Bioconjugate Chem. 1991, 2, 367-374
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Reversed-Phase High-pressure Liquid Chromatographic Tryptic Peptide Mapping for the Comparison and Study of Monoclonal Antibodies Geoffrey F. Lee+ and D. C. Anderson' Biochemistry Department, NeoRx Corporation, Seattle, Washington 98119. Received February 4, 1991
We have examined and optimized several parameters to generate efficient, high-resolution, highinformation tryptic peptide maps of monoclonal antibodies and their Fab fragments, without separating the H and L chains, using reversed-phase high-pressure liquid chromatography. Use of a high proteaseto-substrate ratio with optimized digestion time and HPLC gradient conditions led to a reproducible mapping of the reduced, carboxymethylated Fab fragments of two antibodies. The technique was then used to screen Fab lots for batch-to-batch consistency, and for examining the effect of 10 mM cysteine on papain cleavage of whole antibody. The technique was modified by labeling cysteine with chromophoric analogues of iodoacetamide instead of radiolabeled iodoacetamide, resulting in a threedimensional peptide map. With multiwavelength detection, this consisted of simultaneous observation of all chromophores at 214 nm, those with aromatic residues at 280 nm, and those with cysteine at 422 nm, without collecting and counting each peak to identify cysteine-containing peptides.
INTRODUCTION Proteins and protein conjugates produced for use in human pharmaceutical products are closely scrutinized by the United States Food and Drug Administration to meet stringent quality control criteria, including lot-tolot consistency. Many laboratories use peptide mapping on reversed-phase high-pressure liquid chromatography (RP-HPLC)columns as a key analytical tool to characterize the primary structure of their protein products (1-5) and to thereby determine primary structural consistency in serially produced lots. The use of peptide mapping of recombinant tissue plasminogen activator (rt-PA)has been published (1, 2 ) , and it is increasingly evident that peptide mapping is becoming astandard test for guaranteeing primary structure consistency (6). A single amino acid difference in an rt-PA variant was identified by tryptic mapping (2),and Chloupek and colleagues have identified glycosylated and cysteine-containing peptides of rt-PA using this technique (I). This method should be applicable to a similar analysis of purified, defined conjugates constructed with proteolytically inert moieties such as drugs or carbohydrate carriers attached to the protein. Monoclonal antibodies (mAbs) and their conjugates are of significant interest in biotechnology for use as imaging and therapeutic agents. Many companies have products in development which use mAbs for the screening and treatment of cancer, toxic shock, graft versus host disease, and cardiovascular illnesses. These complex glycoproteins and their fragments need to be examined for constancy of primary structure. We report here on the development of high-resolution RP-HPLC tryptic peptide mapping for the analysis of proteolytically derived Fab fragments from two monoclonal antibodies and additional mapping studies on one of the precursor whole antibodies. The effects of digestion time and proteaseto-substrate ratio, as well as the effect of HPLC gradient slope on map resolution were investigated. We also report on studies of the reproducibility of the mapping assay,
* Author to whom correspondence should be addressed: Somatogen Inc., 5797 Central Ave., Boulder, CO 80301. + Current address: Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164. 1043-100219 1l2902-0307$02.50/ 0
and on the lot-to-lot comparison of Fab fragments produced by papain digestion in the presence or absence of cysteine as a reducing agent. Results from this experiment are relevant to explaining the effect of cysteine in enhancing the yield of papain-mediated production of Fab fragments. To avoid the process of radiolabeling cysteine with reagents such as iodoacetic acid, which requires the collection and counting of individual peaks from the peptide map, cysteine-containing peptides were identified with a chromophoric labeling reagent. EXPERIMENTAL PROCEDURES
Antibodies and Reagents. Two IgG2b antibodies with melanoma (7) and pancarcinoma (8) reactivities (designated NRML-05 and NRLU-10, respectively) were used. Fab fragments of these antibodies were generated by using immobilized papain on agarose beads (9). Each antibody and Fab fragment was purified by ion-exchange chromatography to a single peak. For lots that had been frozen, their purity was checked on the analytical scale by anionexchange HPLC on a Synchrom strong-anion-exchange column; each Fab fragment and whole antibody used here gave a single peak under isocratic elution conditions. Iodoacetic acid (IAA), tris(hydroxymethy1)aminomethane (Tris) buffer, and calcium chloride (CaClz) were purchased from Sigma Chemical Co. (St. Louis, MO). Hydrochloric acid was from J. T. Baker (Phillipsburg,NJ). Urea was from EM Sciences (Cherry Hill, NJ), and 2-mercaptoethanol (electrophoresis grade) was from Bio-Rad Laboratories (Richmond, CA). Lucifer yellow iodoacetamide (LYIA) was from Molecular Probes, Inc. (Eugene, OR). HPLC-grade water and acetonitrile were from Burdick & Jackson (Muskegon, MI) or J. T. Baker. HPLCgrade trifluoroacetic acid (TFA)was from Pierce Chemical Co. (Rockford, IL). [14C]Iodoacetate (1.25 mg, 50 pCi) was from ICN (Irvine, CA). Equipment. A Hewlett-Packard (Palo Alto, CA) HP1090M chromatography system, equipped with column oven, diode-array detector, and autosampler, was used for mapping digests. Vydac 4.1 X 250 mm CISreversed-phase columns (300Apores,5 "particles) and Cuguard column cartridges obtained from Alltech Associates (Deerfield, IL) were used. A Varian DMS 100 dual-beam spectro0 1991 Amerlcan chemical Society
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Figure 1. Peptide maps showing optimization of digestion time and protease to substrate ratio using NRML-05 Fab. S-carboxym-
ethylation used iodoacetate. The eluting gradient was from 100%A (water + 0.05%TFA) to 60%B (acetonitrile + 0.05%TFA) from 5 to 35 min. The flow rate was 1 mL/min and the oven temperature was 40 "C. Twenty-five microliters (0.65 nmol) of antibody was injected. Different columns of the graphs list the different initial protease to substrate ratios: (A) 1:lq (B) 1:2@(C)1:40. The rows of the graphs list different digestion times: (1) 2, (2) 4,(3)6,(4) 8 h. The X axis of individual maps gives the retention time in minutes, the Y axis milli absorbance units (mAU) at 214 nm.
photometer was used for protein A2m measurements. Integration of HPLC peak areas and calculation of peak retention times was done with standard ChemStation software supplied by Hewlett-Packard. The baselines for the peaks were assigned by using the macro THINKING-G, supplied by Dr. Tom Keel (Hewlett-Packard), which constructed the peak baselines by drawing a straight line from valley to valley of each peak. Sample Preparation. Reduction of antibody chains and alkylation of cysteines were performed by using modifications of the method of Crestfield et al. (IO). Antibody (10 mg/mL) in degassed chain-separationbuffer [0.1 M Tris, 0.1 or 0.01 M dithiothreitol (DTT), and 6 M urea, pH 8.01 was incubated for 1h at 60 "C under argon to effect reduction of disulfide bonds. For NRLU-10whole and Fab antibodies the DTT concentration was 10 mM, whereas for the more stable NRML-05 Fab, 100 mM DTT was used to reduce disulfide bonds. A 2-fold molar thiol excess of alkylating agent (iodoacetate or LYIA) was then added to the reaction, and the mixture was incubated in the dark for another hour at 60 "C with constant stirring. 2-Mercaptoethanolwas then added to quench excess alkylating agent. The antibody preparation was then dialyzed, or buffer exchangedby ultrafiltration, into digestion buffer (0.1 M Tris, 10 mM CaC12, 6 M urea, pH 8.0), and the protein concentration was determined from absorbance measurements at 280 nm. LYIA (97.2% pure by RPHPLC) alone absorbed strongly a t 280 nm, causing overestimation of protein concentration for antibodies treated with these agents. To circumvent this problem, we S-carboxymethylated an additional antibody sample with IAA as a control and assumed that its A2m protein concentration was similar to that of the LYIA-labeled samples. Tryptic Digestion. After chain reduction and alkylation of cysteines, antibodies were diluted to 2 mg/mL in digestion buffer and then digested with trypsin (TPCKtreated, type XIII, Sigma Chemical Co., St. Louis, MO) at 37 "C with constant stirring. The protease-to-substrate ratios used varied by experiment (see the text for details). After 2 h, a second equal-volume aliquot of trypsin was
added, and incubation continued at 37 "C. The digestion was terminated by the addition of l/10 volume 1 M HC1. Peptide Mapping. Peptide digests (25 pL, 0.65 nmol) were separated with water + 0.05% TFA (buffer A) to acetonitrile + 0.05%TFA (buffer B) linear gradients (see text for exact gradients used) at 1mL/min and 40 OC. The diode-array detector was set to gather absorption signals at 214 and 280 nm in all experiments (detection of peptide bonds and aromatic amino acids, respectively), and also at 422 nm when the chromophoric labeling agent lucifer yellow iodoacetamide was used. The reproducibility of each peak retention time and area was calculated by using the coefficient of variation ( I I ) , which was calculated from the standard deviation u and the mean value from n analyses, X,, as (a/X,) X 100%. This assumed a normal (Gaussian) error distribution. RESULTS
Optimization of Map Resolution. Effect of Digeation Conditions. Whole antibody and Fab fragments were both analyzed with respect to the effect of digestion time and protease-to-substrate ratio on map resolution. The general protocol involved reduction of antibody chains, S-carboxymethylation of cysteines with iodoacetate, and digestion of antibody chains at l:lO, 1:20, and 1:40 (w/w) initial protease to substrate ratios, followed by sampling of digests at 2,4,6,8, and 24 h and separation of peptides by HPLC on Cle columns using gradients of 2% buffer B/min. Figure 1 shows a matrix of mapping results for the Fab fragment of the anti-melanoma antibody, NRML05. Although the maps changed over time and protease to substrate ratio, there were time periods where the number, position, and height of peaks changed little at a given ratio by visual examination. We defined optimal digestion conditions as being the combination of protease to substrate ratio and time span at which few peaks changed and where the number of peaks was at a maximum. For NRML-05 Fab, this combination was achieved at a 1:lO (w/w) protease to substrate ratio at 6 h (Figure 1).
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Figure 2. Optimization of peptide mapping gradient conditions using NRML-05 Fab. S-carboxymethylation was with iodoacetate. Digestion was for 6 h at a 1:lO initial protease to substrate ratio, and 25 wL (0.65 nmol) of antibody was injected. The X axes of individual maps give retention time in minutes, the Y axes milli absorbance units (mAU)at 214 nm. Gradients were (A) 2.0% B/min over 30 min, (B) 1.0% B/min over 60 min, (C)0.5% B/min over 120 min, and (D)0.33% B/min over 180 min. The flow rate was 1 mL/min and the oven temperature was 40 O C .
For NRLU-10, both whole and Fab, digestion at a 1:lO ratio for 5 h produced the best results (data not shown). These data show that trypsin is active on these substrates in 6 M urea at 37 "C. Due to the high protease to substrate ratio used here, trypsin was checked for autolysis. A trypsin solution was mapped that had been incubated without substrate for 5-6 h a t 37 "C in digestion buffer at concentrations duplicating the 1 : l O and 1 5 protease to substrate ratios. No additional peaks were generated in the presence of the 10 mM calcium used here. Effect of HPLC Gradient Slope. Due to the large number of peaks expected, the gradient slope was optimized by running aliquots from a single digest. The digestion was performed at an optimal protease to substrate ratio and optimal time for a limit digest. HPLC gradient slopes which were tested ranged from 0.33 to 2.0% buffer B/min at a flow rate of 1 mL/min and a column temperature of 40 "C. Previous tryptic maps have used gradients as low as 0.22 % B/min (12). Decreasinggradient slope (and lengthening gradient time) from 2.0 to 0.5% B/min enhanced gradient resolution as measured visually by the number and sharpness of peaks for all tested
antibodies. Figure 2 illustrates this effect with NRML-05 Fab antibody fragments. With the highest gradient slope conditions (2 % / m i d , the mapping generated about 66 peaks. Reduction of the gradient slope to 1%B/min increased the number of peaks by 7 to ca. 73, and further reduction of the slope to 0.5% B/min increased the number of peaks to ca. 89. Decreasing the slope to 0.33% B/min, however, did little to improve resolution, although the peaks appeared slightly more dispersed in time. This pattern of increasing peak number with decreasing gradient slope also occurred in the optimization of NRLU10 Fab and whole antibody maps (Figure 3). As the slope decreased from 2 % to 1% to 0.5 % B/min, the number of Fab peaks increased from 53 to 60 to 69, while the number of peaks generated from whole antibody went from 58 to 73 to 89. All of the peaks from each sample were eluted from the column by the time the gradient reached 40% acetonitrile. The optimized HPLC slope was thus determined to be one where buffer B went from 0 to 40% in 80 min after an initial 5 min at 100% buffer A. Peptide Map Reproducibility and Comparison of Antibody Lots. The reproducibility of our procedure was checked since more peaks than predicted by the
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Figure 3. Optimization of peptide mapping gradient conditions using the NRLU-10 whole antibody and Fab fragment. S-carboxymethylation was with iodoacetate,digestion was for 5 h at 1:lO initial protease to substrate ratio and 25 p L (0.65nmol) of antibody was injected. The X axis of individual maps gives retention time in minutes, the Y axis milli absorbance units (mAU) at 214 nm. Gradients were (A) 2.0% B/min over 30 min, (B) 1.0% B/min over 60 min, and (C) 0.5% B/min over 120 min. The flow rate was 1 mL/min, and the oven temperature was 40 "C.
consensus (Lys + Arg) content of IgG2b were observed. About 35-37 peaks were expected for the Fab fragments, and about 63-65 were expected for the whole antibodies. These numbers are estimates since the sequences of only the Fv regions of these antibodies are known. Multiple consecutive mapping runs of a single digest under optimized conditions appeared identical with one another at both 214 or 280 nm (data not shown). Two samples from one vial of antibody which were simultaneously digested and mapped also appeared to give visually identical maps. The reproducibility of the tryptic mapping procedure and of NRLU-10 Fab production was also examined more quantitatively. Under optimized conditions for digestion and mapping, several lots of NRLU-10 Fab were compared. Two of the lots of Fab (E and F, the latter produced by a second manufacturer) were produced by using papain in the absence of cysteine, while four other lots (A-D) were produced by using 10 mM cysteine as a reducing agent. Cysteine has been found in the past to increase the yield of Fab produced from while IgGl antibodies by preactivating papain (13),and we have noticed this effect for
these IgG2b antibodies as well (data not shown). Samples of these lots were subjected to the optimized reduction, S-carboxymethylation,digestion, and mapping procedures. The peptide maps of the six lots tested appeared to be very similar at both 214 nm (Figure 4A) and at 280 nm (Figure 4B). In only one case, lot F, was a difference seen: the enhanced appearance of a very small peak eluting at 78 min that was not present to as large an extent in any other sample. The coefficient of variation was calculated for lots A-F for the retention times and peak areas of the 35 largest peaks observed at 214 nm, approximately the number of peaks expected from the tryptic digest. This coefficient ranged from 0.0051 to 0.23 oio with an average of 0.0375% for retention times of maps from the six lots. For the corresponding peak areas, the coefficient of variation ranged from 2.6 to 20.4% with an average of 10.7%. Thus the peak positions were highly reproducible, while the areas were somewhat less so. This may be due in part to difficulties in reproduciblyassigning parameters important
Bioconlugete Chem., Vol. 2, No. 5, 1991
Monoclonal Antibody Tryptic Peptide Mapping I
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Figure 4. Comparison of peptide maps of six different lota of NRLU-10Fab. Samples were S-carboxymethylatedwith iodoacetate and then digested for 5 h at an initial 1:lO protease to substrate ratio. Injections were 25 pL (0.65 nmol) of antibody, the gradient was 0.5 76 B/min over 80 min, and the flow rate was 1 mL/min at 40 "C. The X axis gives retention time in minutes, the Y axis milli absorbance units (mAU) at 214 nm (left) or at 280 nm (right). Rows 1 and 2 have lota A-F listed from top to bottom.
for integration of the HPLC chromatogram such as the baseline for each peak. The coefficient of variation in the retention times and areas was also calculated for lots A-D. For peak retention times and areas, this coefficient ranged from 0.0040 to ' ,and from 2.40 to 29.0 7% 0.28 7% with an average of 0.044 % with an average value of 10.8%,respectively. On the basis of the similarities of the average values for the coefficients of variation for lota A-F vs lots A-D as well as the visual similarities of the tryptic maps, there appears to be little difference between NRLU-10Fab prepared in the presence or absence of cysteine during digestion by papain. Similar results were obtained when several lots of NRML-05 Fab were compared to one another, with lower resolution 2.0% B/min gradients (data not shown). Identification of Cysteine-Containing Peptides. Cysteine-containing peptides were identified by substituting lucifer yellow iodoacetamide, a highly water soluble chromophoric analogue of iodoacetamide, for iodoacetic acid in the labeling procedure (see Experimental Procedures). This and similar haloacetyl compounds react most rapidly with thiols between pH 7 and 8, and significant reactivity with amines begins to occur a t pH values higher than about 8.5 (14). To avoid potential additional reaction with lysines, histidines, or methionines, the stoichiometric excess over the expected number of cysteines in both the heavy and light chains was kept low (14). Numerous such iodoacetyl chromophores have been used as thiol-
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specific reagents (151,including such relatively apolar ones as 2-(bromoacetamido)-4-nitrophenol(16). The labeled protein was digested and mapped under optimized conditions. Detection of LYIA-labeled peptides was accomplished by monitoring elution at 422 nm simultaneously with 214 and 280 nm wavelengths by using a photo-diode array detector. NRLU-10 Fab labeled with LYIA gave high-resolution maps containing ca. 70 peaks at 214 nm (Figure 5; column A), and 9-12 major peaks at 422 nm (Figure 5; column B). The large peak a t 16 min in the 422-nm maps coelutes with free LYIA. A comparison of several lots of NRLU-10 Fab labeled with LYIA (Figure 5) showed that the maps appeared very similar at both 214 and 422 nm as would be expected on the basis of the similarity of maps of IAA-labeled Fabs. The reproducibility of the elution times and peak areas (from four different lots) of the 422-nm map was estimated by calculating the coefficient of variation as before. For the areas from the 10 largest peaks other than the free LYIA peak, the coefficient of variation ranged from 0.045
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Figure 6. (A) Comparison of peptide maps of IAA- and LYIAlabeled samples from one lot of NRLU-10 Fab at 214 nm, with the IAA map inverted. (B)Comparisonof peptide mapsof LYIAlabeled sample at 214 and 422 nm, with the latter map inverted. In both cases,the gradientwas 0.5% B/min over 80 min, the flow rate was 1 mL/min at 40 "C. The X axis shows retention time in minutes, the Y axis milli absorbance units. sequent ultrafiltration to remove excess free reagent) with a 2-fold excess of LYIA. Only 12% of the control 82 800 cpm were incorporated, suggesting that LYIA competes with iodoacetate for labeling these antibody chains. 5
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Figure 5. Comparison of peptide maps of four lots of NRLU-10. The Fab fragment was labeled with lucifer yellow iodoacetamide and then digested for 5 h at an initial 1:lO protease to substrate ratio. Injections were 25 rL (0.65 nmol) of labeled antibody,the gradient was 0.5% B/min over 80 min, and the flow rate was 1 mL/min at 40 O C . The X axis gives retention time in minutes, the Y axis milli absorbance units (mAU) at 214 nm (bottom)and 422 nm (top). Free lucifer yellow iodoacetamide elutes at ca. 16 min in these chromatograms. to 0.38% with an average value of 0.23 '32. This coefficient ranged from 9.6 to 26 7% with an average value of 187% for the areas of these peaks. Thus the peak retention times for the 422-nm map were highly reproducible while the peak areas were less reproducible. These maps appeared to be different from those labeled with IAA (Figure 6A). This is not unexpected, since the LYIA-labeled peptides may shift to earlier retention times with the addition of charged groups present on the chromophoric label. Direct comparison of 214 and 422-nm signals (Figure 6B) indicated the peaks that are expected to contain cysteine. Papain-produced mouse IgGzb Fab fragments are expected to contain 10 cysteines (9, 17-18), and NRLU-10 has an additional cysteine in a heavy chain hypervariable loop (unpublished data). The number of major peaks (9-12) thus agrees well with the expected number (I1)of cysteinecontaining peptides generated by tryptic digestion of this mouse IgG2b Fab fragment. In a separate experiment, NRLU-10 Fab was reacted with a 2-fold excess of [ Wliodoacetate under standard conditions with and without a prior reaction (and sub-
DISCUSSION In this paper we have examined the application of tryptic peptide mapping techniques to two IgG2b monoclonal antibodies and their Fab fragments which have gone through Phase 111clinical trails as *Tc-labeled imaging agents for melanoma and small cell lung cancer. This technique should ')e useful for quality assurance groups to monitor batches of incoming whole antibody and processed Fab fragments for consistency and to monitor different production techniques to identify those which may modify residues of the final product. One of our goals was to develop a reproducible procedure generating a large number of peptide fragmentsto facilitate a detailed comparison of different antibody lots. On the basis of estimation of the reproducibility by the coefficient of variation, this has been achieved for the map of NRLU10 Fab alkylated by either iodoacetic acid or by lucifer yellow iodoacetamide. Due to their larger coefficient of variation, the peak areas appear in general to contain more variability than the peak retention times with the HPLC hardware and integration algorithms used here. The peak retention times appear to be remarkably consistent from run to run, with one standard deviation of the elution time of a NRLU-10 Fab eluting after 40 min, typically in the range of 1 s. This may be due in part to the use of a precise automated injection system, reliable microbore HPLC pumps, a good algorithm for peak detection, and a flow-cell volume (ca. 7 pL) which is less than the volume of a narrow peak (ca. 200 pL).
Monoclonal Antlbody Tryptic Peptide Mapping
A single substitution of a norleucine for the N-terminal methionine in a bovine somatotropin 17-mer tryptic peptide has been shown to result in a 30-40 s increase in this peptide’s 53-min retention time (19). This suggests that any change in the amino acid composition of a specific peptide might be detectable by an altered peak retention time (as well as the appearance of a new peak) in our system. Factors derived from the tryptic digest itself, from the properties of the generated peptides such as solubility or susceptibility to different reactions, and perhaps from the peak integration protocol, may limit the reproducibility of the peak areas. With an HPLC system such as that used here, the most sensitive way to detect changes in the final antibody may be detection of changes in the peak retention times. Consensus sequences exist for the mouse K light chain and y 2b heavy chains (17,18,20).By including the known Fv-region sequences of the two antibodies used here, we expect that 35 tryptic sites exist in the NRML-05 Fab fragment, that 37 exist in the NRLU-10 Fab fragment, and that an additional 28 sites (excluding peptides resulting from adjacent basic residues) exist in the Fc region of the heavy chain. Including minor peaks, at the highest resolution our maps contained 83-89 peaks (Fab fragments) or ca. 89 peaks (NRLU-10 whole antibody), suggesting that either incomplete digestion or overdigestion may have occurred. Incomplete digestion is known to occur with other proteins, for example at Arg-Pro or Lys-Pro bonds (1). Since high levels of urea (6 M) were used to solubilize the denatured alkylated chains, and since trypsin is thought to be less active at higher levels of urea (23),this may occur here as well. The facts that we obtain distinct and reproducible tryptic maps of different antibodies and that control experiments with trypsin alone show no evidence of autolysis (due perhaps to the 10 mM calcium added for its suppression (21)suggest that trypsin is in fact proteolytically active on these substrates. Further, conditions were developed so that this would be a limit digest, and little change was seen in the map for NRLU-10 Fab after 6 h. Thus incomplete digestion is perhaps not the only explanation for the number of peaks seen here. Due to the high 1:lO enzyme:substrate weight ratio employed here [1:100 is normally used (2111,it could be argued that an overdigestionis responsible for the observed number of peaks. For example, trypsin is known to cleave and this could generate extra at chymotryptic sites (1,21), fragments. It is also possible that the high urea levels result in a loss of the normal specificity of trypsin for cleavage on the C-terminal side of Lys or Arg. However LYIA-labeled maps of NRLU-10 Fab contain close to the expected number of 11peaks, thus trypsin appears toretain significant specificity. Other possible reasons for the observation of extra peaks could include the presence of glycosylated residues known to occur in IgG2b antibodies (22).Without the N-terminal sequences of a number of the peaks generated in these tryptic maps, we cannot at present distinguish these possibilities. In spite of the number of peaks observed, the maps appear reproducible both upon multiple injections or upon mapping of different antibody lots. Besides their previous use for sequence comparisons of antibodies or individual antibody chains (21,24,25), we have used tryptic maps here to examine modifications in the papain fragmentation procedure used to produce Fab fragments. The Fab peptide maps compared here with and without 10 mM cysteine appear essentially identical, suggesting that the papain cleavage sites, located in the
Bioconjugftte Chem., Voi. 2, No. 5, 1991 373
hinge region in the sequence (241,have probably not changed in the presence of cysteine. Although this has not been seen by Parham for other IgG2b antibodies (13), one or more of the hinge region disulfides, all three of which are within about 16 residues of the papain cleavage sites, may be reduced, with a concomitant improvement of the substrate properties of the antibody. In order to facilitate the detection of cysteine-containing peptides, we have used lucifer yellow iodoacetamide as an analogue of iodoacetate to react with cysteines and thus label cysteine-containing peptides. This approach is similar to the use of other chromophores to label cysteine, such as DTNB or 4-vinylpyridine (17). The advantages of the chromophore used here are that it is highly watersoluble and has an absorption band with an extinction coefficient of 13 OOO M-l cm-l at 426 nm (15), which is separate from the bands at 214 or 280 nm. Lucifer yellow iodoacetamide is also strongly fluorescent, which should if necessary allow a more sensitive detection of these peptides. We have used the LYIA alkylation of cysteines to replace the carboxymethylationby iodoacetate which may prevent the refolding of very stable disulfide-containing proteins such as antibodies before trypsinization. Due to the appearance of a lucifer yellow labeled tryptic map of similar complexity to that generated after S-carboxymethylation, alkylation with lucifer yellow iodoacetamide appears to be as successful as iodoacetate in preventing this refolding of the reduced H and L chains. Results presented here demonstrate the reproducibility of mAb scale-up production runs and fragmentation processes, and the utility of three-wavelength data collection for rapid mapping of cysteine-containingproteins. Additional dimensions might be added to these maps, for example by specific labeling of arginines with l-pyreneor by glyoxal or 7-(diethylamino)coumarin-3-glyoxal(22), labeling aspartates and glutamates with chromophoric carboxylate-specific reagents. Application to the characterization of immunoconjugates can also be envisioned wherein the number and type of modified residues, which alter the chromatographic properties of the peptides produced, can be identified after isolation and sequencing. ACKNOWLEDGMENT
We acknowledge Dr. Alan Fritzberg for his support of this work, numerous helpful suggestions, and careful reading of the manuscript, and John Reno for suggesting a comparison of the Fab fragments produced in the presence and absence of cysteine. LITERATURE CITED (1) Chloupek, R. C., Harris, R. J., Leonard, C . K., Keck, R. G., Keyt, B. A., Spellman, M. W., Jones, A. J. S., and Hancock, W. S. (1989) J. Chromatog. 463, 375-396. (2) Garnick, R. L., Solli, N. J., and Papa, P. A. (1988) Anal. Chem. 60,2546-2557. (3) Hartman, P.A., Stodola, J. D., Harbour, G. C., and Hoogerheide, J. G. (1986) J. Chromatogr. 360, 385-395. (4) Harris, R. J., Chamow, S. M., Gregory, T. J., and Spellman, M. W. (1990) Eur. J . Biochem. 188, 291-300. (5) Kohr, W. J., Keck, R., and Harkins, R. N. (1982) Anal. Biochem. 122, 348-359. (6) Hancock, W.S.(1986) Chromatogr. Forum 1, 57-59. (7) Woodhouse, C. S.,Bordonaro, J. P., Beaumier, P. L., and Morgan, A. C. (1990) In Human Melanoma, from Basic Research to Clinical Application (S. Ferrone, et al., Eds.) pp 413-429, Springer-Verlag, New York. (8) Varki, N. M.,Reisfeld, R. A., and Walker, L. E. (1984) Cancer Res. 44, 681-687.
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(9) Parham, P. (1986)In Handbook of Experimental Zmmunology, Volume 1,Zmmunochemistry (D. M. Weir, Ed.) Chapter 14,Blackwell Scientific Publications, London. (10) Crestfield, A.M., Moore, S.,and Stein,W. H. (1963)J.Biol. Chem. 238,622427. (11) Bendat, J. S.,and Piersol, A. G. (1986)Random Data, Analysis and Measurement Procedures, 2nd ed., pp 252-256,
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(16) Gardner, J. A., and Matthews, K. S. (1987)Anal. Biochem. 167,140-144. (17) Kabat, E. A,, Wu, T. T., and Bilofsky, H. (1979)Sequences of Immunoglobulin Chains, U S . Dept. of Health, Education
and Welfare, Public Health Service, National Institutes of Health, Washington, DC. (18) Kabat, E. A., Wu, T. T., Bilofsky, H., Reid-Miller, M., and Perry, H. (1983)Sequences of Proteins of Immunological
Interest, U.S.Dept. of Health and Human Services, Public Health Service, National Institutes of Health, Washington,
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netics Inc., Mountain View, CA).
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