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In-depth characterization of the commonly observed variants is critical to the successful development of recombinant monoclonal antibody therapeutics. Multiple peaks of a recombinant monoclonal antibody were observed when analyzed by hydrophobic interaction chromatography and imaged capillary isoelectric focusing. The potential modification causing the heterogeneity was localized to F(ab')2 region by analyzing the antibody after IdeS digestion using hydrophobic interaction chromatography. LC-MS analysis identified asparagine deamidation as the root cause of the observed multiple variants. While the isoelectric focusing method is expected to separate deamidated species, the similar profile observed in hydrophobic interaction chromatography indicates that the single site deamidation caused differences in hydrophobicity. Forced degradation demonstrated that the susceptible asparagine residue is highly exposed, which is expected as it is located in the light chain complementarity determining region. Deamidation of this single site decreased the mAb binding affinity to its specific antigen.

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.

An efficient strategy to characterize recombinant monoclonal antibody charge variants was established using weak anion exchange chromatography, LC-MS and IdeS digestion to allow subunit level characterization. Significantly higher resolution was achieved at subunit levels by weak anion exchange chromatography and LC-MS. In addition, subunit analysis localized potential modifications to either F(ab')2 or Fc fragments to facilitate further characterization. Peptide mapping of fractions from various charge variants after IdeS digestion identified aspartate isomerization, asparagine deamidation and glycation as the modifications. Although, aspartate isomerization does not generate net charge difference directly, it does generate antibody basic species. Antibodies with either isoaspartate or aspartate from deamidation showed different retention times by chromatography. Even more interestingly, the antibody contained succinimide as the isomerization intermediate, which though more basic compared to aspartate, eluted off the weak anion exchange column as an acidic species. The results demonstrated not only the utility of subunit level characterization but also the unpredictable chromatographic behavior of antibody charge variants.

Therapeutic recombinant monoclonal antibodies are subject to various modifications during cell culture, and to a lesser degree, during purification. These modifications are expected to remain relatively constant during storage with the protection of appropriate formulations. However, after administration to patients, the levels of modifications may vary over time in circulation, where the recombinant monoclonal antibodies are exposed to the physiological conditions. Scientific understanding of those in vivo modifications can help drug candidate selection to choose the most stable molecules and set appropriate specifications for product release, which ultimately ensures safety and efficacy.

Charge variants, especially acidic charge variants, of recombinant monoclonal antibodies have been challenging to fully characterize despite the fact that several posttranslational modifications have already been identified. The acidic species of a recombinant monoclonal antibody were collected using weak cation exchange (WCX)-10 chromatography and characterized by LC-MS at multiple levels. In this study, methionine oxidation and asparagine deamidation are the only two modifications identified in the acidic species. Incubation of the collected main chromatographic peak with hydrogen peroxide generated acidic species, which confirmed that acidic species were enriched in oxidized antibody. Differences observed between the original acidic species and the oxidization-induced acidic species indicate that different mechanisms are involved in the formation of acidic species. Additionally, acidic species were generated by thermal stress of the collected main peak from the original sample. Thermal stress of the collected main peak in pH 9 buffer or ammonium bicarbonate generated chromatograms that are highly similar to those from the analysis of the original molecule. LC-MS analysis identified oxidation of the same methionine residue and deamidation of the same asparagine in the corresponding acidic fractions generated by thermal stress; however, relatively lower levels of methionine oxidation and higher levels of asparagine deamdiation were observed. The results support the use of stressed conditions to generate low abundance species for detailed characterization of recombinant monoclonal antibody charge variants, but with caution.

Glycosylation of the conserved asparagine residue in the CH2 domain is the most common posttranslational modification of recombinant monoclonal antibodies. Ideally, a consistent oligosaccharide profile should be maintained from early clinical material to commercial material for the development of recombinant monoclonal therapeutics, though variation in the profile is a typical result of process changes. The risk of oligosaccharide variation posed to further development is required to be thoroughly evaluated based on its impact on antibody structure, stability, efficacy and safety. The variation should be controlled within a range so that there is no detrimental impact on safety and efficacy and thus allowing the use of early phase safety and efficacy data to support project advancement to later phase. This review article focuses on the current scientific understanding of the commonly observed oligosaccharides found in recombinant monoclonal antibodies and their impact on structure, stability and biological functions, which are the basis to evaluate safety and efficacy. It also provides a brief discussion on critical quality attribute (CQA) assessment with regard to oligosaccharides based on the mechanism of action (MOA). © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1173-1181, 2017.

Deamidation of glutamine (Gln) proceeds at a much slower rate than deamidation of asparagine (Asn) residues at peptide level. However, deamidation of Gln residues in native proteins may occur faster because of the impact of protein structure and thus plays a significant role in affecting protein stability. Gln deamidation of a recombinant monoclonal IgG1 antibody was investigated in the current study. Deamidation was determined by a molecular weight increase of 1 Da, a retention time shift on reversed-phase chromatography and tandem mass spectrometric (MS/MS) analysis of the peptides. As expected, Gln residues at different locations in the three-dimensional structure had different susceptibilities to deamidation. Gln deamidation was highly pH dependent with the highest level detected in the sample incubated at pH 9, and lowest level at pH 6 in the pH range from 5 to 9. The detection of significant levels of Gln deamidation suggested that it may play an important role in affecting heterogeneity and stability of recombinant monoclonal antibodies.

Methionine (Met) is one of the most susceptible amino acids to oxidation. Met256 (CH2-Met15.1) and Met432 (CH3-Met107) of a recombinant humanized monoclonal IgG1 antibody are located in the CH2 and CH3 domains, respectively. In three-dimensional structure, these two Met residues are close to the CH2-CH3 interface. In close proximity, oligosaccharides on the conserved asparagine (Asn) residues are enclosed in the CH2 domains. The relationship of Met oxidation with oligosaccharides and their effect on the structure of the antibody was investigated. Removal of oligosaccharides did not alter the oxidation rates of Met256 and Met432, however it caused significant structural changes as evidenced by the susceptibility of the deglycosylated antibody to trypsin and chymotrypsin. Oxidation of Met256 and Met432 did not cause significant conformational changes of the antibody with oligosaccharides, however oxidation of these Met residues accelerated degradation of the deglycosylated antibody. Analysis by mass spectrometry indicated that most of the protease cleavage sites were in the CH2 domains, which suggested that conformational changes induced by the removal of oligosaccharides and further by Met oxidation were local to the CH2 domains.

Nitration of a recombinant human monoclonal antibody was carried out in vitro by incubating the antibody with the nitrating reagent tetranitromethane (TNM). The susceptible sites of nitration were identified using high-performance liquid chromatography/mass spectrometry (HPLC/MS). In general, tyrosine residues in the variable domains of the antibody are more susceptible to nitration, while tyrosine residues in the constant domains are relatively resistant to nitration. However, one tyrosine residue in the CH1 domain and one tyrosine residue in the CH2 domain are highly susceptible to nitration. Interestingly, the susceptible tyrosine residue in the CH2 domain is followed by the conserved asparagine residue that is glycosylated.

The sites and levels of Asn deamidation in proteins are often determined by LC-MS analysis of peptides obtained from enzymatic digestion. However, deamidation that occurs during sample preparation steps results in overestimation of the original level of deamidation. The inherent deamidation and those introduced by sample preparation can be differentiated by preparing samples in (18)O water. When using H(2)(18)O, the formation of isoAsp and Asp by Asn deamidation during sample preparation results in a molecular weight increase of 3 Da due to the incorporation of the (18)O atom to the side chains of isoAsp or Asp; in contrast, inherent deamidation only results in a molecular weight increase of 1 Da. In addition, up to two (18)O atoms can also be incorporated into the peptide C-terminal carboxyl group during enzymatic digestion. Therefore, the 2 Da molecular weight difference at the deamidation sites can only be used to differentiate deamidation that occurs prior to or during sample preparation under conditions that a fixed number of (18)O atoms are incorporated into the peptide C-terminal carboxyl groups. Otherwise, it is challenging to apply this procedure because of the resulting complicated isotopic distributions. Here, a new procedure of using (18)O-water for sample preparation coupled with tandem mass spectrometry (MS/MS) was established to calculate the deamidation artifacts. In this method, b ions were used for the calculation of Asn deamidation that occurred prior to or during sample preparation, which eliminated the complicated factor of various number of (18)O-atoms to the peptide carboxyl groups. This procedure has the potential to be applied under the general peptide mapping conditions.

Nonenzymatic asparagine (Asn) deamidation is one of the commonly observed posttranslational modifications of proteins. Recent development of several specific analytical methods has allowed for efficient identification and differentiation of the deamidation products (i.e., isoaspartate [isoAsp] and aspartate [Asp]). Isotope labeling of isoAsp and Asp that are generated during sample preparation by 18O has been developed and can differentiate isoAsp and Asp as analytical artifacts from those present in the samples prior to sample preparation for an accurate quantitation. However, the 18O labeling procedure has a limitation due to the additional incorporation of up to two 18O atoms into the peptide C-terminal carboxyl groups. Variability in the incorporation of 18O atoms into the peptide C-terminal carboxyl groups results in complicated mass spectra and hinders data interpretation. This limitation can be overcome by the dissection of the complicated mass spectra using a calculation method presented in the current study. The multiple-step calculation procedure has been successfully employed to determine the levels of isoAsp and Asp that are present in the sample prior to sample treatment.

To determine the relative importance of direct hydrolysis and beta-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.A recombinant monoclonal antibody was incubated in buffers of various pH at 40 degrees 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).Direct hydrolysis was accelerated by acidic and basic pH, while beta-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.Antibody fragmentation level, sites and mechanisms were affected by pH. Oligosaccharides only affected the rate of fragmentation at pH 4.

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.