Accelerated Art ides Anal. Chem. 1994,66,585-595
Characterization of Humanized Anti-TAC, an Antibody Directed Against the Interleukin 2 Receptor, Using Electrospray Ionization 'Mass Spectrometry by Direct Infusion, LC/MS, and MS/MS Derf A. Lewls, Andrew W. Guzzetta,' and Wllllam S. Hancock Medicinal and Analytical Chemistry, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California 94080-4990 Maureen Costello Department of Quality Control, Hoffmann-La Roche, Inc., 340 Kingsland Street, Nutley, New Jersey 071 10
Characterization of a humanized monoclonal antibody (Huanti-TAC) directed against a surface protein expressed on T-lymphocytes was performed with an electrospray mass spectrometer. Capillaryreversed-phaseliquid chromatography (LC)/mass spectrometry (MS) and direct infusion MS were utilized along with tandem MS/MS analysis to confirm the sequence and to determine the sources of heterogeneity in Huanti-TAC. The MS analysis was performed on disulfidereduced and trypsin-digested samples of the antibody. Two forms of diantennary carbohydrate structures were identified and found to be consistent with those reported for the human IgGl framework. The analysis demonstrated that the N-terminus was modified by conversion of a glutamine residue to pyroglutamic acid. Another source of heterogeneity was the partial removal of the C-terminal lysine residue and was confirmed by mass calculationsof tryptic peptides followed by MS/MS sequencing. This study demonstrates that the high sensitivity of electrospray mass spectrometry when combined with capillary chromatography can allow detailed characterization of microgram samples of high molecular weight proteins such as antibodies.
residues from the complementarity-determining regions of the mouse protein into a human IgGl framework. This incorporation is a means of retaining the unique specificity of the murine immunoglobulin (IgG), while reducing any potential antigenicity of the therapeutic molecule. The analysis of this and other protein products of biotechnology has required the development of new analytical techniques to manage both the increasing complexity of the therapeutic entities and the intensifying regulatory scrutiny. While no single method to date is adequate for the complete analysis of complex glycoproteins, this study utilized an electrospray mass spectrometer as the primary tool. The use of such a powerful new technology can provide pertinent and timely information on small quantities of a given sample. Analysis of the individual polypeptide chains as both intact and fragmented species has allowed for examination of minor populations of variants and sources of microheterogeneity. Proteolytic digestion, such as with trypsin, in combination with reversed-phasehigh-performance liquid chromatographic separation (RP-HPLC) has been shown to be of importance as a means of characterizing proteins of interest and monitoring for minor alterations in a population of molecule^.^ Appli-
The present study involves the characterization of a recombinant DNA- (rDNA-) derived humanized monoclonal antibody raised against the human interleukin 2 receptor (Huanti-TAC). This antibody is under development as an immunosuppressive agent*''2 The humanization process Of a murine antibody involves the incorporation of amino acid
(1) Queen, C.; Schneider, W. P.; Selick, H. E.; Payne, P. W.; Landolfi, N. F.; Duncan, J. F.; Avdalovic, N. M.; Levitt, M.; Junghans, R. P.;Waldmann. T. A. Proc. Nafl. Acad. Sci. U.S.A. 1989, 86, 10029-10033. (2) Brawn, P. S., Jr.; Parenteau, G. L.; Dirbas, F. M.; Garsia, R. J.; Goldman, C. K.; Bukowski, M. A.; Junghas, R. P.; Queen, C.; Hakimi, J.; Benjamin, W. R.; Clark, R. E.; Waldmann, T. A. Proc. Narl. Acad. Sci. U.S.A. 1991, 88, 2663-2667. (3) Hancock, W. S.; Bishop, C. A,; Hearn, M. T. W. Anal. Biochem. 1978.89, 203-212.
0003-2700/94/0368-0585$04.50/0 0 1994 American Chemical Society
AnaiyticaIChemlstry, Vol. 66, No. 5, March 1, 1994 585
1548.5 cation of RP-HPLC using liquid chromatography electrospray It15 1659.1 1 1786.8 ionization mass spectrometry (LC/ESI-MS) to record a tryptic map has also been described and was shown to add a new dimension to the usual UV absorbance data."6 In addition to providing mass information to aid in the identification of peptides, easy identification of glycosylated peptides is possible for those sites with heterogeneous carbohydrate structures by the observation of a characteristic diagonal pattern formed in the mass to charge (m/z)vs scan number or contour p l ~ t . ~ , ~ With the advent of packed capillary columns for LC separations, smaller sample sizes and lower flow rates can result +33 in lower levels of detection, making mass spectrometry an a obvious next step for routine analysis of capillary LC separations. Numerous reports have discussed the application of FAB-MS in conjunction with capillary LC.8,9 However, matrix interference and inefficient ionization combine to limit the mass range, while fragmentation during ionization can complicate the analysis of samples containing mixtures of proteins or peptides.'&13 The application of electrospray ionization techniques for 1.91 intact IgG has been reported in the literature, showing that 1460 1470 1480 1490 1500 1510 1520 1530 1540 neither size nor extent of glycosylation is a hindrance to their mlz e~amination.'~ Several states of highly charged ions were Figure 1. Electrospray spectrum of reduced Huantl-TAC. Aliquots of observed, allowing correlation for mass calculations and IgG were reduced wlth 10 mM D l l for 30 mln at 37 OC, then acidifled wlth acetlc acid to a final concentration of 30 mM, and mlxed wlth significantly more precise estimates than standard electroacetonitrile (30% v/v). Infuslon Into the mass spectrometer at 3bLl phoretic methods; however, glycosylation and other subtle min generated mumply charged Ions of the llght chain wlth charges of structure variants for some samples remained unresolved. A +11 to +18 (upper panel.) The heavy-chain Ions are visible as the lower level ions: the lower panel focuses on the +33 to +34 chargemore recent report used collisionally assisted dissociation state ions. Differing glycosylation states may contribute to the (CAD) as a means of examining structural characteristic^.'^ heterogeneity Observed. While limited structural information of IgG was obtained by dissociation of a single light chain, the degree of detail was severelylimited by the complexity of the intact species. Other of these bioengineered molecules will greatly facilitate subreports of tandem MS studies with large, single-chain proteins sequent clinical developments. showed the significance of collisional"heating" at the interface by the proper setting of nozzle to skimmer voltage potential EXPERIMENTAL SECTION and its importance in obtaining sequence information.16 Materials. Humanized monoclonal antibodies against the However, the limited amount of sequence data provided was TAC protein were produced and provided by Hoffmann-La from the amino and carboxyl terminals of albumin; thus, the Roche as described previously.' Dithiothreitol (DTT) and four polypeptide chains of an IgG would severely complicate iodoacetic acid were obtained from Sigma. TPCK-trypsin such an analysis. (225 units/mg of protein) was purchased from Cooper This study extends the use of LC/ESI-MS to mapping Biomedical. N-Glycanase (6.4 units/mg protein) was from studies of peptide digests of antibodies, which when combined Genzyme. HPLC/Spectro Grade trifluoroacetic acid (TFA) with results of MS analysis of the reduced molecule can allow supplied in 1-g ampules was from Pierce. Acetonitrile and for detailed characterization of the protein structure. Such water (UV-grade) were from Burdick & Jackson. advances in the ability to rapidly characterize small amounts Tryptic Digestion. The method and materials for reduction and S-carboxymethylation and the tryptic digestion are as Huang, E. C.; Henion, J. D. J . Am. Soc. Mass Specrrom. 1990, I , 158. detailed previou~ly.'~Briefly, aliquots of Hu-anti-TAC were Ling, V.; Guzzetta, A. W.; Canova-Davis, E.; Stults, J. T.; Hancock, W. S. Anal. Chem. 1991.63, 2909-2915. reduced in 10 mM DTT for 30 min at 37 OC and then Covey, T. R.; Huang, E. C.; Henion, J. D. Anal. Chem. 1991,63,1193-1200. S-carboxymethylated by incubation with 25 mM iodoacetic Guzzetta, A. W.; Basa, L. J.; Hancock, W. S.: Keyt, B. A,: Bennett, W. F. Anal. Chem. 1993, 65, 2953-2962. acid for 30 min at ambient temperature, protected from light. Henzel, W. J.; Bourell, J . H.; Stults, J. T. Anal. Biochem. 1990,187,228-233. After exchanging the Hu-anti-TAC to a 100mM ammonium Stults, J. T. B.; James H.; Canova-Davis, E.; Ling, V. T.; Laramee, G . R.; Winslow, J. W.; Griffin, P. R.: Rinderknecht, E.; Vandlen, R. L. Biomed. bicarbonate buffer, two additions of trypsin at 20 pg/mg of Enuiron. Mass Spectrom. 1990, 19, 655-664. IgG (2%w/w) were made at least 12 h apart, incubating at Gross, M. L.; Tomer, K. B.;Cerney, R. L.;Giblin, D. E. In MassSpectrometry in the Analysis o f h r g e Molecules; McNeal, C . J., Ed.; Wiley: Chichester, ambient temperature for a total of 24 h. Aliquots were then U.K., 1986; pp 171-190. frozen and stored at -60 OC until analyzed. Caprioli, R. M. Anal. Chem. 1990, 62, 477A-485A. Hemling, M. E.; Roberts, G. D.; Johnson, W.: Carr, S. A,; Covey, T. R. N-GlycanaseDigestion. Removal of asparagine or N-linked Biomed. Enuiron. Mass Spectrom. 1990, 19, 6 7 7 6 9 1 . oligosaccharides was performed with N-glycanase using 6.4 Neumann, G. M.; Shiel, M. M.; Derrick, P. J . Z . Naturforsch. 1984, 39A, 584-592. units per milligram of reduced and S-carboxymethylated HuFeng, R.; Konishi, Y. Anal. Chem. 1992, 64, 209G2095.
"1
Feng, R.; Konishi, Y. Anal. Chem. 1993, 65, 645449. Loo, J . A.; Edmonds, C. G.;Smith, R. D. Anal. Chem. 1991,63,2488-2499.
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(17) Kohr, W. J.: Keck, R.; Harkins, R. N. Anal. Biochem. 1982,122,348-359.
anti-TAC for a minimum of 12 h at 37 OC, in 250 mM potassium phosphate buffer, pH 8.6,lO mM EDTA, and 0.02% (w/v) sodium azide. A second 6.4 units/mg addition of the enzyme was made and the resultant mixture allowed to incubate a total of 24 h. Aliquots were then frozen and stored at -60 OC until analyzed. Chromatography. Capillary separation was carried out at ambient temperatures with a C18, 5-pm (Nucleosil) support material packed into a glass-lined stainless steel 0.32 mm i.d. X 150 mm column. The capillary column was custom packed by LC Packings. Pumping and gradient formation was performed by a Hewlett-Packard 1090 liquid chromatograph at a flow rate of 100 pL/min. An LC Packings Acurate Microflow Processor was used for flow reduction to a Rheodyne manual injector with a 5-pL sample loop. The flow rate through the column was 3-5 pL/min and was fed directly into the electrospray nebulizer. Standard chromatography was performed using a 2.1 mm i.d. X 250 mm, (218, 5-pm (Vydac) reversed-phasecolumn heated to 40 OC with a flow rate of 0.2 mL/min. The column effluent was split 1:17 with a Valco tee, to give a flow rate of about 11 pL/min into the electrospray nebulizer. The bulk of the flow was run through the diode array detector on the HP1090 for collection of peaks as measured by absorbance at 214 nm. Solvent A was 0.05% TFA (v/v) in water; solvent B was 0.05%TFA in acetonitrile. Separation of the tryptic peptides was effected with a gradient of 0-5095 B in 90 min for the 2.1-mm4.d. column and 80 min for the capillary column. Mass Spectrometry. All electrospray ionization mass spectrometrywas performed using the Perkin-Elmer/SCIEX API I11 triple-quadrupole mass spectrometer in the positive ion mode. For direct infusion analyses, samples were acidified with 10-30 mM acetic acid and 10-30% acetonitrile or methanol prior to being infused at 3-5 pL/min using a Harvard Apparatus syringe pump. Quadrupole 1 (Ql) was scanned over various ranges of mass to charge using steps of 0.5 Da or less and dwell times of 1.0 ms or more. For the LC/MS analysis, Q1 was scanned from m/z 250 to 2200 with a scan duration of 4.13 s, using a step size of 0.5 and a 1-0-msdwell time per step. Tandem MS analyseswere obtained for infused samples using Q1 to select for the parent ion, high-purity argon as the collision gas in 4 2 , and scanning over various m/z ranges with 4 3 . The mass spectrometer was set to the following parameters: ion spray voltage (ISV) 4800 V, interface plate voltage (IN) 650 V, orifice lens voltage (OR) 70 V, ac entrance rod offset (RO) 30 V, quadrupole one rod offset ( R l ) 27 V, R2 = -35 V, R3 = -3.5 V, and a collision gas density (CGT) of -340 X 10l2 molecules/cm2.
RESULTS AND DISCUSSION Direct Infusion MS Studies. The humanized anti-TAC monoclonal antibody samples were provided with a known DNA and translated amino acid sequence provided that both the N- and C-termini were not subjected to additional enzymatic processing. In addition, the molecule would be expected to possess features in common with the welldocumented framework of IgG1.l On the basis of a model of the IgGl molecule and the amino acid sequence, one would predict Hu-anti-TAC to consist of four polypeptide chains, two pairs of heterodimers containing a light chain (Mr =
Trbh 1. M a n Dotormlnrtkn ol Horvy and LI#M Chrlm ol HwntCTAC’
reduced Hu-anti-TAC
expected mass
observed mass
light chain heavy chain, minus Lys446,O Gal heavy chain, minus Lys446,l Gal
23 214.9 60 163.7 50 326.9
50 166.4
A
23 213.0
-1.9 1.7 -1.1
60 324.8
RCM and N-glycanase treatment
expected mass
observed mass
A
light chain heavy chain, minus Lye446
23 606.1 49 366.4
23 603.0 49 239.6
-2.1 -116.Bb
0
Observed and expected masses are expressed as aver e maeeee.
b Large discrepancy of mass due to incomplete reductionsdieulfide
bridge. See text for discussion. Ught Chain
OJ
600
WO
(OW
1100
1ZW
1300
1400
1MO
1600
1700
Heavy Chain
m/z Flgm2. Eelectrosprayspectrumof reducedand scarboxymethyleted Hu-antLTAC after Mglycanase treatment. After reductbn with DlT and carboxymethylatlon with Iodoacetic acid, the IgQ was incubated with two additions of 1 unit of Mglycanase/ nmol of protein for 24 h at 37 OC. The upper panel shows the multiply charged ions of the llght chain. The lower panel shows a single series of muttlply charged ions for the heavy chain of Hu-anti-TAC.
23 215.9 Da) and a heavy chain (M, = 48 846.2 Da) linked both intra- and intermolecularly by disulfide bonds. Additionally, the heavy chain would be predicted to have a carbohydrate structure covalently bound to Asp296. An initial study of Hu-anti-TAC was conducted to examine the individual, intact heavy and light chains by reducing the disulfide linkages with 10 mM DTT for 30 min at 37 OC. A partial mass spectrum of an infused sample of reduced Huanti-TAC is shown in Figure 1. The spectrum represents the summation of 74 scans from m/z 1000 to 2200 in 0.5-unit steps and consuming a total of 291 pmol of sample. A Analytical Chemistry, Vol. 66,No. 5, March 1, 1994
607
E
1 0.0
201 13.8
601 41.3
401 27.5
801 55.1
1001 68.9
1201 82.6
Scan/Time (min) Figure 3. Reversed-phase HPLC tryptic map of Hu-anti-TAC with the total ion current (TIC) as measured by eiectrospray mass spectrometry. Allquots of the IgG were reduced and carboxymethyiatedprior to digestionwith two additions of trypsin, 150 (w/w). Tryptic peptkles. as determined by mass spectrometry, are numbered from the N-terminus and end with a letter designation for the heavy (H) or light (L) chain of the IgG. Two glycoforms of T24H are observed as well as a modified amino-termlnus-contalning peptide T1H. The Cn3 region of the heavy chain is observed as large partially digested fragments, designated T31-41H along with partially dlgested peptides T8-9H and T18-19H. The TIC shown has had background subtracted.
dominant series of ions is observed (upper panel) which correspond to the light chain of Hu-anti-TAC at charge states +11 to +18, along with less abundant ions displayed by different forms of the heavy chain at higher charge states (+24 to +39). Mass correlation is provided in Table 1, which demonstrates close agreement between expected and observed mass values for the individual polypeptide chains of Hu-antiTAC upon simple reduction. The light chain has an observed mass of 23 213.0 Da which is 1.9 Da less than expected mass (
