monoclonal antibodies can be introduced by intra-cellular processes2,4,11,25,49,50or extracellular pro-cesses2,4,17,25,49,50taking place in serum,ascites,and cell culture medium.In addition,heterogeneity can be introduced by incubation with buffers,during purification process,storage,2,17,25,31,51and under dif-ferent stressed conditions17,21,26,27,29,30,32,39,40,44,51–57 such as elevated temperature,exposure to intense light,or exposure to chemicals.

Heterogeneity has been commonly observed when monoclonal antibodies were analyzed using charge based analytical techniques such as IEF,4,9,11,14–17,21,22,26,31–33,36,39,41,45,46,50,57,58 capillary isoelectric focusing(cIEF),27,30,31,36,39 chromatofocusing,7two-dimensional(2-D)electro-phoresis,5,6,8,10,12,13,19,23–25and ion-exchange chro-matography.9,17,22,26,29,32,34–38,40,41,44,46–48,57,59In addition,heterogeneity of monoclonal antibodies has also been observed on hydrophobic interaction chromatography(HIC)18,40,60,61and reverse-phase chromatography(RP).42,43,62,63Separation by HIC and RP is directly related to sample hydrophobicity,which is also sensitive to surface charge.Size heterogeneity has been determined using size-exclusion chromatography(SEC),61,64 sodium dodecyl sulfate–polyacrylamide gel elec-trophoresis(SDS–PAGE),21,51,53,57,65–67sodium dodecyl sulfate capillary electrophoresis(SDS–CE),53,68and most accurately by mass spec-trometry.

A summary of the current knowledge regard-ing observed monoclonal antibody heterogeneity caused by chemical modifications,noncovalent interactions,conformational diversity and aggre-gation is presented in this article. CHEMICAL MODIFICATIONS

Disulfide Bonds

IgG molecules are composed of two heavy chains and two light chains.Each light chain is connected to each heavy chain by one disulfide bond.Each heavy chain is connected to the other heavy chain by two to four disulfide bonds depending on the subtypes of the antibodies.Each IgG has12domains(one variable(V L)and one constant domain(C L)of each light chain,one variable(V H)and three constant domains(CH1, CH2,and CH3)of each heavy chain),and each domain contains one intrachain disulfide bond. Disulfide bond formation is important for the assembly and maintenance of the structural integrity of antibodies.

Heterogeneity related to disulfide bonds can be introduced at different stages.Free sulfhydryl and reactive cysteine(Cys)residues have been reported for IgGs of different species.69Zhang and Czupryn70reported approximately0.02and 0.1moles of free sulfhydryl per mole of recom-binant monoclonal antibodies of IgG1,IgG2,and IgG4under native or denaturing conditions, respectively.Taylor et al.71reported that for recombinant humanized monoclonal IgG4anti-bodies,no free sulfhydryl can be detected in the native forms,whereas0.2–0.3moles of free sulfhydryl per mole of molecule can be detected under denaturing conditions.Thesefindings suggest that the presence of free sulfhydryl is probably due to incomplete formation of intra-chain disulfide bonds.And indeed,unpaired Cys22and Cys96of the heavy chain of a recombinant antibody have been reported.61,72 Interchain disulfide bonds are the most suscep-tible to reduction,which supports the notion that the incomplete intrachain disulfide bonds are due to incomplete formation and not due to reduc-tion.73,74Incomplete formation of intrachain disulfide bonds provides a source of free sulfuhy-dryl,which will catalyze disulfide bond scram-bling.71In addition,disulfide bond breakage due to b-elimination is normally accelerated under basic conditions.75–77

A single incomplete disulfide bond results in two free sulfhydryl groups and increases the molecular weight by2Da,which can be detected by mass spectrometry with or without chemical modifications.At neutral pH,free sulfhydryl is not charged,therefore it may not contribute to charge heterogeneity directly.However,incom-plete formation of disulfide bonds will likely cause structural disturbances,which may also be re-flected as size and charge heterogeneity.Unpaired Cys residues have been reported to result in a longer retention of the molecule on a HIC column.61,72On RP,Dillon et al.42observed multiple peaks of a recombinant IgG2molecule. The authors proposed these peaks were due to different disulfide bond structures.

The presence of incomplete disulfide bonding can trigger disulfide bond scrambling,especially under denaturing conditions,that can result in the formation of disulfide bond related frag-ments as observed on SDS–PAGE and SDS–CE. These artifacts are not fragments from peptide bond cleavage.They are different combinations

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

HETEROGENEITY OF MONOCLONAL ANTIBODIES2427