Anal. Chem. 1995, 67, 3626-3632
Monitoring of IgG Antibody Thermal Stability by Micellar Electrokinetic Capillary Chromatography and Matrix-Assisted Laser Desorption/lonization Mass Spectrometry Anthony J. Alexander* and David Emyln Hughes* Bristol-Myers Squibb, Analytical Research & Development, Pharmaceutical Research Institute, P.0. Box 4755, Syracuse, New York 13221-4755
Monitoring the stability of immunoglobulin G (IgG) type antibodies is a crucial analytical issue spanning a wide variety of immunologicaVbiotechologid studies, which includes the analysis of conjugated IgG's for drug delivery. Capillaryelectrophoresis (CE) has proven valuable for the analysis of proteins and has the potential to separate and detect native antibody components. An ideal complement to CE, which is capable of providing the desired detection specificity to provide species identification information, is matrix-assisted laser desorptiodionimtion mass spectrometry (MALDI-MS). Utilizing these two techniques we have developed an antibody examination procedure and monitored the degradation of an internalizing chimeric (humadmouse) monoclonal antibody (BR96). Electropherograms of the antibody after up to 166 h of thermal stress are presented; MALDI mass spectra of the stressed antibody were acquired at the same time points. At 166 h, the percentage of ionization carried by the intact antibody molecular ions M+, M2-, etc., had clearly decreased, while that due to additional ion species had signifjcantly increased. Ions corresponding in mass to loss of one light Chain, loss Of an Fab arm to yield an type fragment, and formation of separated heavy-chain and light-chainmoieties were observed. Several of these fragments result from simple disulfide linkage disruption. In addition, species less in mass than common antibody subunits were also observed, demonstrating peptide as well as disulfide bond cleavage. The observation that a small number of well-defined species were formed during the study suggests that the cleavage induced by thermal stress is very site-specificwithin the IgG. The analytical examination of an immunoglobulin (IgG) antibody represents a substantial challenge. These large proteinaceous species consist principally of two larger heavy-chain strands covalently attached to two smaller lightchain strands by disulfide bonds, as depicted in Figure 1. The variable carbohydrate content is typically present only on the heavy chain.' Since IgG antibodies possess an average molecular mass of 150 000 Da and multiple forms varying only slightly in amino acid and carbohydrate content, examinations based on molecular weight (1) Goading, K. M.; Regnier, F. E. In HPLC ofBiological Macromolecules; Marcel Dekker: New York. 1990; pp 487-528.
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Analytical Chemistry, Vol. 67, No. 20, October 75, 7995
(MW) differences alone would not be expected to be useful. Liquid chromatographic separatory procedures, principally size exclusion, ion-exchange, aflinity, hydrophobic interaction, and hydroxylapatite chromatography, have proven useful for antibodiesS2 Of these techniques, ion-exchange chromatography, is possibly the most selective procedure for large proteins, perhaps implying that chargedependent separations show substantial promise. Capillary electrophoresis (CE), which separates species on the basis of molecular mass and net charge, has been used for analysis of peptides and a few proteins such as adrenocorticotropin, transferrin, ribonuclease, insulin, and growth h o r m ~ n e . ~ . ~ CE peptide separation studies have principally involved synthetic (or known) specie^^,^ and peptides resulting from protein digestion. CE tryptic mapping procedures are available for human recombinant insulin-like growth factor I (rhIgG-I),6 my~globin,~ hemoglobins,8 a-1-acid glycoprotein, and human growth hormone OIGH) .9J0 Comparisons of enzymatic digestion protocols have previously been performed," as well as alternative forms of detection.'* Comparison of the selectivity of CE peptide separations with the more established liquid chromatographic techniques has generally supported the primary or complimentary use of the CE t e c h n i q ~ e . ~ , ~ . ~ J ~ Large proteins, glycoproteins, and metal-associated proteins require particularly selective techniques of analysis to represent the multiple, closely related forms present in actual samples. Importantly, protein studies performed by CE have been shown to be able to separate the major species arising from degradative deamidation and amino acid and glycan mi~roheterogeneity.'~ Whereas peptide CE separations have often been adequately Gutbnan, A; Paulus, A; Cohen, A S.; Karger, B. L.; Rodriques, H.; Hancock, W. S. In Electrophoresis 88; VCH Publishers: New York, 1988; pp 151160. Colbum, J. C.; Lauer, H. H.; Nielson, R G.; Riggin, R. Grossman, P. D.; M.; Sittampalam, G. S.: Rickard, E. C. Anal. Chem. 1989,61, 1186. Zhang, Y. IC; Chen, N.; Wang, L. Biomed. Chromatogr. 1993,7 (21, 7577. Gaus, H. J.; Beck-Sickinger, A. G.; Bayer, E.Anal. Chem. 1993,65,13991405. Hilser, V. J.; Worosila, G. D.; Rudnick, S. E. J. Chromatogr. 1993,630, 329-336. Cassiano, L.; Rabine, R; Rossetti. D. V.; Bassi, F. A. J , Chromatogr. 1991, 572, 51-58. Ross, G. A.; Lorkin, P.; Perrett, D. J. Chromatogr. 1993,636, 69-79. Nashabeh, W.; el Rassi. Z. J. Chromatogr. 1991,536,31. Nielsen, R. G.; Rickard. E. C. J. Chromatogr. 1990,516, 99. Cobb, K. A; Novotny, M. V.Anal. Chem. 1992,64, 879. Lee, T. T.; Lillard, S. J.; Yeung, E. S. Electrophoresis 1993,24, 429. Sutcliffe, N.; Corran, P. H. J. Chromatogr. 1993,636, 95. Compton, B. J.: O'Grady, E. A. Anal. Chem. 1991,63, 2597. 0003-2700/95/0367-3626$9.00/0 0 1995 American Chemical Society
Antigen Binding Site
Antigen Binding Site
Heavy Chain (H)
4
-
Human H a y Chain Murine Human Light Chain
@
Carbohydrate
Figun I. Schematic representation of chimeric BR96 (humadmouse) monmlonal antibody.
described by simple charge and mass elemophoretic models'5 (although the exponent of the mass in the defming equation is not firmly established), proteins require a more complex separ;t tion model. Even in peptide separations, the importance of hydrophobicity and conformation has been observed? Specificity related to conformation presumed to ocfur due to different hydrodynamic pro6les for different conformers has been observed in protein studies. The unfolding of human serum bansfenin has been studied by CE and found to be dependant on iron content, but independant of carbohydratecontentl6 S i i l y , free solution CE has been used to monitor the temperaturedependent unfolding of i y s o q n ~ eat~low ~ pH. Although CE studies on antibodies are not abundant, one combined theoretical and empirical study18 demonstrated that a single amino acid difference in a chimeric IgG antibody form was detectable. In a second study. a micellar CE procedure was developed that was capable of separating IgG monoclonal antibody, alkaline phosphatase (MW 140000), and an alkaline phosphatase-IgG ~0njugate.l~ Micellar CE procedures have also been developed for the analysis of recombinant proteins in fermentation broth' and a chimeric monoclonal antibody-cyte toxin conjugateF Matrkassisted laser desorptiodionization mass spectromehy (MALDI-MS) generates molecular ions of predominantly low charge state (MH" with I = 1-4), which are generally detected (15)
Wu.S. L; Teshim. G.;Caeia. 1.: H a n m k , W.S J.
Chlmmnfogr. 1991.
559. 357.
Kilar. E;Hkrten. S. J. Chmtogr. 1993,638,289. Hilser. V. I.; WomsiQ G. D.; Freire, E.And. Bimhm. 1993,208. 125. Compton. B. 1.J Chmnrotoa: 1991.559.357. HaningIon. S. 1.; Varm. R E.T. M.1. Chmm-. 1991,559,385, (20) Hughes. D. E.; Nchberg, P.J C h m t w . 1993,635,313. (16) (17) (18) (19)
with a mass accuracy of *O.l%, or better, using a timeof-flight mass M ~ Y Z C ? ~ The technique is exhpmely sensitive, requiring low picomole to subpicomole amounts of material, and has a mass range in excess of 200 WO Da It is also particularly well suited to the analysis of large biomolecules, as it is relatively insensitive to the presence of buffering agents,salts,and denaturants, which are often essential to maintain sample stability.n A s w c a n t exception to this is the innuence of sodium dodecyl sulfate (SDS),which causes spectral degradation at levels as low as 0.01%," which is unfortunate, as this detergent is used extensively in CE separations. For this reason, no attempt was made in this study to directly peak collect and analyze the CE fractions by MALDI-MS. Several off-line CE/MALDI-MS studies have been reported in the lite~ature;"-*~however, these have predominantly addressed the separation and detection of peptides andsmallproteins (
