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Glycosylation is a common posttranslational modification and a key quality attribute of recombinant monoclonal antibodies. In particular, the level of core-fucosylation is critical to several Fe mediated biological functions. However, it is challenging to accurately determine the level of afucosylation due to its low level and possible distribution in multiple oligosaccharide species. Methods that can accurately determine the level of afucosylation were developed in the cunent study. Digestion of monoclonal antibodies with Endoglycosidases F2 and H, which cleave the glycosidic bond between the two primary GlcNAc residues, reduced complicated oligosaccharide population into two groups with either only GlcNAc or GlcNAc and the core-fucose residue. Analysis of the digested antibodies by LC-MS provided a quick estimate of the level of afucosylation. Alternatively, PNGaseF released oligosaccharides were labeled with 2-aminobenzamide, digested with Endoglycosidases F2 and H followed by normal phase HPLC with fluorescence detection. The results demonstrated that the latter method can provide an accurate quantitation of the level of afucosylation. [ABSTRACT FROM AUTHOR]

One of the basic structural features of human IgGi is the arrangement of the disulflde bond structure, 4 inter chain disulfide bonds in the hinge region and 12 intra chain disulfide bonds associated with twelve individual domains. Disuffide bond structure is critical for the structure, stability, and biological functions of IgG molecules. It has been known that inter chain disulfide bonds are more susceptible to reduction than intra chain disulfide bonds. However, a complete ranking of the susceptibility of disulfide bonds in IgGi molecules is lacking. A method including reduction, differential alkylation, and liquid chromatography-mass spectrometty (LC-MS) analysis was developed and employed to investigate the complete ranking order of the susceptibility of disulfide bonds in two recombinant monoclonal antibodies. The results confirmed that inter chain disulfide bonds were more susceptible than intra chain disulfide bonds. In addition, it was observed that the disuffide bonds between the light chain and heavy chain were more susceptible than disulfide bonds between the two heavy chains. The upper disuffide bond of the two inter heavy chain disuffide bonds was more susceptible than the lower one. Furthermore, disullide bonds in the CR2 domain were the most susceptible to reduction. Disulfide bonds in VI, CL, Vii, and CH1 domains had shuilar and moderate susceptibility, while disulfide bonds in the CH3 domain were the least susceptible to reduction. Interestingly, a difference between IgGit and IgGl.% was also observed. The difference in the susceptibility of inter light heavy chain disulfide bonds and inter heavy chain disuffide bonds was smaller in lgGlic than in IgG1L The intra chain disuffide bonds in the Fab region of IgGlic were also less susceptible than disulfide bonds in the Fab region of lgGl2. [ABSTRACT FROM AUTHOR]

Abstract Purpose  To determine the relative importance of direct hydrolysis and β-elimination, two common mechanisms of antibody hinge region fragmentation, and the impact of the conserved N-linked oligosaccharides in affecting antibody fragmentation under various pH. Methods  A recombinant monoclonal antibody was incubated in buffers of various pH at 40°C for 5 weeks. The level of fragmentation was measured using size-exclusion-chromatography (SEC). The specific sites of fragmentation were determined by analyzing SEC fractions using liquid chromatography mass spectrometry (LC-MS). Results  Direct hydrolysis was accelerated by acidic and basic pH, while β-elimination contributed to hinge region fragmentation at pH 7 and above. In addition, a shift of the major peptide bond hydrolysis sites in the hinge region towards the C-terminal direction with the decrease of sample pH from 9 to 5 was observed. At pH 4, the major cleavage site shifted outside the hinge region and was localized in the CH2 domain. Oligosaccharides did not affect hinge region fragmentation in the pH range of 5–9, however, at pH 4 oligosaccharides slowed down fragmentation in the CH2 domain. Conclusions  Antibody fragmentation level, sites and mechanisms were affected by pH. Oligosaccharides only affected the rate of fragmentation at pH 4. [ABSTRACT FROM AUTHOR]

Asparagine deamidation in the complementarity determining regions of recombinant monoclonal antibodies has been extensively studied and shown to have a negative impact on antigen binding. Those asparagine residues are typically exposed and thus have a higher tendency toward deamidation, depending also on local structure and environmental factors such as temperature and pH. Deamidation rates and products of a susceptible asparagine residue in the complementarity determining regions of a recombinant monoclonal antibody free in solution or in the antibody-antigen complex were studied. The results demonstrated that incubation of the antibody or its antigen complex generated a similar amount of aspartate. The expected amount of isoaspartate product was detected in free antibody, but it was completely lacking in the antibody-antigen complex. [ABSTRACT FROM AUTHOR]

A low percentage of free sulfhydryl is a common feature of recombinant monoclonal antibodies although, in theory, all cysteine residues should be involved in disulfide bonds. A differential alkylation method was developed to determine the percentage of free sulfhydryl at each cysteine residue of four recombinant monoclonal antibodies. Free sulfhydayl was first alkylated with 12C iodoacetic acid. Free sulfhydryl, resulting from the reduction of disulfide bonds, was then alkylated with 13C iodoacetic acid. Cysteine containing peptides that were modified by 13C iodoacetic acid showed a molecular weight that was 2 Da higher than the same peptide that was modified by 12C iodoacetic acid. Peptides, containing the same cysteine residues that were modified with both alkylation reagents, coeluted on reversed-phase chromatography. Analysis by mass spectrometry resulted in two partially overlapped m/z series for each cysteine containing peptide, corresponding to modification by iodoacetic acid with 12C or 13C. The percentage of free sulfhydryl was then calculated using the two mix series at each cysteine site. A low percentage of free sulfhydryl was detected at every cysteine residue in the four antibodies studied. Although different antibodies contained different levels of free sulfhydryl, similar distribution of free sulfhydryl in the domain structures was observed in the four antibodies. [ABSTRACT FROM AUTHOR]

Each human IgG1 antibody contains a total of thirty-two cysteine residues. In theory, all of them are involved in disulfide bonds, and no free sulfhydryl should be detected. However, literature has suggested that the presence of low levels of free sulfhydryl groups is likely a common feature of recombinant and wild type IgG1 antibodies. Currently, there is little information correlating the presence of free sulfhydryl to specific cysteine residues. The presence of free sulfhydryl groups in five recombinant monoclonal antibodies and their locations were further investigated in the current study. Free sulfhydryl groups were first modified using 5-idoacetamidofluorescein (5-IAF), which was followed by reduction of disulfide bonds and alkylation with iodoacetic acid (IAA). This procedure allowed differentiation of free cysteine residues from cysteine residues that were involved in disulfide bonding. In addition, it allowed a sensitive fluorescence detection of peptides with free sulfhydryl groups. The locations of the free sulthydryl groups were determined using mass spectrometry after fraction collection. The results indicated that different antibodies had different levels of free sulfhydryl due to multiple unpaired cysteine residues commonly in the constant domains. Furthermore, free sulfhydryl due to unpaired cysteine residues in the variable domains varied for different antibodies. Interestingly, free sulfhydryl was rarely associated with cysteine residues that were involved in interchain disulfide bonds. [ABSTRACT FROM AUTHOR]

A recombinant monoclonal antibody with trisulfide bonds and cysteinylation was thoroughly characterized in the current study. Trisulfide bonds and cysteinylation were first detected when the recombinant monoclonal antibody was analyzed by LC-MS to determine the molecular weights of the intact antibody and its F(ab')2 fragment generated from IdeS digestion. LC-MS analysis of nonreduced tryptic peptides indicated trisulfide bonds are associated with the interchain disulfide bonds of both A isoform and A/B isoform and cysteinylation is associated only with the A isoform. A low percentage of trisulfide bonds was detected in between the light chain and heavy chain disulfide bond of the A and A/B forms. While the majority of trisulfide bonds and cysteinylation is associated with the hinge region peptide that involves the four closely spaced cysteine residues of the heavy chain. The locations of trisulfide bond and cysteinylation were determined using a combination of Edman sequencing and LC-MS. In the A isoform, the major site of the trisulfide bond and cysteinylation is between the first disulfide bond in the hinge region. In the A/B isoform, the trisulfide was also located in between the disulfide bond that is formed by the second pair of cysteine residues. [ABSTRACT FROM AUTHOR]

Recombinant monoclonal antibody charge heterogeneity has been commonly observed as multiple bands or peaks when analyzed by charge-based analytical methods such as isoelectric focusing electrophoresis and cation or anion exchange chromatography. Those charge variants have been separated by some of the above-mentioned methods and used for detailed characterization. The utility of a combination of OFFGEL fractionation and weak anion exchange chromatography to separate the charge variants of a recombinant monoclonal antibody was demonstrated in the current study. Charge variants were separated into various fractions of high purity and then analyzed dioroughly by liquid chromatography mass spectrometry. Analysis of intact molecular weights identified the presence of heavy chain leader sequence, C-terminal lysine, and C-terminal amidation. The identified modifications were further localized into different regions of the antibody from analysis of antibody fragments obtained from FabRICATOR digestion. Analysis of tryptic peptides from various fractions further confirmed the previously identified modifications in the basic variants. Asparagine deamidation and aspartate isomerization were identified in acidic fractions from analysis of tryptic peptides. Basic variants have been fully accounted for by the identified modifications. However, only a portion of the acidic variants can be explained by deamidation and isomerization, suggesting that additional modifications are yet to be identified or acidic variants are an ensemble of molecules with different structures. [ABSTRACT FROM AUTHOR]

Oligosaccharides are critical for structural integrity, stability, and biological functions of recombinant monoclonal antibodies. It is relatively easy to characterize, quantify, and determine the impact of major glycoforms. While challenging to detect and quantify, certain low abundance oligosaccharides are highly relevant to the stability and functions of recombinant monoclonal antibodies. Methods were established in this study based on enzymatic digestion to consolidate peaks of the same type of oligosaccharides by removing heterogeneity and thus increase detectability of low abundance peaks. Endo H was used to collapse high mannose oligosaccharides to a single peak of GlcNAc for ease of detection and quantitation. β-Galactosidase and β-N-acetylhexosaminidase were used to convert complex oligosaccharides into two peaks containing either GlcNAc2Man3Fuc or GlcNAc2Man3, which simplified the chromatograms and data analysis. More importantly, low abundance hybrid oligosaccharides can only be detected and qualified after β-galactosidase and β-N-acetylhexosaminidase digestion. Detection and quantitation of low abundance oligosaccharides can also be achieved using a combination of all three enzymes. These methods can be applied to the development of recombinant monoclonal antibody therapeutics. [ABSTRACT FROM AUTHOR]

Heterogeneity is common among protein therapeutics. For example, the so-called acidic species (charge variants) are typically observed when recombinant monoclonal antibodies (mAbs) are analyzed by weak-cation exchange chromatography (WCX). Several protein post-translational modifications have been established as contributors but still cannot completely account for all heterogeneity. As reported herein, an unexpected modification by methylglyoxal (MGO) was identified, for the first time, in a recombinant monoclonal antibody expressed in Chinese hamster ovary (CHO) cells. Modifications of arginine residues by methylglyoxal lead to two adducts (dihydroxyimidazolidine and hydroimidazolone) with increases of molecular weights of 72 and 54 Da, respectively. In addition, the modification by methylglyoxal causes the antibody to elute earlier in the weak cation exchange chromatogram. Consequently, the extent to which an antibody was modified at multiple sites corresponds to the degree of shift in elution time. Furthermore, cell culture parameters also affected the extent of modifications by methylglyoxal, a highly reactive metabolite that can be generated from glucose or lipids or other metabolic pathways. Our findings again highlight the impact that cell culture conditions can have on the product quality of recombinant protein pharmaceuticals. [ABSTRACT FROM AUTHOR]

Asparagine (Asn) deamidation is a major source of antibody instability and micro heterogeneity. For this reason, it is critical to accurately characterize both the levels and the sites of Asn deamidation in therapeutic antibodies. Asn deamidation is normally quantified by analyzing antibodies at the peptide level by liquid chromatography-mass spectrometry. This requires denaturation, reduction, alkylation, and enzyme digestion of the antibody prior to analysis. These steps in sample preparation may directly contribute to the total levels of Asn deamidation detected. Therefore, to obtain accurate levels and sites of Asn deamidation, it is important to determine if any deamidation occurred during the sample preparation steps. However, this could be challenging because deamidation that occurred prior to and during sample preparation resulted in peptides with the same retention times and the same molecular weight increase of 1 Da. Sample preparation was carried out in 18O-water in the current study to differentiate between the two events of Asn deamidation. Using this method, deamidation that occurred during sample preparation resulted in a molecular weight increase of 3 Da instead of 1 Da. This molecular weight difference was readily detected by inspection of the isotopic peak cluster of the peptides containing the deamidation products, isoAsp and Asp residues. It enabled discrimination of deamidation that was due to analytical artifacts and thus determination of the level of deamidation that was present in the samples. [ABSTRACT FROM AUTHOR]