of human IgG in healthy subjects and increas-ed level in diabetic patients has been widely studied.188–193Glycation of monoclonal and poly-clonal immunoglobulins of different species can also occur in vitro188,189,194–203Andya et al.56 reported that storage of the spray-dried powder of a recombinant monoclonal antibody with a molar ratio of300:1lactose to antibody at308C for 9months caused a100%glycation of this antibody as assayed by binding to a boronate affinity column.Harris61reported that glycation of lysine residues of the light chain and the Fab part of the heavy chain with a molecular weight increase of 162Da is often observed by mass spectrometry analysis.A minor peak detected in a deglycosy-lated humanized monoclonal antibody by mass spectrometry was also hypothesized to be par-tially due to glycation.47Glycation can mask the positive charge on the N-terminal primary amino group or the side chain of lysine residues,which will make the antibody more acidic.

Peptide Bond Cleavage

The hinge region is the least structured and thus most susceptible region of an antibody for enzy-matic and chemical cleavage.Cleavage in the hinge region results in fragments of different sizes and different p I s.Hence,cleavage introduces both size and charge heterogeneity.

Many different enzymes have been shown to cleave IgGs in the hinge region.Papain is the most common enzyme that cleaves antibody hinge region to generate Fab and Fc fragments. Plasmin,204Cathepsin L,205–207and Lys-C63can also cleave peptide bonds in the hinge region. Peptide bonds close to the hinge region but in the CH2domains are cleaved by both pepsin and metalloproteases.208More sites around the hinge region,the CH2–CH3interface,and within the domain structures become susceptible to enzymatic cleavage after a brief exposure to low pH.209–214

Direct hydrolysis of peptide bonds under acidic or basic conditions around the weak spots such as the hinge region and the CH2–CH3interface is common especially for monoclonal antibodies in liquid formulation at high temperature.Jiskoot et al.21reported cleavage of the hinge region of mouse monoclonal antibodies at a higher pH range.Similar hinge region cleavage of mouse monoclonal IgG2a antibody,OKT3,51,215chimeric

Table5.Oxidation of Met and Other Amino Acids in Monoclonal Antibodies

Molecules Oxidation Sites Domain Location References Murine monoclonal IgG2a(2–88C14months–3years)STYSMÃ(174)SSTL CL51

TRYTMÃ(34)HWV HC-CDR1

QDWMÃ(316)SGK CH2

PPEEEMÃ(360)TKK CH3

DGSYFMÃ(408)YSK CH3

DDHYCÃ(105)LDY HC-CDR3

Humanized IgG1SDYYMÃ(34)HWVR HC-CDR1114

Humanized IgG1,HER2(Incubation with tBHP or Higher temperature,light)KDTLMÃ(255)ISRTP

SCSVMÃ(431)NEAL

CH2

CH3

183,184

Humanized IgG1QGLWÃ(102)FDSD HC-CDR3119

DIQMÃ(4)TQSPSSL VL

Fully human IgG1(incubation with tBHP, higher temperature)KDTLMÃ(256)ISRTP

SCSVMÃ(432)NEAL

CH2

CH3

185

Humanized monoclonal IgG1(UV or tBHP)Met4VL186

Met34HC-CDR1

Met101HC-CDR3

Met255CH2

Met361CH3

Met431CH3

Trp54HC-CDR2

Trp55HC-CDR2

Trp105HC-CDR3

Fully human IgG2Trp103HC-CDR3187 DOI10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.97,NO.7,JULY2008

HETEROGENEITY OF MONOCLONAL ANTIBODIES2433