Heterogeneity of Monoclonal Antibodies(7)

antibodies with aspartate residues,40and this observation indicates chromatographic behavior is not due to the direct charge effect but rather an indirect effect by modulating the structures. The structural effect was also evidenced by the fact that antibodies containing isoaspartate eluted later on HIC than antibodies containing aspartate.60,166Isomerization,like the formation of isoaspartate from asparagine deamidation, introduces a methyl group to the peptide backbone,so a structural change is expected. Succinimide,also an asparagine deamidation intermediate,is more basic and less polar than aspartate and isoaspartate and proteins contain-ing it eluted later on cation exchange40and HIC.60,166

In addition to isomerization of aspartate residues, cis proline is also common in antibodies.180,181 In vitro studies indicate that trans/cis proline isomerization is involved in the folding of Fab and CH3domains at different stages.181,182Proline residues in the cis or trans positions may affect the structure of antibodies and their chromato-graphic behavior.

Oxidation

Methionine(Met)is one of the most susceptible residues to oxidation.Oxidation of Met to sulf-oxide increases the mass by16Da and makes the side chain of Met more polar.Roberts et al.114 reported oxidation of Met34of a humanized monoclonal antibody.Matamoros Fernandez et al.119reported that Met4of the light chain of a humanized monoclonal antibody was oxidized. It is unclear at what stage these residues are oxidized.Under most circumstances,oxidation can be readily detected in monoclonal antibodies after long-term storage,incubation at elevated tempera-tures or incubation with oxidizing reagents.Kroon et al.51reported oxidation of several Met residues of a murine monoclonal antibody after storage at 2–88C for3years.Met255and Met431in the Fc region of a recombinant human IgG1are the two most susceptible sites for oxidation when incubated with tert-butyl hydroperoxide(tBHP),183exposed to intense light or incubated at elevated tempera-tures.184Oxidation of the Met residues of the equivalent sites have also been reported when a fully human monoclonal antibody was incubated with tBHP or after long-term storage at258C for 6months.185Wei et al.186reported that exposure of two humanized monoclonal antibodies to ultra-violet(UV)light or tBHP led to oxidation of Met101,Met255,and Met431of the heavy chain, and to a lesser extent oxidation of Met4of the light chain and Met34and Met361of the heavy chain. The susceptibility of Met255and Met431or Met residues at the equivalent positions to oxidation is expected as they are close to the CH2–CH3 interface and are exposed in the three-dimensional (3-D)structure.180

The Fc region with oxidized Met residues elutes earlier relative to the nonoxidized molecule on HIC column.183,184On weak cation exchange chromatography,antibodies with oxidized Met residues elute later.185As expected,oxidized Met containing peptides eluted earlier than the same peptides with unoxidized Met residues on RP.51,183,185

In addition to Met residues,Matamoros Fernandez et al.119reported oxidation of a tryptophan(Trp) residue of a humanized monoclonal antibody. Oxidation of a Trp residue of a fully human IgG2 results in an earlier elution of the intact antibody on SEC column as a front shoulder and earlier elution of the reduced heavy chain on the reversed-phase column.187The shorter retention time of the anti-body with oxidized Trp on SEC may suggest less nonspecific hydrophobic interations of the molecules with the column matrix rather than a size difference. Exposure of two humanized IgG1antibodies to UV light leads to oxidation of Trp residues in the heavy chain CDRs.186It is interesting to note that all the oxidized Trp residues of humanized monoclonal antibodies that have been reported so far are located in the CDR regions,119,186,187which may reflect the fact that they are the only solvent-exposed Trp residues.Cys is another amino acid that is susceptible to oxidation.Kroon et al.51reported oxidation of an unpaired Cys residue of a murine monoclonal antibody after storage at2–88C for3 years.However,Cys residues of the antibodies are most likely involved in disulfide bonds.In general, the presence of extra Cys residues is avoided for the development of recombinant monoclonal antibodies to prevent instability caused by the highly reactive free sulfhydryl.Oxidation of several monoclonal antibodies with sites identified is summarized in Table5.

Glycation

Unlike glycosylation,glycation results from the nonenzymatic reaction between reducing sugars and the N-terminal primary amine or the amine group of lysine side chains.In vivo glycation

JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.97,NO.7,JULY2008DOI10.1002/jps 2432LIU ET AL.