mouse/human monoclonal antibodies,55,216and humanized monoclonal antibodies42,43have also been reported.Cordoba et al.217studied none-nzymatic cleavage at the hinge region of four humanized IgG1monoclonal antibodies at dif-ferent temperatures with different buffers.They found that EDTA,protease inhibitors,or host cell proteins had no effect on this hinge region cleavage.Liu et al.158reported several additional peptide cleavage sites including cleavage sites around the hinge region and around the CH2–CH3interface of a fully human monoclonal antibody after storage at408C for6months in formulation buffer of pH5.2.Cu2þhas been also shown to cleave antibody in the hinge region most likely through a hydrolysis mechanism.218,219 Nonreducible Cross-Linking

Cross-linking of light chain and heavy chain by nonreducible covalent bonds has been observed on several occasions.Jiskoot et al.21reported that incubation of a mouse IgG1and a mouse IgG2a monoclonal antibodies at378C led to the formation of a nonreducible band on SDS–PAGE containing both light chain and heavy chain.The intensity of this band increased with the increase of the incubation pH.Kroon et al.51reported a similar cross-linked light chain and heavy chain on SEC of murine monoclonal antibody,OKT3,after storage at5–88C for3years.The cross-linking site was between amino acids46–52of the light chain and99–121of the heavy ami et al.53observed a cross-linked band of a human monoclonal antibody on SDS–PAGE after incuba-tion at a basic pH in the presence of hydrogen peroxide.Tous et al.64carried out a detailed study of a nonreducible species of a humanized mono-clonal IgG1antibody.When analyzed by reducing SDS–PAGE and SDS–CE,the nonreducible spe-cies was observed,and the amount of this species increase in the accelerated stability sample. Analysis of the SEC fraction of this species by mass spectrometry indicated that it was formed because of a thioether linkage between the light chain and the heavy chain in place of the original inter light chain and heavy chain disulfide bond. As has been demonstrated,75–77disulfide bonds can be broken by a b-elimination reaction with the formation of dehydroalanine and thiocysteine, which is unstable and decomposes into elemental sulfur and Cys.Dehydroalanine can react with several amino acid side chains such as lysine,cysteine,serine,and threonine to form covalent bonds.220Acceleration of the formation of non-reducible species of recombinant monoclonal antibodies under high pH is in agreement with this mechanism,which may also suggest that cross-linking may be a common feature of recombinant monoclonal antibodies.

Mutation,Insertion,and Truncation

Other than modifications involving the side chains of amino acids,modifications on the peptide back-bone such as amino acid mutation,insertion,and truncation have also been observed.Harris et al.163 reported a mutation of tyrosine376replaced by glutamine during the transfection step of a recombinant humanized monoclonal antibody.Lyu-barskaya et al.47reported incomplete removal of the leader sequence from the heavy chain of a recombinant humanized antibody.Beck et al.46 reported a translation of intron sequence between the variable region and the constant region1of the heavy chain when a humanized monoclonal IgG1 was expressed in CHO,but not in NS0cell line. C-terminal lysine encoded in the DNA sequence can be partially or completely removed by enzy-matic activity of a carboxypeptidase as has been discussed earlier.Interestingly,Johnson et al.48 reported that the amino acid glycine,which is on the N-terminal side of lysine,is further removed in a humanized IgG1antibody expressed in CHO cells.The new C-terminal amino acid pro-line is amidated.As discussed by Johnson et al. that enzymatic amidation requires a consensus sequence of XGB,where X is the potential amino acid for amidation,which is followed by glycine and then followed by a basic amino acid.As this consensus sequence is present in human IgG1at the C-termini,unless C-terminal lysine is deleted from the gene sequence,removal of glycine and amidation of proline may be a common phenom-enon,in addition to the removal of C-terminal lysine.

Other Chemical Modifications

Several modifications have been reported,but are limited to special cases.In the characterization of a fully human monoclonal antibody expressed by transgenic goat,Santora et al.38found that a significant amount of acidic species observed on WCX-10chromatography is due to the modifica-tion of the N-termini of either light chain or heavy

JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.97,NO.7,JULY2008DOI10.1002/jps 2434LIU ET AL.