Heterogeneity of Monoclonal Antibodies(4)

moieties and the amino acids of the CH2 domains.147,148Therefore,even neutral oligosac-charides may affect the chromatographic behavior of monoclonal antibodies by modulating local surface charge.

N-Terminal Pyroglutamate Formation

The N-terminal residue of human IgG heavy chains is either glutamine or glutamate.149 Lambda light chains usually start with glutamine with the occasional exception of serine,arginine, or leucine.150Kappa light chains start with glutamate or aspartate,and less frequently with alanine,asparagine,or valine.151–153Most of the N-terminal glutamine of heavy chains of human IgGs is cyclized to pyroglutamate.154 As shown in Table1,the N-terminal glutamine can be partially29,47or comple-tely22,46,51,62,91,113,114,119,122,155,156cyclized in monoclonal antibodies.Dick et al.157reported that90%of the heavy chain N-terminal gluta-mine of a recombinant monoclonal antibody was cyclized to pyroglutamate in the bioreactor at day 15,while the other10%was cyclized during purification,formulation,and sample handling. Nonenzymatic formation of pyroglutamate from N-terminal glutamate has also been reported for recombinant monoclonal antibodies158–160 (Table1),but this chemical change occurs usually under much harsher conditions than those re-sulting in the spontaneous formation of pyroglu-tamate from glutamine.

Formation of N-terminal pyroglutamate from glutamine and glutamate decreases the molecular weight by17and18Da,respectively.More significantly,cyclization of glutamine to form pyroglutamate causes a loss of the N-terminal primary amine,which is positively charged at neutral pH,therefore results in antibodies with more acidic species.Monoclonal antibodies with pyroglutamate elute earlier than antibodies with glutamine on cation exchange.29,47The heavy chain with pyroglutamate elutes later than the heavy chain with glutamine of a recombinant human monoclonal antibody on RP.62

C-Terminal Lysine Processing

C-terminal lysine processing of the heavy chain is one of the most common modifications of recombi-nant monoclonal antibodies.The C-terminal lysine residue of the heavy chain is encoded in the gene sequence,161but this residue can be comple-tely22,40,122,162or partially removed22,26,29,30,34–36,43,44,46–48,60,63,113,114,119,126,156,163,164by the acti-

vity of basic carboxypeptidases165(Table2). Removal of one C-terminal lysine residue decreases the molecular weight by128Da and the positive charge by1unit.Partial removal of the C-terminal lysine residues results in a mixed population of antibodies with zero,one, or two C-terminal lysine residues,that can be separated into multiple bands/peaks by IEF,22,26 cIEF,26,30,36and ion exchange chromatogra-phy.22,29,34–38,44,46–48Antibodies with different

Table1.N-Terminal Pyroglutamate of Monoclonal Antibodies

Molecules Formation of N-Terminal PyroE References Human monoclonal IgG Heavy chain Q to PyroE91 Murine monoclonal IgG2a Heavy chain Q to PyroE22,51 Humanized IgG1Heavy chain Q to PyroE113 Humanized IgG1Heavy chain Q to PyroE114 Human IgG2Heavy chain and light chain Q to PyroE155 Mouse monoclonal IgG1Heavy chain Q to PyroE156 Mouse/human chimeric Heavy chain and light chain Q partially to PyroE29 Humanized monoclonal IgG1Heavy chain Q to PyroE119 Humanized IgG1Heavy chain Q to PyroE122 Humanized IgG1Heavy chain Q to PyroE46 Humanized IgG Heavy chain Q partially to PyroE47 Recombinant IgG Heavy chain Q to PyroE62 Recombinant monoclonal IgG Heavy chain Q to PyroE157 Recombinant fully human IgG1Heavy chain E partially to PyroE158 Recombinant monoclonal antibody Heavy chain and light chain E partially to PyroE159 Recombinant monoclonal antibodies Heavy chains or light chains E partially to PyroE160 DOI10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.97,NO.7,JULY2008

HETEROGENEITY OF MONOCLONAL ANTIBODIES2429