chain by maleuric acid.Gadgil et al.63reported that an extra Cys other than those conserved disulfide bonded Cys residues of a recombinant monoclonal IgG1is partially modified by cystei-nylation,a reaction between the extra Cys and a free Cys to form disulfide bond.As discussed earlier,Johnson et al.48reported that a basic peak observed on WCX-10chromatogram of a recombi-nant monoclonal IgG1is due to either incomplete removal of the C-terminal lysine residue or amidation of a newly exposed C-terminal proline after removal of both C-terminal lysine and glycine residues.

NONCOVALENT INTERACTION

In addition to covalent modifications,monoclonal antibodies can also associate with other molecules noncovalently.For example,immunoglobulin is one of the major proteins that can bind to riboflavin.221Two IgG2antibodies that can bind riboflavin and its derivatives tightly were found in patients of multiple myeloma.155,222,223As ribo-flavin is bound to the antigen combining site,it is still arguable whether the riboflavin is the actual antigen of the IgG molecules or just a cofactor.224

Immunoglobulin has been reported to be sepa-rated from bovine serum using metal chelate chromatography.225Immobilized metal affinity chromatography has been used for the purification of humanized and murine IgG1antibodies,226and the binding sites were localized to the C-terminal region of heavy chains.A copper binding IgG was found in a myeloma patient with hyper-cupremia,227where copper is bound to Fab region with the involvement of a light chain free Cys. Dower et al.228reported that rabbit IgG has two tight binding sites in the Fc region and two weak binding sites in the Fab region for lanthanides (Gd).

There are no reports on the contribution of noncovalently associated molecules on the hetero-geneity of monoclonal antibodies.Presumably, binding can change the charge properties of monoclonal antibodies and make them more acidic or more basic.

CONFORMATIONAL HETEROGENEITY Protein exists as an ensemble of different con-formations in equilibrium instead of a single rigid structure.119By studying the complex binding kinetics of hapten at various concentrations to three specific antibodies,Foote and Milstein229 proposed that monoclonal antibodies exist as more than one conformation,at least at the combining site,in equilibrium.Different conformations of a monoclonal IgE were observed by X-ray crystal-lography and kinetics studies.230In the study of a murine monoclonal IgG2a antibody using cryo-electron tomography,Sandin et al.231reported that in regard to the dispositions of Fab and Fc, none of the antibody molecules are identical.Fab–Fc angles are in the range of638–1568.Fab–Fab angles are in the range of888–1418.

In addition,IgG molecules are not symmetri-cal.231,232Modifications of the same nature on the same residue but on different light chains or heavy chains can have different effects on the structures of monoclonal antibodies.

It is believed that ion exchange chromatography can recognize more than just the net charges of antibodies.Peaks with different retention times on ion exchange chromatography have been shown to have the same migration times on IEF gels.26,41Antibodies with the same number of pyroglutamate residues or the same amount of deamidation,but on different sides of the mole-cules result in different retention times.26,29 Therefore,ion exchange chromatography cannot necessarily respond to the net overall charges,but rather local charges especially in the regions interacting with column matrix. AGGREGATION

Monoclonal antibodies like all other proteins are prone to aggregation.Aggregates are formed mainly because of intermolecular interactions of hydrophobic regions as a result of partial or transient unfolding of the proteins.233–235Condi-tions such as incubation at high temperature, contact with hydrophobic surfaces,exposure to extreme pH,and exposure to shear forces that induce protein unfolding and promote protein–protein interactions will most likely increase the amount of aggregates.235,236Aggregates can be classified based on different physical/chemical properties such as size,reversibility,and covalent or noncovalent interactions.237

It is reasonable to assume that the presence of aggregates is a universal phenomenon for recombinant monoclonal antibodies.Producing

DOI10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.97,NO.7,JULY2008

HETEROGENEITY OF MONOCLONAL ANTIBODIES2435