Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity

An alternative method to traditional 2-dimensional gel electrophoresis (2D-PAGE) and its application in characterizing the inherent charge heterogeneity of chromatographically isolated monoclonal antibody heavy and light chains is described. This method, referred to as ChromiCE, utilizes analytical size-exclusion chromatography (SEC), performed under reducing and denaturing conditions, followed by imaged capillary isoelectric focusing (icIEF) of the chromatographically separated heavy and light chains. Under conditions suitable for the subsequent icIEF analysis, the absolute and relative SEC elution volumes of the heavy and light chains were found to be highly pH dependent, a phenomenon that can be exploited in optimizing chromatographic separation. Compared to 2D-PAGE, the ChromiCE method substantially decreases the time and labor needed to complete the analysis, improves reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown.     

Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells

7H2HM is a new humanized recombinant monoclonal antibody (MAb) directed against insulin-like growth factor-1 receptor and produced in CHO cells. Homogeneity of intact antibody, reduced light and heavy chains, Fab and Fc fragments were investigated by analytical methods based on mass (SDS-PAGE, SEC), charge (IEF, C-IEX) and hydrophobicity differences (RP-HPLC, HIC) and compared side-by-side with A2CHM, produced in NS0 cells. Primary structures and disulfide bridge pairing were analyzed by microsequencing (Edman degradation), mass spectrometry (MALDI-TOF, ES-TOF) and peptide mapping after enzymatic digestion (Trypsin, endoprotease Lys-C, papain). The light chains demonstrated the expected sequences. The heavy chains yielded post-translational modifications previously reported for other recombinant humanized or human IgG1 such as N-terminal pyroglutamic acid, C-terminal lysine clipping and N-glycosylation for asparagine 297. More surprisingly, two-thirds of the 7H2HM heavy chains were shown to contain an additional 24-amino-acid sequence, corresponding to the translation of an intron located between the variable and the constant domains. Taken together these data suggest that 7H2HM is a mixture of three families of antibodies corresponding (i) to the expected structure (17%; 14,9297 Da; 1330 amino acids), (ii) a variant with a translated intron in one heavy chains (33%; 15,2878 Da; 1354 amino acids) and (iii) a variant with translated introns in two heavy chains (50%; 15,4459 Da; 1378 amino acids), respectively. RP-HPLC is not a commonly used chromatographic method to assess purity of monoclonal antibodies but unlike to SEC and SDS-PAGE, was able to show and to quantify the family of structures present in 7H2HM, which were also identified by peptide mapping, mass spectrometry and microsequencing.     

Characterization of a recombinant monoclonal antibody by mass spectrometry combined with liquid chromatography

In this report, we present the characterization of a humanized monoclonal antibody specific for the human epidermal growth factor receptor (hEGFR). Direct analysis by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) of peptide mixtures and chromatographically isolated fractions allowed identification of 94.0% and 85.4% of the amino acid sequence of light and heavy chains, respectively. Microheterogeneity sources were identified in light and heavy chains and a previously unreported posttranslational modification for immunoglobulins was found. One N-glycosylation site was identified in the heavy chain with non-sialylated bianntenary fucosylated structures. This study is one of the first to assess the potential of MALDI-MS in combination with more conventional protein chemistry techniques for the characterization of monoclonal antibodies.     

Idiotypic analysis of myeloma proteins: anti-DNA activity of monoclonal immunoglobulins bearing an SLE idiotype is more common in IgG than IgM antibodies

The anti-idiotype 3I which recognizes a determinant on kappa-chains of anti-DNA antibodies in SLE patients also recognizes a determinant on kappa-chains of 82/706 myeloma proteins tested. Twenty-nine of these 82 proteins bind to double-stranded DNA, including two monoclonal IgM, one monoclonal IgA, and 26 monoclonal IgG proteins. DNA binding is much more frequent in the IgG than in the IgM myeloma proteins (p less than 0.001), and is also associated with cationic antibody charge. Two-dimensional gel electrophoresis reveals markedly increased charge heterogeneity of both heavy and light chains of the monoclonal IgG as compared with the monoclonal IgM proteins. We postulate that the increased charge heterogeneity of the IgG-associated 3I-reactive kappa-light chains may reflect somatic mutation, and that DNA specificity within the 3I idiotype system arises by somatic mutation of germ-line genes found in normal individuals. DNA binding may be associated with those mutations that give rise to a cationic immunoglobulin charge.     

Characterization of monoclonal antibody size variants containing extra light chains

Size exclusion chromatography (SEC) is the most commonly used method to separate and quantify monoclonal antibody (mAb) size variants. MAb-A is an IgG1 subtype humanized monoclonal antibody recombinantly produced in Chinese hamster ovary (CHO) cells. SEC analysis of MAb-A resolved a peak, named Peak 1, which elutes between monomer and dimer peaks. MAb-A lots produced from different clones and production scales all have 0.2–0.3% of SEC Peak 1. Electron spray ionization—time of flight mass spectrometry (ESI-TOF MS), microfluidics capillary electrophoresis and sodium dodecyl sulfate-PAGE (SDS PAGE) results demonstrated that SEC Peak 1 contains two structural variants: MAb-A with one extra light chain (2H3L) and MAb-A with two extra light chains (2H4L). The C-terminal Cys of the extra light chain in Peak 1 variants is either a free thiol, capped by glutathione, cysteine, or another light chain. Both electrophoresis and LC/MS analyses of non-reduced and reduced samples suggested that the extra light chains are linked to the MAb-A light chain through disulfide bonds. Isolated SEC Peak 1 fraction had a potency of 50% relative to MAb-A reference material. The 50% potency loss may result from the reduced accessibility to the antigen-binding site caused by the extra light chain(s)’ steric hindrance.     

N端测序作为单克隆抗体常规放行分析方法的探讨

旨在探讨N端测序作为单克隆抗体常规放行分析方法的适用性。应用Edman降解法、质量肽图法对两个针对不同靶点的单抗进行N端测序,用肽图法寻找二者的特征性鉴别肽段,用离子色谱、毛细管区带电泳和成像毛细管等点聚焦电泳进行异质性分析。Edman降解法显示两个单抗轻链和重链的15个氨基酸分别完全一致,质量肽图法显示二者轻重链的T1肽段分别完全一致,而肽图法和3种异质性分析方法则可对两个抗体进行有效鉴别。由于人源化或人源单抗序列框架数量较为有限,两个单抗的N末端序列完全相同,运用Edman降解法进行N端测序是否能作为单抗的常规放行分析方法值得进一步商榷,同时上述多种方法可运用于单抗的鉴别分析,并可对其异质性进行控制,较N端测序分析更具有客观性。     

Controlling trisulfide modification in recombinant monoclonal antibody produced in fed-batch cell culture

Molecular heterogeneity was detected in a recombinant monoclonal antibody (IgG1 mAb) due to the presence of a trisulfide linkage generated by the post‐translational insertion of a sulfur atom into disulfide bonds at the heavy–heavy and heavy–light junctions. This molecular heterogeneity had no observable effect on antibody function. Nevertheless, to minimize the heterogeneity of the IgG1 mAb from run‐to‐run, an understanding of the impact of cell culture process conditions on trisulfide versus disulfide linkage formation was desirable. To investigate variables that might impact trisulfide formation, cell culture parameters were varied in bench‐scale bioreactor studies. Trisulfide analysis of the samples from these runs revealed that the trisulfide content in the bond between heavy and light chains varied considerably from <1% to 39%. Optimizing the culture duration and feeding strategy resulted in more consistent trisulfide levels. Cysteine concentration in the feed medium had a direct correlation with the trisulfide level in the product. Systematic studies revealed that cysteine in the feed and the bioreactor media was contributing hydrogen sulfide which reacted with the IgG1 mAb in the supernatant leading to the insertion of sulfur atom and formation of a trisulfide bond. Cysteine feed strategies were developed to control the trisulfide modification in the recombinant monoclonal antibody. Biotechnol. Bioeng. 2012; 109: 2523–2532. © 2012 Wiley Periodicals, Inc.     

Comparative proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate

We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant monoclonal antibody production (qMab) by engineered murine myeloma (NS0) cells. Four homogeneous NS0 cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab datasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant monoclonal antibody production.     

Production of an anti-mouse MHC class II monoclonal antibody with biological activity in transgenic tobacco

To produce a monoclonal antibody specific to a mouse major histocompatibility complex (MHC) class II protein, we synthesized the complementary DNAs for the heavy and light chains of a monoclonal antibody by PCR amplification. These cDNAs were then introduced separately into tobacco plant cells. After performing Northern blot analysis to confirm the expression of each of the chain genes in the transformed plants, we constructed transgenic plants expressing both the heavy and light chains by sexual crossing. The expression of the heavy and light chain genes in the sexually crossed plant was confirmed by Northern and Western blot analyses, respectively. Fluorocytometric analysis showed that the plant-derived antibodies, which we purified using a protein G affinity column, bound specifically to target cells that expressed the cognate MHC class II molecules on their cell surfaces. The results of this study demonstrate that a monoclonal antibody against mouse MHC class II proteins can be expressed in transgenic plants. They also show the specific binding activity of plant-derived antibodies to cognate antigens.     

Identification of a 34-kD polypeptide as a light chain of microtubule- associated protein-1 (MAP-1) and its association with a MAP-1 peptide that binds to microtubules

We examined the association of a 34-kD light chain component to the heavy chains of MAP-1 using a monoclonal antibody that specifically binds the 34-kD component and labels neuronal microtubules in a specific and saturable manner. Immunoprecipitation of MAP-1 heavy chains together with the 34-kD component by the antibody indicates that the 34-kD polypeptide forms a complex with MAP-1 heavy chains. Both major isoforms of MAP-1 heavy chains (MAP-1A and MAP-1B) were found in the immunoprecipitate. Digestion of MAP-1 with alpha-chymotrypsin and analysis of the chymotryptic peptides reveals a 120-kD fragment of the MAP-1 heavy chain that binds to microtubules and is precipitable with the 34-kD light chain antibody, suggesting that the 34-kD light chain also binds to this domain of the molecule. Since microtubules that contain the 120-kD fragment lack the long lateral projections characteristic of microtubules with intact MAP-1, the 34-kD light chains may be localized at or near the microtubule surface.     

Precise quantification of mixtures of bispecific IgG produced in single host cells by liquid chromatography-Orbitrap high-resolution mass spectrometry

Bispecific IgG are heterotetramers comprising 2 pairs of heavy and light chains. Co-expression of the 4 component chains in a single host cell typically yields the desired bispecific IgG plus up to 9 additional incorrect chain pairings. Several protein engineering strategies have been reported to facilitate the heterodimerization of antibody heavy chains or cognate pairing of antibody heavy and light chains. These technologies have been used to direct the efficient assembly of bispecific IgG in single host cells and minimize unwanted chain pairings. When purifying bispecific IgGs, the identification and quantification of low levels of closely related IgG contaminants are substantial analytical challenges. Here we have developed a robust high-throughput method for quantitative analysis of bispecific IgG preparations using novel online liquid chromatography in conjunction with an extended mass range Orbitrap-based high-resolution mass spectrometer. A mathematical method was developed to estimate the yields of the 2 isobaric species, namely the desired bispecific IgG and the light chain-scrambled IgG. The analytical methods described herein are anticipated to be broadly applicable to the development of bispecific IgG as drugs and potentially to other complex next-generation biotherapeutics.     

Monoclonal antibodies demonstrate limited structural homology between myosin isozymes from Acanthamoeba

We used a library of 31 monoclonal and six polyclonal antibodies to compare the structures of the two classes of cytoplasmic myosin isozymes isolated from Acanthamoeba: myosin-I, a 150,000-mol-wt, globular molecule; and myosin-II, a 400,000-mol-wt molecule with two heads and a 90-nm tail. This analysis confirms that myosin-I and -II are unique gene products and provides the first evidence that these isozymes have at least one structurally homologous region functionally important for myosin's role in contractility. Characterization of the 23 myosin-II monoclonal antibody binding sites by antibody staining of one-dimensional peptide maps and solid phase, competitive binding assays demonstrate that they bind to at least 15 unique sites on the myosin-II heavy chain. The antibodies can be grouped into six families, whose members bind close to one another. None of the monoclonal antibodies bind to myosin-II light chains and polyclonal antibodies against myosin-II light or heavy chain bind only to myosin-II light or heavy chains, respectively: no antibody binds both heavy and light chains. Six of eight monoclonal antibodies and one of two polyclonal sera that react with the myosin-I heavy chain also bind to determinants on the myosin-II heavy chain. The cross-reactive monoclonal antibodies bind to the region of myosin-II recognized by the largest family of myosin-II monoclonal antibodies. In the two papers that immediately follow, we show that this family of monoclonal antibodies to myosin-II binds to the myosin-II tail near the junction with the heads and inhibits both the actin-activated ATPase of myosin-II and contraction of gelled cytoplasmic extracts of Acanthamoeba cytoplasm. Further, this structurally homologous region may play a key role in energy transduction by cytoplasmic myosins.     

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.     

The association between murine beta 2-microglobulin and HLA class I heavy chains results in serologically detectable conformational changes of both chains

HLA-A3-, HLA-B7-, and HLA-CW3-transfected L cells, maintained in medium supplemented with murine serum so as to ensure that the human heavy chains were associated with murine beta 2-microglobulin, were subjected to a systematic serologic analysis for an evaluation of the structural consequences of such an heterologous association. The hybrid molecules exhibited alterations of their serologic reactivities that suggest the occurrence of structural modifications of both light and heavy chains. Thus, reactivity of HLA-A3-, HLA-B7-, and HLA-Cw3-transfected L cells with a monoclonal antibody (B1.1G6) directed at a human beta 2-microglobulin specific antigenic determinant was observed; this implies structural modifications of murine beta 2-microglobulin after its association with HLA class I heavy chains. Conversely, a profound reduction of the reactivity of the same transfectants with a monoclonal antibody (W6/32) directed at a monomorphic heavy chain related epitope was observed. The W6/32 reactivity was restored after replacement of the murine by the human light chain, indicating that the conformation adopted by the HLA class I heavy chain depends on the origin of the beta 2-microglobulin associated. Therefore it appears that the complex interactions that develop between the extracellular domains (including the one formed by the light chain) markedly influence the overall structure and the antigenic properties of HLA class I molecules.     

Investigations into charge heterogeneity of wool intermediate filament proteins

Genomic studies have shown that there are four abundant type I and type II intermediate filament proteins (IFPs) in wool. When separated using 2D-PAGE, the type I IFPs separated into four clearly defined major rows. The type II IFPs separated into two distinct staggered rows. The large number of spots seen by 2D-PAGE has previously been attributed to charge heterogeneity caused by post-translational modification of the protein. However, analysis of wool IFPs by 2D-PAGE techniques and mass spectrometry suggested an absence of phosphorylation or glycosylation modifications. Investigations with both the type I and type II IFPs showed that when single protein spots from a 2D-PAGE separation are eluted, re-focused and re-electrophoresed, several spots are formed on both the acidic and basic side of the original spot. Amino acid analysis, mass spectrometry and Ellman's assay support the hypothesis that the proteins have the same sequence but vary in isoelectric charge, due to differences in exposure of charged residues on the molecular surface. The cause of IFP charge heterogeneity is thus proposed to be a conformational equilibrium between several different forms of the same protein in the rehydration solution used for the first dimension.     

离子交换色谱对抗VEGFR2单抗的电荷异质性分析

目的采用离子交换高效液相色谱(CEX-HPLC)分析抗VEGFR2(抗血管内皮生长因子受体2)单抗制品的电荷异质性。方法应用CEX-HPLC技术,对VEGFR2单抗进行电荷异质性分析,并结合羧肽酶B(CpB)和N-糖酰胺酶F(EndoF2)酶切,初步研究其电荷异质性的成因。结果用CEX-HPLC分析CpB酶切前后单抗,证明其碱性变异体主要由C末端赖氨酸不均一性引起;分析EndoF2酶切前后处理的单抗,证明其酸性变异体部分由N-糖末端上的唾液酸修饰所引起。通过2种酶的顺序酶切可相对准确地分析含C-末端赖氨酸以及含唾液酸单抗的比例。结论采用CEX-HPLC可较好地分析单抗的电荷异质性;并结合合适的酶切处理,可判断单抗主要电荷异质性的来源,为保证单抗制品生产工艺的稳定性及质量控制提供了有效手段。     

Comparison of methionine oxidation in thermal stability and chemically stressed samples of a fully human monoclonal antibody

Methionine (Met) oxidation is a major degradation pathway of protein therapeutics. Met oxidation of a fully human recombinant monoclonal antibody was investigated under both chemically stressed conditions using tert-butylhydroperoxide (tBHP) and thermal stability conditions where the sample was incubated in formulation buffer at 25 degrees C for 12 months. This antibody has one Met residue on each of the light chains and four Met residues on each of the heavy chains. In the thermal stability sample, only Met residues 256 and 432 in the Fc region were oxidized to form methionine sulfoxide, while Met residues in the Fab region were relatively stable. The susceptibility of Met residues 256 and 432 was further confirmed by incubating samples with tBHP, which has been shown to induce Met oxidation. Further analysis revealed that the susceptible Met residues of each heavy chain were randomly oxidized in samples incubated with tBHP, while in the thermal stability sample, the susceptible Met residues of one heavy chain were preferentially oxidized.     

Separation of four class II molecules from DR2 and DRw6 homozygous B lymphoid cell lines

Four human class 11 molecules, one FA, one DC1, and two DR-like molecules, were isolated from DR2 and DRw6 homozygous cell lines by means of a variety of monoclonal antibodies and were compared with each other by two-dimensional (2-D) gel electrophoresis. Anti-DR2 or anti-DR3 + 5 + w6 sera immunoprecipitated two distinct light chains (L1 and L2) and one heavy chain (H1) from a DR2 or DRw6 homozygous cell line, respectively. One or both of these two class II molecules were also immunoprecipitated by DR-specific monoclonal antibodies and were considered to constitute a DR family of molecules. Three DC1-specific monoclonal antibodies, SDR4.1, Tu22, and PLM5, immunoprecipitated a set of heavy (H2) and light (L3) chains distinct from those of two DR-like molecules. The heavy chains of the DC1 antigens from DR2 and DRw6 cell lines were indistinguishable, whereas the light chains were structurally distinct from each other. A fourth molecule, FA, was identified by a novel monoclonal antibody and was also detected by two additional antibodies, Tu39 and SG171, that blocked the SB-specific T-cell proliferative response. The FA light chain (L4) was distinct from those of the former three antigens on both cell lines, whereas the FA heavy chain was indistinguishable from the DC1 heavy chain (H2) in this 2-D gel analysis. Thus, four light chains and two heavy chains were isolated from both DR2 and DRw6 homozygous cell lines. A possible gene-antigen organization of the DC-like antigens was also discussed.     

Biochemical and functional characterization of anti-HIV antibody-ELP fusion proteins from transgenic plants

The stability and recovery of recombinant proteins expressed in plants are improved by fusion to elastin-like peptides (ELPs). In order to test the suitability of ELP for the production of pharmaceutical proteins, transgenic plants were created that individually expressed the light and heavy chains of the broadly neutralizing anti-human immunodeficiency virus type 1 (anti-HIV-1) monoclonal antibody 2F5, which is being evaluated as a microbicide component. The antibody chains were expressed both with and without a C-terminal ELP fusion. Crossing these plants in all combinations resulted in transgenic lines producing the full antibody in four formats, with ELP on either the light or heavy chains, on both or on neither. Characterization of the affinity-purified antibodies by surface plasmon resonance spectroscopy showed that the kinetic binding parameters were identical to those of a Chinese hamster ovary (CHO) cell counterpart lacking ELP. N -Glycan analysis showed that all four derivatives contained predominantly oligo-mannose-type N -glycans and that the ELP fusions had no significant effect on N -glycan structure. It was concluded that ELP fusion to the light chain, heavy chain or both chains of a plant-derived antibody had no adverse affects on protein quality, but had a positive impact on the yield. ELP fusions do not interfere with folding, assembly, trafficking in the secretory pathway or post-translational modification, but enhance stability whilst at the same time simplifying recovery.     

Cloning and characterization of cDNAs coding for heavy and light chains of a monoclonal antibody (MabB23) specific for human plasma apolipoprotein B-100

We have determined the nucleotide sequences encoding the heavy and light chains of the Fab fragment of murine monoclonal antibody MabB23 (γ2b, λ), which is specific for human plasma apolipoprotein B-100 of low-density lipoproteins. The sequence analyses revealed that the variable regions of the heavy and light chains are members of mouse heavy-chain subgroup I(B) and λ light-chain, respectively. A few unusual amino acids in the framework and constant regions of the heavy-chain were also noticed