additional negative charge to antibodies and generates acidic species,26,32,40,41,44,47,51,57which results in an earlier elution on cation exchange chromatography and faster migration towards the acidic direction on IEF.From the structural point of view,formation of isoaspartate by deamidation or aspartate isomerization is more significant because it introduces a methyl group into the peptide backbone,which may cause structural changes.

Isomerization

The aspartate residue in the sequence of Asp-Gly is prone to isomerization through the formation of a succinimide intermediate.169There are a few examples of this isomerization of aspartate residues of recombinant monoclonal antibodies (Table4).Isomerization of aspartate residue 102of the heavy chain CDR3of recombinant monoclonal antibody,HER2,was observed in a late elution peak on a cation exchange column.40 Incubation at elevated temperatures can promote isomerization of aspartate residues in CDR1of two humanized monoclonal antibodies60,166and the rate of isomerization can be significantly affected by the presence of different excipients.179 The effect of isomerization of an aspartate residue to an isoaspartate on charge heterogene-ity may be more subtle.Chemically,isoaspartate is slightly more acidic than aspartate.Antibodies containing isoaspartate are predicted to elute earlier than antibodies containing aspartate on cation exchange chromatography.However, antibodies with isoaspartate eluted later than

Table3.Deamidation of Asparagine Residues in Monoclonal Antibodies

Molecules Deamidation Sites Domain Location References CD4-IgG fusion protein QDWLNÃ(275)GK CH2162

EWESNÃ(344)GQPENNY CH3

Mouse IgG2a WTNNÃ(386)GKTE CH351

SERQNÃ(156)GVLN CL

NWERNÃ(423)SYSC CH3

Murine IgG1SAAQTNÃ(141)SMVT CH126

GSERQNÃ(161)GVLN CL

Humanized,HER2QDVNÃ(30)TAVA LC-CDR140

IYPTNÃ(55)GYTR HC-CDR2

Humanized IgG1QDWLNÃ(316)GKEY CH2122

WESNÃ(385)GQPENNY CH3

WESNGQPENÃ(390)NY CH3

Recombinant human IgG QDWLNÃ(313)GKEYK CH252

WESNÃ(382)GQPENÃ(387)NYK CH3

WESNGQPENÃ(387)NYK CH3

VCLLNÃ(137)NFYPR CL

Monoclonal antibody ILPGNÃ(55)GNINY HC-CDR244

Two N followed by G CH2and CH3

Murine IgG1SLAHSNÃ(33)GNTYL LC-CDR1123 Humanized antibody Asn55HC-CDR247

Asn387,Asn392or Asn393CH2and CH3

Recombinant human IgG1DNAKNÃ(77)SLYLQ VH158

CLLNÃ(137)NFYP CL

KVSNÃ(329)KALP CH2

Table4.Isomerization of Aspartate Residues in Monoclonal Antibodies

Molecules Isomerization Sites Domain Location References Humanized monoclonal IgG1SVDYDÃ(32)GDSY LC-CDR160 Humanized monoclonal IgG1WGGDÃ(102)GFYAMD HC-CDR340 Humanized monoclonal IgG1s SVDYDÃ(32)GDSY LC-CDR1166

SKPVDÃ(30)GEGD LC-CDR1

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

HETEROGENEITY OF MONOCLONAL ANTIBODIES2431