Identification of metallothionein isoforms on capillary zone electrophoresis by adding anti-metallothionein antibody

The aim of this study was to identify metallothionein (MT) isoforms in mouse liver by using capillary zone electrophoresis (CZE). Purified MT-1 and MT-2 isoforms were completely separated by CZE using a polyacrylamide-coated tube at physiologic pH. There were two peaks in the cytosol fraction prepared from zinc-injected mouse liver, in which the migration times corresponded with those of purified MT-1 and MT-2 isoforms. When anti-MT monoclonal antibody was added with the purified MT-1 or MT-2 solution, the peaks decreased. Furthermore, the two peaks in the cytosol prepared from Zn-injected mouse liver decreased in a time-dependent manner from the electropherogram after the addition of the antibody. Therefore, those peaks were identified as MT-1 and MT-2 isoforms, respectively. In conclusion, the addition of anti-MT monoclonal antibody to the cytosol fraction of tissues is an effective method for identification of MT isoforms after separation using CZE.     

Characterization of the glycosylation state of a recombinant monoclonal antibody using weak cation exchange chromatography and mass spectrometry.

Recombinant monoclonal antibody heterogeneity is inherent due to various enzymatic and non-enzymatic modifications. In this study, a recombinant humanized monoclonal IgG1 antibody with different states of glycosylation on the conserved asparagine residue in the CH(2) domain was analyzed by weak cation exchange chromatography. Two major peaks were observed and were further characterized by enzymatic digestion and mass spectrometry. It was found that this recombinant monoclonal antibody contained three glycosylation states of antibody with zero, one or two glycosylated heavy chains. The peak that eluted earlier on the cation exchange column contained antibodies with two glycosylated heavy chains containing fucosylated biantennary complex oligosaccharides with zero, one or two terminal galactose residues. The peak that eluted later from the column contained antibodies with either zero, one or two glycosylated heavy chains. The oligosaccharide on the antibodies eluted in the later peak was composed of only two GlcNAc residues. These results indicate that conformational changes in large proteins such as monoclonal antibodies, caused by different types of neutral oligosaccharides as well as the absence of oligosaccharides, can be differentiated by cation exchange column chromatography.     

Cysteinylation of a monoclonal antibody leads to its inactivation

Post-translational modifications can have a signification effect on antibody stability. A comprehensive approach is often required to best understand the underlying reasons the modification affects the antibody's potency or aggregation state. Monoclonal antibody 001 displayed significant variation in terms of potency, as defined by surface plasmon resonance testing (Biacore), from lot to lot independent of any observable aggregation or degradation, suggesting that a post-translational modification could be driving this variability. Analysis of different antibody lots using analytical hydrophobic interaction chromatography (HIC) uncovered multiple peaks of varying size. Electrospray ionization mass spectrometry (ESI-MS) indicated that the antibody contained a cysteinylation post-translational modification in complementarity-determining region (CDR) 3 of the antibody light chain. Fractionation of the antibody by HIC followed by ESI-MS and Biacore showed that the different peaks were antibody containing zero, one, or two cysteinylation modifications, and that the modification interferes with the ability of the modified antibody arm to bind antigen. Molecular modeling of the modified region shows that this oxidation of an unpaired cysteine in the antibody CDR would block a potential antigen binding pocket, suggesting an inhibition mechanism.     

Bivalent monoclonal IgY antibody formats by conversion of recombinant antibody fragments

Monoclonal IgY have the potential to become unique tools for diagnostic research and therapeutic purposes since avian antibodies provide several advantages due to their phylogenetic difference when compared to mammalian antibodies. The mechanism of avian immunoglobulin gene diversification renders chicken an excellent source for the generation of recombinant scFv as well as Fab antibody libraries of high diversity. One major limitation of these antibody fragments, however, is their monovalent format, impairing the functional affinity of the molecules and, thereby, their applicability in prevalent laboratory methods. In this study, we generated vectors for conversion of avian recombinant antibody fragments into different types of bivalent IgY antibody formats. To combine the properties of established mammalian monoclonal antibodies with those of IgY constant domains, we additionally generated bivalent murine/avian chimeric antibody constructs. When expressed in HEK-293 cells, all constructs yielded bivalent disulfide-linked antibodies, which exhibit a glycosylation pattern similar to that of native IgY as assessed by lectin blot analysis. After purification by one step procedures, the chimeric and the entire avian bivalent antibody formats were analyzed for antigen binding and interaction with secondary reagents. The data demonstrate that all antibody formats provide comparable antigen binding characteristics and the well established properties of avian constant domains.     

Solution properties ofEscherichia coli-expressed VH domain of anti-neuraminidase antibody NC41

The V_H domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAG^TM), has been expressed in high yield (15–27 mg/L) inEscherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The V_H-FLAG was composed of three isoforms (pI values of ～4.6, 4.9, and 5.3) and the V_H molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the V_H isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20°C and concentrations of 5–10mg/ml the V_H domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its¹H NMR spectrum. Reagents such as CHAPS,n-octylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the V_H molecule, prevented dimerization of the V_H and permitted good-quality NMR spectra on isotope-labeled protein to be obtained.     

Purification of transgenic tobacco-derived recombinant human monoclonal antibody

Purification of recombinant monoclonal antibody from transgenic plant extract is technically challenging as it involves the processing of large volume of material, containing low titre of antibody, present along with large quantities of native proteins and other impurities. The conventional approach of capturing antibody from a clarified extract using packed-bed chromatography is therefore not particularly suitable. This study evaluates the suitability of using a combination of ultrafiltration and chromatography for purifying transgenic tobacco-derived human monoclonal antibody. A two-stage cascade ultrafiltration process removed about 97% impurities while ensuring almost complete recovery of antibody, providing 32-fold antibody enrichment in the process. The primary objective of the ultrafiltration step was to reduce the burden on the subsequent chromatographic steps. A two-step chromatographic process was then used to eliminate remaining impurities. Using this approach, recombinant human antibody expressed in tobacco could be purified to greater than 95% purity with 50% overall recovery (ca. 12.5 mg antibody/kg tobacco tissues).     

Alz 50, a monoclonal antibody to Alzheimer's disease antigen, cross-reacts with tau proteins from bovine and normal human brain

Microtubule-associated protein tau from bovine brain reacted on immunoblots and on enzyme-linked immunosorbent assay with a monoclonal antibody, Alz 50, which has previously been found to bind to an Alzheimer disease-specific antigen. The apparent affinity of binding of Alz 50 to tau was 2.1 X 10(-9) M on competitive enzyme-linked immunosorbent assay, and it was in the same range as for Tau-1 (0.5 X 10(-9) M), an antibody raised against purified bovine tau proteins. Immunoblotting of trypsin-digested tau revealed differences between Alz 50 and Tau-1 binding sites. The binding of both antibodies to tau was not affected by prior treatment with phosphatase, indicating that the cross-reactivity of Alz 50 with tau is due to the presence of phosphate-independent epitope. This epitope then differs from phosphate-dependent tau epitopes often shared with other cytoskeletal proteins. Alz 50 and Tau-1 binding sites were present in all isoelectric (pI 6-8) and molecular weight variants of tau. In contrast, phosphate-dependent epitopes recognized by another tau-reactive antibody (NP14) were found mostly in acidic tau variants. Similarly to tau proteins from bovine brain, tau-enriched preparations from normal human brain contained Alz 50 and Tau-1 reactive sites in all isoelectric (pI 6.5-8.5) and molecular weight variants. Our observation of Alz 50 cross-reactivity with tau suggests a relationship between tau and the novel protein identified recently in Alzheimer brains.     

Loss of N-terminal charged residues of mouse CD3 epsilon chains generates isoforms modulating antigen T cell receptor-mediated signals and T cell receptor-CD3 interactions

The antigen T cell receptor (TCR)-CD3 complexes present on the cell surface of CD4(+) T lymphocytes and T cell lines express CD3 epsilon chain isoforms with different isoelectric points (pI), with important structural and functional consequences. The pI values of the isoforms fit the predicted pI values of CD3 epsilon chains lacking one, two, and three negatively charged amino acid residues present in the N-terminal region. Different T cells have different ratios of CD3 epsilon chain isoforms. At a high pI, degraded CD3 epsilon isoforms can be better recognized by certain anti-CD3 monoclonal antibodies such as YCD3-1, the ability of which to bind to the TCR-CD3 complex is directly correlated with the pI of CD3 epsilon. The abundance of CD3 epsilon isoforms can be modified by treatment of T cells with the proteinase inhibitor phenanthroline. In addition, these CD3 epsilon isoforms have functional importance. This is shown, first, by the different structure of TCR-CD3 complexes in cells possessing different amounts of isoforms (as observed in surface biotinylation experiments), by their different antigen responses, and by the stronger interaction between low pI CD3 epsilon isoforms and the TCR. Second, incubation of cells with phenanthroline diminished the proportion of degraded high pI CD3 epsilon isoforms, but also the ability of the cells to deliver early TCR activation signals. Third, cells expressing mutant CD3 epsilon chains lacking N-terminal acid residues showed facilitated recognition by antibody YCD3-1 and enhanced TCR-mediated activation. Furthermore, the binding avidity of antibody YCD3-1 was different in distinct thymus populations. These results suggest that changes in CD3 epsilon N-terminal chains might help to fine-tune the response of the TCR to its ligands in distinct activation situations or in thymus selection.     

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.     

Liquid-liquid phase separation of a monoclonal antibody and nonmonotonic influence of Hofmeister anions

Liquid-liquid phase separation was studied for a monoclonal antibody in the monovalent salt solutions of KF, KCl, and KSCN under different pH conditions. A modified Carnahan-Starling hard-sphere model was utilized to fit the experimental data, establish the liquid-liquid coexistence curve, and determine antibody-antibody interactions in the form of T_c (critical temperature) under the different solution conditions. The liquid-liquid phase separation revealed the complex relationships between antibody-antibody interactions and different solution conditions, such as pH, ionic strength, and the type of anion. At pH 7.1, close to the pI of the antibody, a decrease of T_c versus ionic strength was observed at low salt conditions, suggesting that the protein-protein interactions became less attractive. At a pH value below the pI of the antibody, a nonmonotonic relationship of T_c versus ionic strength was apparent: initially as the ionic strength increased, protein-protein interactions became more attractive with the effectiveness of the anions following the inverse Hofmeister series; then the interactions became less attractive following the direct Hofmeister series. This nonmonotonic relationship may be explained by combining the charge neutralization by the anions, perhaps with the ion-correlation force for polarizable anions, and their preferential interactions with the antibody.     

Thermodynamic basis for antibody binding to Z-DNA: comparison of a monoclonal antibody and its recombinant derivatives

Antibody engineering represents a promising area in biotechnology. Recombinant antibodies can be easily manipulated generating new ligand and effector activities that can be used as prototype magic bullets. On the other hand, an extensive knowledge of recombinant antibody binding and stability features are essential for an efficient substitution. In this study, we compared the stability and protein binding properties of two recombinant antibody fragments with their parental monoclonal antibody. The recombinant fragments were a monomeric scFv and a dimeric one, harboring human IgG1 CH2-CH3 domains. We have used fluorescence titration quenching to determine the thermodynamics of the interaction between an anti-Z-DNA monoclonal antibody and its recombinant antibody fragments with Z-DNA. All the antibody fragments seemed to bind DNA similarly, in peculiar two-affinity states. Enthalpy-entropy compensation was observed for both affinity states, but a marked entropy difference was observed for the monomeric scFv antibody fragment, mainly for the high affinity binding. In addition, we compared the stability of the dimeric antibody fragment and found differences favoring the monoclonal antibody. These differences seem to derive from the heterologous expression system used.     

Multiforms of mammalian adenylate kinase and its monoclonal antibody against AK1

An attempt has been made to determine the intracellular distribution of the multiforms of the adenylate kinase (AK) isoenzymes in mammalian tissues, to shed some light on their physiological roles, especially in energy metabolism. The adenylate kinase zymograms obtained from isoelectric focusing yielded two typical isoform patterns: (1) with a pI greater than or equal to 9 and 8.6, specific for bovine skeletal muscle, heart, aorta and brain, and (2) with a pI = 7.9 and 7.1, specific for liver and kidney. Pattern (1) was attributed to the cytosolic isoenzyme (AK1) as demonstrated by immunostaining with anti-AK1. Pattern (2) was attributed to the mitochondrial isoenzyme (AK2). These results were largely confirmed by chromatofocusing experiments. The AK1 isoenzyme was partially purified from the cytosol fraction of bovine aortic smooth muscle and had an apparent Mr of 23.5 kilodaltons. Its kinetic features are discussed from a comparative standpoint. Finally, the human serum AK1 isoform was also detected by Western blotting with a monoclonal antibody directed against crystalline porcine muscle AK1. These results are to form the basis of further studies on the 'aberrant' adenylate kinase isoenzyme from the serum of Duchenne muscular dystrophics.     

Identification of cysteinylation of a free cysteine in the Fab region of a recombinant monoclonal IgG1 antibody using Lys-C limited proteolysis coupled with LC/MS analysis

MAB007, an IgG1 monoclonal antibody, is unique because of the presence of a free cysteine residue in the Fab region at position 104 on the heavy chain in the CDR3 region. Mass spectrometric analysis of intact MAB007 showed multiple peaks varying in mass by 120-140 Da that cannot be fully attributed to glycosylation isoforms typically present in IgG molecules. Limited proteolysis of MAB007 with Lys-C led to a single cleavage at the C-terminus of a lysine residue in the hinge region of the heavy chain at position 222, generating free Fab and Fc fragments. Reversed-phase liquid chromatography/mass spectrometry analysis of the Fab and Fc fragments revealed several modifications. The Fab fraction showed cysteinylation of a free cysteine in the CDR3 region resulting in a mass shift of 119 Da. Using limited proteolysis, we also identified modifications resulting in a mass increase of 127 Da in the Fc region, corresponding to C-terminal lysine variants in the heavy chain. Other modifications, such as oxidation (+16 Da) and succinimide formation (-17 Da), were also detected in the Fab fragment. The cysteinylation observed after limited proteolysis was confirmed by peptide mapping coupled with tandem mass spectrometry analysis.     

Solution properties ofEscherichia coli-expressed VH domain of anti-neuraminidase antibody NC41

The V_H domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAG^TM), has been expressed in high yield (15–27 mg/L) inEscherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The V_H-FLAG was composed of three isoforms (pI values of 4.6, 4.9, and 5.3) and the V_H molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the V_H isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20°C and concentrations of 5–10mg/ml the V_H domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its¹H NMR spectrum. Reagents such as CHAPS,n-octylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the V_H molecule, prevented dimerization of the V_H and permitted good-quality NMR spectra on isotope-labeled protein to be obtained.     

Kinetics and thermodynamics of dimer formation and dissociation for a recombinant humanized monoclonal antibody to vascular endothelial growth factor.

The recombinant humanized antibody (rhuMAb) VEGF has a high affinity for vascular endothelial growth factor and is currently being evaluated in clinical trials as a cancer therapeutic. Under acidic pH and low ionic strength conditions, the antibody was predominantly present as monomer. Under physiological conditions, the appearance of significant amounts of a noncovalent, reversible dimer were observed by size-exclusion chromatography. The kinetics and thermodynamics of the reversible self-association for rhuMAb VEGF monomer were investigated as a function of pH, temperature, and ionic strength by size-exclusion chromatography using the concentration jump method. The rate constant for dimer formation ranged 23-112 M(-)(1) min(-)(1) under the conditions studied, values that are significantly lower than those reported in the literature for other proteins that self-associate. The rate constant for dissociation ranged 0.0039-0.021 min(-)(1). Gibbs' free energies, enthalpies, entropies, and activation energies were determined and revealed that dimer formation is optimal at pH 7.5-8.0, which may be reflective of charge shielding occurring near the pI of the protein. There was a negative change in entropy for dissociation (values from -18.1 to -12.8 cal/mol K). In the presence of D(2)O or 1 M NaCl, dimerization was enhanced. The results of the kinetic and thermodynamic analysis of this study indicate that rhuMAb VEGF dimerization occurs primarily through hydrophobic interactions.     

Micro isoelectric point heterogeneity of a murine monoclonal antibody (L6) originating from cell cultivation conditions

Summary Isoelectric point (pI) microheterogeneity of an IgG monoclonal antibody (L6) was studied by rapid affinity chromatography, isoelectric focusing on polyacrylamide gels, and rapid ion-exchange chromatography. An abiotic or strictly chemical basis for observed changes is postulated. The described methods are useful for monoclonal antibody process monitoring since they are rapid, automated assay procedures.     

Motility-dependence of the heterogenous staining of culture cells by a monoclonal anti-tropomyosin antibody

A monoclonal antibody (CG1) which recognizes tropomyosin isoforms 1 and 3 of chicken embryo fibroblasts was used to detect what is a motility-dependent change in the availability of the antigenic determinant in tropomyosin molecules along microfilaments. Immunofluorescence microscopy with this antibody revealed a heterogenous staining pattern among chicken embryo fibroblasts cells such that a population (17%) of cells showed only background staining. Stress fibers in about half the population of the cells stained weakly with this antibody, while the stress fibers in another population of cells (35%) showed very strong staining. After glycerination or cytochalasin B treatment, all of the cells became positive in reaction to CG1 antibody, suggesting that the antigenic determinant was present in every cell. On the other hand, all of the cells after brief nonionic detergent treatment became negative to CG1 antibody. The CG1 staining pattern was not significantly changed in cells at different stages after release from colcemid blockage, nor was a brief treatment of cells with buffer containing 2 M urea, mild trypsin, chymotrypsin, or V.8 protease effective in changing the reactivity. However, most of the cells with a morphology typical of movement, and all of the contracted, glycerinated cells were strongly positive to CG1 antibody. These results suggest that the unmasking of the CG1 determinant may be motility-dependent. Immunoblot analysis showed that forced modification on the cysteine residue of tropomyosin molecules, caused either by performic acid oxidation or by disulfide cross-linking with the chemical 5,5'-dithiobis (2-nitrobenzoate), results in drastic changes in the reactivity of the different isoforms to CG1 antibody. These results indicate that the cysteine residue is involved in the CG1 determinant. The motility-dependent unmasking of this determinant may suggest an important role for nonmuscle tropomyosin in regulating cell motility.     

Multispecificity of a recombinant anti‐ras monoclonal antibody

Recombinant monoclonal antibodies (Ab's) have widespread application as research tools, diagnostic reagents and as biotherapeutics. Whilst studying the cellular molecular switch protein m‐ras, a recombinant monoclonal antibody to m‐ras was generated for use as a research tool. Antibody genes from a single rabbit B cell secreting IgG to an m‐ras specific peptide sequence were expressed in mammalian cells, and monoclonal rabbit IgG binding was characterized by ELISA and peptide array blotting. Although the monoclonal Ab was selected for specificity to m‐ras peptide, it also bound to both recombinant full‐length m‐ras and h‐ras proteins. The cross‐reactive binding of the monoclonal Ab to h‐ras was defined by peptide array blot revealing that the Ab showed preference for peptide sequences containing multiple positively charged amino acid residues. These data reinforce the concept of antibody multispecificity through multiple interactions of the Ab paratope with diverse polypeptides. They also emphasize the importance of immunogen and Ab selection processes when generating recombinant monoclonal Ab's.     

Changes in monoclonal antibody productivity of recombinant BHK cells immobilized in collagen gel particles

Animal cell perfusion high density culture is often adopted for the production of biologicals in industry. In high density culture sometimes the productivity of biologicals has been found to be enhanced. Especially in immobilized animal cell culture, significant increase in the productivity has been reported. We have found that the specific monoclonal antibody (MAb) productivity of an immobilized hybridoma cell is enhanced more than double. Several examples of enhancing productivities have been also shown by collagen immobilized cells. Immobilized cells involve some different points from non-immobilized cells in high density culture: In immobilized culture, some cells are contacted together, resulting in locally much higher cell concentration more than 10⁸ cells/ml. Information originating from a cell can be easily transduced to the others in immobilized culture because the distance between cells is much nearer. Here we have performed collagen gel immobilized culture of recombinant BHK cells which produce a human IgG monoclonal antibody in a protein-free medium for more than three months. In this high density culture a stabilized monoclonal antibody production was found with around 8 times higher specific monoclonal antibody productivity compared with that in a batch serum containing culture. No higher MAb productivity was observed using a conditioned medium which was obtained from the high density culture, indicating that no components secreted from the immobilized cells work for enhancing monoclonal antibody production. The MAb productivity by the non-immobilized cells obtained by dissolving collagen using a collagenase gradually decreased and returned to the original level in the batch culture using a fresh medium. This suggests that the direct contact of the cells or a very close distance between the cells has something to do with the enhancement of the MAb productivity, and the higher productivity is kept for a while in each cell after they are drawn apart.     

Proton nuclear magnetic resonance study of a selectively deuterated mouse monoclonal antibody: use of two-dimensional homonuclear Hartmann-Hahn spectroscopy

A 1H NMR study of a selectively deuterated mouse anti-dansyl monoclonal antibody is reported. Two-dimensional homonuclear Hartmann-Hahn (2D-HOHAHA) spectroscopy was found to be effective for establishing the connectivity between the C2-H and C4-H protons of His residues in the antibody molecule. It has been concluded that 1) even in the case of large proteins such as an antibody, HOHAHA peaks can be observed for amino acid residues that are located in a flexible environment, and 2) deuterium labeling is effective in reducing the efficiency of spin relaxation and makes it possible to increase the number of observed HOHAHA cross peaks. It was suggested that 2D-HOHAHA can also be used to obtain information concerning the flexible parts of antibody molecules.