recombinant proteins at higher titer may saturate protein-folding machinery,which could result in a higher probability of producing partially folded or incorrectly folded molecules.After secretion, recombinant monoclonal antibodies encounter stress conditions in the cell culture medium and during purification such as high temperature, agitation,shear forces,extreme pH,and various surfaces.Purification processes can normally remove most of the aggregates.However,aggre-gates can be further generated during formula-tion,storage,and various accelerated stability conditions.

Higher temperature and longer incubation time have been shown to increase the amount of aggregates of several monoclonal antibodies of different species in the liquid and in the solid forms.21,216,238–245The solution pH is also critical.As demonstrated by the studies of polyclonal human IgG,246polyclonal rabbit IgG,247–249monoclonal mouse IgG,250–252and a monoclonal chimeric IgG,253antibodies at low pH adopt nonnative structures with lower stability and larger exposed hydrophobic surfaces.The low pH induced conformational changes are only partially reversible after pH neutraliza-tion.248,249,253Antibodies at low pH or after low pH incubation and subsequent pH neutralization are prone to form aggregates.21,250–252Jiskoot et al.21reported that insoluble aggregates were formed when two mouse monoclonal antibodies were incubated at378C at pH3and4,and at48C at pH3.Paborji et al.216reported that aggregates of a chimeric monoclonal antibody were primarily formed at pH values below5at608C.Incubation at pH2.5at room temperature followed by pH neutralization led to a significant increase in the amount of aggregates of a humanized monoclonal antibody.254On the other hand,Ejima et al.255did not detect a significant increase of aggregates of a recombinant humanized IgG4after incuba-tion at pH2.7and3.5at48C.It is clear from these studies that temperature is still a key factor affecting aggregation at low pH.Further-more,different antibodies likely have different pH and temperature stability profiles.Increased amount of aggregation of various kinds of monoclonal antibodies has also been reported by spray-drying,56lyophilization,238freeze/thaw,216 and agitation from stirring or horizontal shak-ing.216,256In addition,protein concentration,salt composition and concentration,and type and concentration of excipients could also affect the nature and level of aggregation.235,236

Certainly,aggregates contribute to size hetero-geneity of monoclonal antibodies as long as they can survive the analytical conditions of different techniques.237Covalent aggregates formed from disulfide bond scrambling result in extra bands on nonreducing SDS–PAGE and CE–SDS.56,245,257The surface properties of aggregates such as hydrophobicity and charge distribution of aggregates are likely different from those of monomers.For example,as shown by Wang et al.,258aggregates are precipitated more efficiently by ammonium sulfate than the monomer of a humanized monoclonal IgG1 antibody,which indicates that the aggregates are more hydrophobic.This conclusion is further supported by the stronger binding of the aggre-gates to polyvinylidenefluoride(PVDF)mem-brane than the monomer.258

SUMMARY

Heterogeneity is common for monoclonal anti-bodies including recombinant monoclonal anti-bodies due to modifications of various natures. Different orthogonal techniques are required to understand the structure and stability of the molecules thoroughly.Characterization experi-ence of one antibody can certainly help to under-stand the heterogeneity of other antibodies, however the information cannot be generalized even though monoclonal antibodies of either humanized or fully human share more than 90%sequence identity.Each molecule has a unique history from the point of synthesis to the point of complete clearance from the subjects, which may result in differences in conformation, charge,size,and hydrophobicity.

Modifications can also be introduced during sample analysis.For example,disulfide bond scrambling under denaturation condition has been shown to result in fragments on SDS–PAGE and SDS–CE.68,70,71Met oxidation185,259and Asn deamidation162can be introduced during sample preparation for peptide ing a model peptide with a N-terminal glutamine residue, Dick et al.157observed that going through the peptide map procedure including denaturation, reduction,alkylation,and trypsin digestion for3h at378C introduced10%of pyroglutamate.Rehder et al.62reported hydrolysis of antibody heavy chain while the sample was bound to a reverse-phase column run at high temperature and with low pH mobile phases.Bailey et al.131

JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.97,NO.7,JULY2008DOI10.1002/jps 2436LIU ET AL.