Glycosphingolipid expression in spontaneously aborted fetuses and placenta from blood group p women. Evidence for placenta being the primary target for anti-Tja-antibodies

A 12-week-old fetus and one 17-week-old fetus + placenta were obtained after spontaneous abortions from two women of blood group p. The 17-week-old fetus was dissected into intestine, liver, brain and residual tissue. Nonacid glycosphingolipid fractions were prepared from the tissues. Glycolipid characterization was carried out using thin layer chromatography immunostained with monoclonal antibodies and bacteria and by¹H NMR spectroscopy and mass spectrometry. In the placental fraction substantial amounts of globotetraosylceramide (P-antigen) and globotriaosylceramide (P^k-antigen) were identified. In contrast, the fetuses contained only trace amounts of these structures, as revealed by immunostaining. These results indicate that the primary target for the antibodies of the anti-Tj^a serum is the placenta tissue, resulting in termination of the pregnancy.     

Blood group type glycosphingolipids of human kidneys. Structural characterization of extended globo-series compounds

Blood group type glycosphingolipids present in kidneys of blood group A and B human individuals have been isolated and structurally characterized by mass spectrometry, proton NMR spectroscopy, degradation studies and by their reactivity with various monoclonal antibodies andEscherichia coli bacteria. The two major complex glycolipids present in the blood group A and B kidneys were globopentaosylceramide (IV³Gal-Gb₄Cer) and the X pentaglycosylceramide (III³Fuc-nLc₄Cer). The major blood group A glycolipid in the blood group A kidneys was based on the type 4 chain (globo-series). There were also small amounts of the type 2 chain and trace amounts of the type 1 and type 3 chain based A glycolipids. In addition, the blood group H type 4 chain structure was present together with Le^a and Le^b compounds. In the blood group B kidneys, the major B glycolipids were monofucosylated hexa- and octaglycosylceramides, where the former were based on the type 2 carbohydrate chain. The blood group B type 4 chain heptaglycosylceramide was found to be a minor component making up only about 1% of the total blood group B structures. Abbreviations: for blood group glycolipid antigens the short hand designation stands for blood group—number of sugar residues—type of carbohydrate chain. Thus A-7-4 means a type 4 chain blood group A heptaglycosylceramide. The sugar types are abbreviated for mass spectrometry to Hex for hexose, HexNAc forN-acetylhexosamine and dHex for deoxyhexose.     

Isolation and characterization of glycosphingolipids with J blood group activity from bovine spleen1,2

Two glycosphingolipids with J blood group activity were found in J-positive bovine spleen. They were tentatively identified as ceramide deca- and dodecahexosides containing galactose, glucose, N -acetylgalactosamine and N -acetylglucosamine in a molar ratio of 5:3:1:1 and 6:3:2:1, respectively. Fucose was not present. Ceramide decahexosides without J activity were also found in J-negative bovine spleen. The principal component fatty acids of the J-active glycosphingolipids were saturated even-numbered long-chain acids with 16 to 24 C atoms. Their principal long-chain bases were sphingosine and dihydrosphingosine with smaller amounts of phy-tosphingosine.
Both J-active glycosphingolipids were readily water-soluble and showed strong activity in the bovine J and in the porcine A blood group system. They exhibited no cross-reactivity in the human A system. However, a J-negatiye glycosphingolipid fraction - also from J-negative spleen - with shorter carbohydrate chain-length showed strong activity in the human A system.     

Ontogeny and expression of chicken A blood group antigens

Expression of chicken red blood cell (RBC) surface antigens was studied by using a monoclonal antibody (ISU-cA) specific for chicken A blood group antigens. Erythrocytes were examined from embryos of 3-18 days of incubation and from chicks at hatch up to 21 weeks of age. Specific antigens were detected on embryonic RBC surfaces by immunofluorescence as early as 3 days of incubation. Antigenic expression was examined by both haemagglutination and immunofluorescence and found to increase with age from embryos to mature birds. The antigen concentration on the cell surface was found to be affected by genotype; heterozygotes had an intermediate level of antigen between that of the two parental genotypes. These data confirm the co-dominance that is observed with most blood group antigens. Flow cytometric analysis allowed confirmation that the entire erythrocyte population gradually increased in antigenic expression over time, rather than having an antigen-negative subpopulation being replaced by a positive subpopulation.     

Analysis of recombinant monoclonal antibody isoforms by electrospray ionization mass spectrometry as a strategy for streamlining characterization of recombinant monoclonal antibody charge heterogeneity

A therapeutic recombinant monoclonal antibody analyzed by cation-exchange chromatography exhibited a heterogeneous profile composed of approximately 10 isoforms. The peaks were isolated and characterized by electrospray quadrupole time-of-flight mass spectrometry (ESI-q-TOF-MS), N-terminal Edman sequencing, peptide mapping, and other techniques. Acidic (lower pI) peaks were found to represent deamidated and sialyated species. Higher pI peaks were found to contain N- and C-terminal heavy-chain variants. Biological activities of the more abundant isoforms were found to be comparable. An approach streamlining the characterization of antibody charge heterogeneity is proposed.     

Structural and functional characterization of the trifunctional antibody catumaxomab

The Triomab^® family of trifunctional, bispecific antibodies that maintain an IgG-like shape are novel tumor targeting agents. These chimeras consist of two half antibodies, each with one light and one heavy chain, that originate from parental mouse IgG2a and rat IgG2b isotypes. This combination allows cost-effective biopharmaceutical manufacturing at an industrial scale since this specific mouse/rat isotype combination favors matching of corresponding antibody halves during production by means of quadroma technology. Whereas every Triomab^® family member is composed of an anti-CD3 rat IgG2b half antibody for Tcell recognition, the antigen binding site presented by the mouse IgG2a isotype is exchangeable. Several Triomab^® antibodies have been generated that bind to tumor-associated antigens, e.g., EpCAM (catumaxomab), HER2/neu (ertumaxomab), CD20 (FBTA05), gangliosides GD2/GD3 (Ektomun^®), on appropriate tumor target cells associated with carcinomas, lymphomas or melanomas. Catumaxomab (Removab^®) was launched in Europe for treatment of malignant ascites in April 2009. Here, we report the structural and functional characterization of this product. Mass spectrometry revealed an intact mass of 150511 Dalton (Da) and 23717 Da, 24716 Da, 51957 Da and 52019 Da of the reduced and alkylated rat light chain, mouse light chain, rat heavy chain, mouse heavy chain chains, respectively. The observed masses were in agreement with the expected masses based on the amino acid sequence obtained from cDNA sequencing. The glycosylation profile was similar to other human IgG consisting of biantennary oligosaccharides with different numbers of terminal galactose. CD spectroscopy showed mainly β-sheets secondary structure that is typical for IgG antibodies. Binding measurement revealed the unique trifunctional features of catumaxomab. Other analytical tools were used to evaluate characteristics of catumaxomab preparations, including the presence of isoforms and aggregates.     

LC-MS对奥马珠单抗与其生物类似物CMAB007的分析与结构特征

哮喘是当今世界威胁公共健康的最主要的慢性肺部疾病,作为治疗中重度和难治性哮喘的特效药奥马珠单抗治疗费用相对昂贵,生物类似药CMAB007的研制可以降低治疗费用。本研究用质谱分析一种奥马珠单抗生物类似药CMAB007与原研药的一致性,分别从氨基酸分析、肽图、N/C端序列,还原质谱,寡糖含量,N糖分析等层面对CMAB007和奥马珠单抗进行了系统的比对研究,为进一步临床研究打下基础。结果表明CMAB007和奥马珠单抗氨基酸序列一致,赖氨酸剪切也基本一致,带唾液酸修饰糖形和带核心岩藻糖形比例相近,高甘露糖形比例差异不大,CMAB007略低于奥马珠单抗。因此CMAB007满足生物类似药在结构比对上一致性的要求,有望成为国内首先上市的重组抗人Ig E单克隆抗体药物。     

Chemical characterization of a blood group H type pentaglycosylceramide of human small intestine

A blood group H type pentaglycosylceramide was isolated in relatively large amounts from human adult small intestine (52mg from one individual) and human meconium (fetal origin). The structure was made likely by mass spectrometry and NMR spectroscopy of non-degraded permethylated and permethylated-LiAlH₄-reduced glycolipid and by degradaton to be Fucα1 → 2GAlβ 1 → 3GlcNAcβ 1 → 3GAlβ 1 → 4Glcβ 1 → l Cer. The ceramide was composed mainly of phytosphingosine and 2-hydroxy 16–24 carbon fatty acids. This novel type 1 chain species (Galβ 1 → 3GlcNAc) was not accompanied by the type 2 chain isomer (Galβ 1 → 4GlcNAc) which in contrast is the sole species in human erythrocyte and dog small intestine.     

Monoclonal antibody differentiates chicken A system alloantigens

A monoclonal antibody (ISU-cA) was produced that recognized certain alloantigens of the chicken A blood group locus. Antigens produced by alleles A3, A4 and A8 were positive, and those produced by A2 and A5 were negative, by haemagglutination. The specificity of ISU-cA for chicken A blood group antigens was demonstrated by serologic analyses, genetic crosses and competitive inhibition of binding by anti-A alloantisera. To our knowledge, this is the first reported monoclonal antibody against a chicken blood group alloantigen system other than the B complex.     

Conformational studies of blood group A and blood group B oligosaccharides using NMR residual dipolar couplings

The conformations of two synthetic trisaccharides of blood group A and B (alpha-L-Fucp-(1-->2)-[alpha-D-GalpNAc-(1-->3)]-alpha-D-Galp and alpha-L-Fucp-(1-->2)-[alpha-D-Galp-(1-->3)]-alpha-D-Galp, respectively) and of a type A tetrasaccharide alditol, Fucp-(1-->2)-[alpha-D-GalpNAc-(1-->3)]-beta-D-Galp-(1-->3)-GalNAc-ol, were studied by NMR measurements of one-bond C-H residual dipolar couplings in partially oriented liquid crystal solutions. The conformations of the three oligosaccharides were analyzed by generating thousands of structures using a Monte-Carlo method. Two different strategies were applied to calculate theoretical dipolar couplings for these structures. In the first method, the orientation of the molecule was calculated from the optimal fit of the molecular model to the experimental data, while in the second method the orientation tensor was calculated directly from the moment of inertia of the molecular model. Both methods of analysis give similar results but with slightly better agreement with experiment for the former one. The analysis of the results implies a single unique conformation for both blood group epitopes in solution in disagreement with theoretical models suggesting the existence of two conformers in solution.     

Identification of the epitope for anti-cystatin C antibody

Human cystatin C (hCC), like many other amyloidogenic proteins, has been shown to form dimers by exchange of subdomains of the monomeric protein. Considering the model of hCC fibrillogenesis by propagated domain swapping, it seems possible that inhibition of this process should also suppress the entire process of dimerization and fibrillogenesis which leads to specific amyloidosis (hereditary cystatin C amyloid angiopathy (HCCAA)). It was reported that exogenous agents like monoclonal antibody against cystatin C are able to suppress formation of cystatin C dimers. In the effort to find a way of controlling the cystatin fibrillization process, the interactions between monoclonal antibody Cyst-13 and cystatin C were studied in detail. The present work describes the determination of the epitope of hCC to a monoclonal antibody raised against cystatin C, Cyst-13, by MALDI mass spectrometry, using proteolytic excision of the immune complex. The shortest epitope sequence was determined as hCC(107-114). Affinity studies of synthetic peptides revealed that the octapeptide with epitope sequence does not have binding ability to Cyst-13, whereas its longer counterpart, hCC(105-114), binds the studied antibody. The secondary structure of the peptides with epitope sequence was studied using circular dichroism and NMR spectroscopy.     

Comprehensive characterization of monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry

The pharmaceutical industry’s interest in monoclonal antibodies (mAbs) and their derivatives has spurred rapid growth in the commercial and clinical pipeline of these effective therapeutics. The complex micro-heterogeneity of mAbs requires in-depth structural characterization for critical quality attribute assessment and quality assurance. Currently, mass spectrometry (MS)-based methods are the gold standard in mAb analysis, primarily with a bottom-up approach in which immunoglobulins G (IgGs) and their variants are digested into peptides to facilitate the analysis. Comprehensive characterization of IgGs and the micro-variants remains challenging at the proteoform level. Here, we used both top-down and middle-down MS for in-depth characterization of a human IgG1 using ultra-high resolution Fourier transform MS. Our top-down MS analysis provided characteristic fingerprinting of the IgG1 proteoforms at unit mass resolution. Subsequently, the tandem MS analysis of intact IgG1 enabled the detailed sequence characterization of a representative IgG1 proteoform at the intact protein level. Moreover, we used the middle-down MS analysis to characterize the primary glycoforms and micro-variants. Micro-variants such as low-abundance glycoforms, C-terminal glycine clipping, and C-terminal proline amidation were characterized with bond cleavages higher than 44% at the subunit level. By combining top-down and middle-down analysis, 76% of bond cleavage (509/666 amino acid bond cleaved) of IgG1 was achieved. Taken together, we demonstrated the combination of top-down and middle-down MS as powerful tools in the comprehensive characterization of mAbs.     

Formation of monoclonal antibody against a major ginseng component, ginsenoside Rb1 and its characterization

The ratio of hapten and bovine serum albumin in an antigen conjugate was determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibody against ginsenoside Rb1 was produced by fusing splenocytes immunized with a ginsenoside Rb1- bovine serum albumin conjugate with HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A very small cross-reaction appeared with ginsenoside Rc and ginsenoside Rd. The full measuring range of the assay extends from 20 ngml-1 to 400 ngml-1 of ginsenoside Rb1. This revised version was published online in July 2006 with corrections to the Cover Date.     

Localized conformational interrogation of antibody and antibody-drug conjugates by site-specific carboxyl group footprinting

Establishing and maintaining conformational integrity of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) during development and manufacturing is critical for ensuring their clinical efficacy. As presented here, we applied site-specific carboxyl group footprinting (CGF) for localized conformational interrogation of mAbs. The approach relies on covalent labeling that introduces glycine ethyl ester tags onto solvent-accessible side chains of protein carboxylates. Peptide mapping is used to monitor the labeling kinetics of carboxyl residues and the labeling kinetics reflects the conformation or solvent-accessibility of side chains. Our results for two case studies are shown here. The first study was aimed at defining the conformational changes of mAbs induced by deglycosylation. We found that two residues in C_H2 domain (D268 and E297) show significantly enhanced side chain accessibility upon deglycosylation. This site-specific result highlighted the advantage of monitoring the labeling kinetics at the amino acid level as opposed to the peptide level, which would result in averaging out of highly localized conformational differences. The second study was designed to assess conformational effects brought on by conjugation of mAbs with drug-linkers. All 59 monitored carboxyl residues displayed similar solvent-accessibility between the ADC and mAb under native conditions, which suggests the ADC and mAb share similar side chain conformation. The findings are well correlated and complementary with results from other assays. This work illustrated that site-specific CGF is capable of pinpointing local conformational changes in mAbs or ADCs that might arise during development and manufacturing. The methodology can be readily implemented within the industry to provide comprehensive conformational assessment of these molecules.     

A DLA-associated polymorphic cell surface determinant defined by the murine monoclonal antibody W3G10

A polymorphic determinant on the surface of canine peripheral blood mononuclear cells (PBMC) has been identified using the murine monoclonal antibody W3G10. The determinant, which has a molecular weight of approximately 39 Kd, is associated with DLA-Dw1 (w = workshop designation), -Dw3, -Dw4, -Dw10, -Dsea1 (sea = Seattle designation) and -Dsea9, but not -Dw8, -Dw9, -Dsea4, -Dsea10, -Dsea16 or -Dsea17, and segregates in Mendelian fashion. There does not appear to be any linkage disequilibrium with currently recognized DLA-A, -B or -C alleles. Cytotoxic effector function was eliminated when W3G10 positive alloantigen primed PBMC were treated with W3G10. However, cytotoxicity was not affected when W3G10 negative PBMC were used, thus further confirming polymorphism. The data suggest that the antigen identified by McAb W3G10 is not a typical class I or class II MHC antigen, but may be encoded by a closely linked gene.     

A monoclonal antibody monitoring band 3 modifications in human red blood cells

Morphologic and methabolic erythrocyte modifications are thought to be the basis of cell removal from circulating blood. A significant role has been ascribed to the immunological network which may remove aged or misshapen erythrocytes through the binding of specific autoantibodies. Along this line recent observations indicate that a senescence antigen appears in consequence of postsynthetic modifications of band 3, one of the most important erythrocyte membrane proteins, which accounts for many functional activities of the red cells. On this basis, we raised a mouse hybridoma anti-band 3 monoclonal antibody (B6 MoAb) of the IgG2a class which monitors band 3 differences among normal red blood cells separated by Percoll density gradient. These differences are outlined by the decrease of B6 MoAb binding to band 3 monomer, the appearance of an 80–90 kDa new band, lighter than band 3, and the increase of low molecular weight fragments in the 4.5 region. The B6 MoAb appears to be very useful in detecting modifications of band 3 since it bind to a 19 kDa Chy-Try fragment estimated to be sensitive to aging.Abbreviations PBS Phosphate Buffer Saline - MoAb Monoclonal Antibody - RBCs Red Blood Cells - PMSF Phenylmethylsulphonyl Fluoride - PVC Polyvinyl Chloride - ACD Acid Citrate Dextrose - HMWP High Molecular Weight Polymers - Chy-Try Chymotrypsin-Trypsin Digested - i.p. intraperitoneum - ELISA Enzyme Linked Immuno Sorbent Assay - Hepes 4-(2-Hydroxyethyl)-piperazine-1-ethane-sulfonic acid. Enzymes: trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), neuraminidase (EC 3.2.1.18)     

Characterization of the co‐elution of host cell proteins with monoclonal antibodies during protein A purification

Protein A chromatography is commonly used as the initial step for purifying monoclonal antibody biotherapeutics expressed in mammalian tissue culture cells. The purpose of this step, as well as later chromatography steps, is, in part, to remove host cell proteins (HCPs) and other related impurities. Understanding the retention mechanism for the subset of HCPs retained during this step is of great interest to monoclonal antibody (mAb) process developers because it allows formation of a guided HCP clearance strategy. However, only limited information is available about the specific HCPs that co‐purify with mAbs at this step. In this study, a comprehensive comparison of HCP subpopulations that associated with 15 different mAbs during protein A chromatography was conducted by a 2D‐LC‐HDMS^E approach. We found that a majority of CHO HCPs binding to and eluting with the mAbs were common among the mAbs studied, with only a small percentage (～10% on average) of a mAb's total HCP content in the protein A (PrA) eluate specific for a particular antibody. The abundance of these HCPs in cell culture fluids and their ability to interact with mAbs were the two main factors determining their prevalence in protein A eluates. Potential binding segments for HCPs to associate with mAbs were also studied through their co‐purification with individual Fc and (Fab′)₂ antibody fragments. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:708–717, 2016     

A monoclonal antibody to swine erythrocytes recognizes the B blood group on the major glycophorin

Recently monoclonal antibodies (mAbs) to swine red blood cells have been described. One of them (1AC11) was specific for the major swine glycoprotein with a molecular weight of 45 kDa and another mAb, 2G2, recognized the B ^a allele in the B system of swine blood groups. Immunoblotting experiments to characterize the mAb 2G2 indicated that it reacts with an antigen of 45 kDa, present on the aqueous phase, glycophorin fraction, of swine red blood cells with the B ^a allele and does not react with B ^bB^b homozygous cells. The antigen recognized by 2G2 has the same characteristics as the major glycophorin recognized by 1AC11, so we can conclude that the B system of the swine blood group is on the major glycophorin of swine erythrocyte membranes.     

Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry

An advanced two-dimensional liquid chromatography/mass spectrometry platform was used to quantify individual host cell proteins (HCPs) present at various purification steps for several therapeutic monoclonal antibodies (mAbs) produced in Chinese hamster ovary cells. The methodology produced reproducible identifications and quantifications among replicate analyses consistent with a previously documented individual limit of quantification of ~13 ppm. We were able to track individual HCPs from cell culture fluid to protein A eluate pool to subsequent viral inactivation pool and, in some cases, further downstream. Approximately 500 HCPs were confidently identified in cell culture fluid and this number declined progressively through the purification scheme until no HCPs could be confidently identified in polishing step cation-exchange eluate pools. The protein A eluate pool of nine different mAbs contained widely differing numbers, and total levels, of HCPs, yet the bulk of the total HCP content in each case consisted of a small subset of normally intracellular HCPs highly abundant in cell culture fluid. These observations hint that minimizing cell lysis during cell culture/harvest may be useful in minimizing downstream HCP content. Clusterin and actin are abundant in the protein A eluate pools of most mAbs studied. HCP profiling by this methodology can provide useful information to process developers and lead to the refinement of existing purification platforms.     

Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry

An advanced two-dimensional liquid chromatography/mass spectrometry platform was used to quantify individual host cell proteins (HCPs) present at various purification steps for several therapeutic monoclonal antibodies (mAbs) produced in Chinese hamster ovary cells. The methodology produced reproducible identifications and quantifications among replicate analyses consistent with a previously documented individual limit of quantification of ~13 ppm. We were able to track individual HCPs from cell culture fluid to protein A eluate pool to subsequent viral inactivation pool and, in some cases, further downstream. Approximately 500 HCPs were confidently identified in cell culture fluid and this number declined progressively through the purification scheme until no HCPs could be confidently identified in polishing step cation-exchange eluate pools. The protein A eluate pool of nine different mAbs contained widely differing numbers, and total levels, of HCPs, yet the bulk of the total HCP content in each case consisted of a small subset of normally intracellular HCPs highly abundant in cell culture fluid. These observations hint that minimizing cell lysis during cell culture/harvest may be useful in minimizing downstream HCP content. Clusterin and actin are abundant in the protein A eluate pools of most mAbs studied. HCP profiling by this methodology can provide useful information to process developers and lead to the refinement of existing purification platforms.