythropoietin was purified from the milk using a multi-step protocol involving milk clarification, two precipitation steps and two affinity chromatographies, with a yield of about 70% and purity over 98%. However, the human erythropoietin expressed in milk was underglycosylated, which seems to be the main cause for its low in vivo hematopoietic activity. Nonetheless, these results demonstrate that through the direct transduction of the mammary epithelium it is possible to produce potentially toxic proteins in milk, at levels high enough for their purification and biological characterization.

Differential in vitro and in vivo glycosylation of human erythropoietin expressed in adenovirally transduced mouse mammary epithelial cells.

The expression of human erythropoietin in the mammary gland is an attractive approach to diminish its current production cost. Previous attempts to produce erythropoietin in the milk of transgenic animals resulted in very low expression levels and in a detrimental effect in the health of the founder animals. Here, we show that the direct transduction of the mouse mammary gland with an adenoviral vector carrying the cDNA of erythropoietin promotes its expression in milk at a level as high as 3.5 mg/ml. The recombinant erythropoietin derived from mouse milk showed a different migration and distribution after SDS-PAGE electrophoresis as well as a low in vivo hematopoietic activity. Enzymatic deglycosylation showed that these molecular weight disparities are in part due to differential glycosylation compared to with its counterpart produced in CHO and HC11 cell lines. The difference between in vivo and in vitro glycosylation of human erythropoietin expressed in adenovirally transduced mammary epithelial cells suggests that key enzymes in the glycosylation pathway may be insufficient during lactation. Thus, the direct transduction of the mammary epithelium seems to be a powerful tool to express toxic proteins in milk at levels high enough for their physical, chemical and biological characterization before undertaking the generation of a transgenic mammal.

An integral approach towards a practical application for a plant-made monoclonal antibody in vaccine purification.

The use of transgenic plants for the production of pharmaceutical compounds has received increasing attention in the last few years. However, many technological and regulatory issues regarding the practical exploitation of this alternative system of production remain to be solved; a situation that explains the lack of commercial products derived from such a system. This paper reports the expression in transgenic plants and cells of a single-chain antibody variable-region fragment (scFv) and a mouse monoclonal antibody to the hepatitis B virus surface antigen (HBsAg). The large-scale purification of the scFv from plants and its use for immunopurification of HBsAg are also described, together with elements concerning regulatory issues and technologies for compliance with good manufacturing and agricultural practices.

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.

Influence of culture conditions on the N-glycosylation of a monoclonal antibody specific for recombinant hepatitis B surface antigen.

MAbs (monoclonal antibodies) are becoming increasingly important as diagnostic tools for pharmaceutical biotechnology, and hence it is crucial that they are produced under controlled conditions to assure their consistency and reproducibility, not only in terms of protein sequence and bioactivity, but also in terms of post-translational modifications, e.g. for N-glycosylation. Hybridoma CB.Hep-1, which secretes an IgG2b mAb, was cultured in vivo in ascites and in vitro in static-flask, spinner-flask, dialysis-membrane and perfusion systems using protein-free, low-serum-containing medium (1% foetal-calf serum) and high-serum-containing medium (8% foetal-calf serum). These CB.Hep-1 mAbs were fully characterized, and insignificant differences in the affinity constant were observed. Glycosylation profiling was performed by labelling the N-glycans released by peptide N-glycosidase F with either of the fluorophore tags 8-aminonaphthalene-1,3,6-trisulphonic acid and 4-aminobenzoic acid. The mAb produced in vivo showed two major biantennary-complex-type N-glycans: monogalactosylated, core-fucosylated and agalactosylated, core-fucosylated. The mAbs produced in vitro in static flasks and spinner flasks were not significantly influenced by the serum content in the culture media and showed a higher degree of N-glycan galactosylation compared with those produced in mouse-ascites, hollow-fibre and membrane systems. The monogalactosylated, core-fucosylated structure was the most abundant N-glycan except for those produced in ascites and hollow fibres, where the agalactosylated, core-fucosylated glycoform was the major specie. MAbs produced in high-cellular-yield systems displayed greater galactosylation heterogeneity influenced by changes in culture media.

The cattle tick antigen, Bm95, expressed in Pichia pastoris contains short chains of N- and O-glycans.

Bm95 is an antigen isolated from Boophilus microplus strains with low susceptibility to antibodies developed in cattle vaccinated with the recombinant Bm86 antigen (Gavac, HeberBiotec S.A., Cuba). It is a Bm86-like surface protein, which by similarity contains seven EGF-like domains and a lipid-binding GPI-anchor site at the C-terminal region. The primary structure of the recombinant (rBm95) protein expressed in Pichia pastoris was completely verified by LC/MS. The four potential glycosylation sites (Asn 122, 163, 329, and 363) are glycosylated partially with short N-glycans, from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) of which, Man(8-9)GlcNAc(2) were the most abundant. O-Glycopeptides are distributed mostly towards the protein N-terminus. While the first N-glycosylated site (Asn(122)) is located between EGF-like domains 2 and 3, where the O-glycopeptides were found, two other N-glycosylated sites (Asn(329) and Asn(363)) are located between EGF-like domains 5 and 6, a region devoid of O-glycosylated Ser or Thr.