Region-Selective Labeling of Antibodies as Determined by

Jun 17, 2000 - Department of Chemistry (9NM), Abbott Diagnostics Division, Abbott ...... Lewis, D. A., Guzzetta, A. W., Hancock, W. S., and Costello, ...
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Bioconjugate Chem. 2000, 11, 557−563

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Region-Selective Labeling of Antibodies as Determined by Electrospray Ionization-Mass Spectrometry (ESI-MS) Maciej Adamczyk,* John Gebler, Kevin Shreder, and Jiang Wu Department of Chemistry (9NM), Abbott Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064-6016. Received December 23, 1999; Revised Manuscript Received March 6, 2000

The electrospray ionization-mass spectrometry (ESI-MS) analysis of three sets of monoclonal antibodyacridinium-9-carboxamide conjugates is described. The conjugates (nine total) were enzymatically digested using papain and the resulting fragments [Fc heavy chain, Fab, or F(ab′)2] were analyzed using liquid chromatography/ESI-MS. The average number of labels per fragment were calculated using Σ nx%, where n is the number of acridinium molecules covalently bound to the fragment and x% is the percent relative area of the corresponding peaks in the mass spectrum. When these values were normalized against the molecular weight of their respective region, antibody-dependent labeling patterns were observed. For antibodies T (anti-L-T4) and F (anti-FITC), there was a preference for conjugation of the Fab region over the Fc region. For antibody B (anti-biotin), the trend was reversed.

INTRODUCTION

Antibody conjugates play a crucial role in diagnostics and medical applications (1). Typically, antibodies are covalently attached to a label which in turn endows the antibody with the ability to generate a signal or carry a biologically destructive payload (e.g., in “magic bullet” therapy). While it is clear that the incorporation ratio of label to protein plays an important role in the performance of a modified antibody (2-4), little more than the average incorporation of label is involved in the characterization of the typical conjugate. In the past, this minimal characterization has been due to the limited number of general techniques to measure other parameters, such as the distribution and location of labels on an antibody conjugate. Traditionally, various spectroscopic techniques are used to determine label load of an antibody conjugate. However, each suffers from certain limitations that makes their use problematic. For example, while UVvis spectroscopy is commonly employed, this technique cannot be readily used unless the label has a strong absorption distinct from that of the antibody. The use of dyes such as 2,4,6-trinitrobenzenesulfonate (TNBS) or Ellman’s reagent [5,5′-dithio-bis(2-nitrobenzoic acid)] to titrate unmodified residues (e.g., amines or thiols, respectively) postconjugation can also be used (5, 6). However, inaccuracies can arise from incomplete titrations and, at best, these colorimetric techniques offer only an indirect look at label load by showing how much of the antibody has not been modified. By contrast, recent advancements in mass spectrometry (MS) have allowed a more informative and direct analysis of antibody conjugates. Once an antibody is labeled, a variety of species corresponding to a range of conjugates that vary by the covalent attachment of a single label can be observed. This distribution can then be used calculate a single average loading value (i.e., ratio of label to antibody). Using electrospray ionization-mass * To whom correspondence should be addressed. Phone: (847) 937-0225. Fax: (847) 938-8927. E-mail: maciej.adamczyk@ abbott.com.

spectrometry (ESI-MS), Bennett et al. determined the label load and distribution of an isolated antibody Fab fragment labeled with biotin (7, 8). Recently, Siegel et al. reported on the use of matrix-assisted laser desorption ionization (MALDI) to characterize the average loading number and distribution of antibody-calicheamicin conjugates (9). Interestingly, Seigel et al. also found that the analysis of intact antibody conjugates by ESI-MS was complicated by the difficult deconvolution of overlapping multiple charged peaks. This multiplicity was attributed to heterogeneity caused by glycosylation and labeling. While issues such as label load and label distribution have been addressed, where labeling preferentially occurs is not part of the routine characterization of antibody conjugates. Because antibodies, like many proteins, can be divided into distinct regions responsible for structure, binding, and physiological effector functions (10), one could propose that different region-selective modifications of the same antibody would yield differences in conjugate performance. Such issues are critical in the immunodiagnostic and immunotheurapeutic fields. For example, modification of Fab residues near the antibody-binding site could adversely affect the affinity of an antibody. Indeed, variation in residues near the binding pocket but not directly contacting a hapten can still modulate the affinity of an antibody (11). Despite such concerns, general methods to characterize this “region-modification” are scarce. Because of the potential implications, we have developed and utilized an ESI-MS strategy to investigate whether an antibody-dependent preference exists for modification of the Fab region or Fc region during the labeling process. RESULTS

Three different antibodies [anti-biotin (B, IgG2a); antiFITC (F, IgG1); and anti-L-T4 (T, IgG2a)] were conjugated with an acridinium label (1) at three different loading levels. The acridinium label was chosen because it provided a spectroscopic handle (visible λmax ) 369 nm) by which quantitative mass spectrometric data could be compared to quantitative UV-vis spectrophotometric data. Use of this label also results in a covalent amide

10.1021/bc990181y CCC: $19.00 © 2000 American Chemical Society Published on Web 06/17/2000

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Figure 1. Figure 3. The total ion count chromatogram from LC/ESI-MS of the papain digested antibody conjugate B1.

Figure 2. Summary of products obtained after papain digestion of antibodies B, T, and F (CHO ) carbohydrate modification).

linkage to lysine residues which facilitates the long term stability of the conjugate. All antibodies were dialyzed exhaustively against PBS (100 mM sodium phosphate and 150 mM NaCl, pH 8.0) prior to conjugation. To each antibody (3.0 mg of mAb in 1.4 mL of PBS) was added 4, 8, or 16 equiv of the acridinium active ester 1 (reactions 1, 2, and 3, respectively) in 0.2 mL of DMF. Upon addition, the solutions were rapidly mixed and then allowed to stand overnight (18 h) in the dark. Each reaction was then purified using gel permeation chromatography, conditions in which the labeled antibody (Rt ≈ 8.3 min) and unconjugated acridinium label (Rt ) 11.1 min) were readily separated. Postpurification, the antibody conjugates were enzymatically cleaved into Fc heavy chains and Fab [or F(ab′)2] using papain (12-14) (Figure 2). Digestion conditions were optimized to minimize nonselective cleavage (i.e., other than that near the hinge region) and disulfide exchange between antibody fragments. Cysteine, a necessary component for papain activation, was found to

promote such exchanges. Consequently, the amount of cysteine present during the digestion was kept to a minimum. In the case of antibodies B and T, the use of a 0.5% solution (w/w) of papain at pH 6-7 for 2 h yielded Fc heavy chains and Fab. For these antibodies, the hinge region was not isolated under the experimental conditions, and its contribution was neglected. In the case of antibody F (IgG1), the use of a 1.5% solution (w/w) of papain under identical conditions of time, temperature, and pH yielded Fc heavy chains and F(ab′)2. Both mouse and rat IgGl antibodies have previously been observed to yield F(ab′)2 upon treatment with papain (15, 16). A reversed-phase HPLC column, interfaced with the ion source of the mass spectrometer, was used for the separation of antibody fragments (17). Gradient elution using a water/acetonitrile mobile phase effectively removed salts and detergents from the whole antibody fragments while offering ready separation of Fc and Fab derived species (Figure 3). In general, baseline resolution was achieved between the Fc heavy chain and Fab for antibodies B and F. In the case of antibody T, there was some overlap between the peaks corresponding to the Fc heavy chain and F(ab′)2. Despite the presence of overlapping peaks, the unambiguous mass analysis of the digestion products was still possible (Table 1). By selection of the appropriate mass window (see Materials and Methods), the mass spectra profiles between the two species were readily distinguished. Importantly, postdigestion, no peak was observed for the native antibody. When injected alone, the whole antibodies eluted with a longer retention time than their postdigestion fragments and generated a readily detectable peak in the ion chromatogram. Therefore, the absence of a peak in the ion chromatogram corresponding to native antibody was taken as an indication that the enzymatic reactions had gone to completion. The raw ESI-MS spectra for the Fc and Fab fragments of the antibody conjugate B3 are shown in Figure 4. This particular conjugate exhibited the second highest level of acridinium incorporation among all the conjugates. Deconvolution of the spectra of multiply charged species to the mass domain for the Fc and Fab fragments of antibody B and its acridinium conjugates is shown in Figure 5. Prior to conjugation, only a single peak is observed for the Fab. Postconjugation with 1, additional peaks corresponding to the acridinium conjugates are observed. The mass difference of each adjacent maxima in the peak modulation is ∼568 Da corresponding to the addition of an acridinium to the anti-biotin antibody. At the acridinium active ester 1 to mAb ratio of 4:1, the native Fab is the most abundant species. As the ratio is increased to 16:1, the Fab covalently modified by two

Region-Selective Labeling of Antibodies by ESI-MS

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Table 1. ESI-MS Characterization of the Papain Digested Native Antibodies T, B, and F

native antibody

a

molecular mass of Fab in daltons (% of total peak area)

molecular mass of F(ab′)2 in daltons (% of total peak area)

anti-L-T4 (T)

47 620 (87%) 47 782 (13%)

n.o.a

anti-biotin (B)

47 777 (100%)

n.o.

anti-F1TC (F)

n.o.

95 664 (100%)

molecular mass of Fc heavy chain in daltons (% of total peak area) 25 710 (37%) 25 872 (54%) 26 035 (9%) 25 715 (44%) 25 878 (49%) 26 038 (7%) 25 527 (5%) 25 715 (49%) 25 878 (29%) 26 038 (4%) 26 187 (8%) 26 350 (4%)

n.o. ) not observed.

Figure 4. The raw ESI-MS spectra of the (a) Fc heavy chain and (b) Fab of antibody B3 (ratio of acridinium label:antibody ∼8.0).

acridinium labels becomes the most abundant species and the addition of as many as five acridinium labels is observed. DISCUSSION

The average number of acridinium labels to each antibody fragment was computed using the equation ∑ nx%, where n is the number of acridinium molecules covalently bound to the fragment and x% is the percent relative area of the corresponding mass spectrum peaks. The calculation of the acridinium labels per region of glycosylated fragments took into account the contribution of all nonglycosylated and glycosylated peaks As expected, the Fc heavy chain was characterized by multiple components due to heterogeneous carbohydrate modification. The observed mass differences are consistent with modification of a heavy chain by a galactose or mannose residue (FW ) 162). Because glycosylation of Fc heavy chains is asymmetric, the “single” heavy chain observed by ESI-MS is really a composite of both Fc heavy chains. For the Fab of antibody T, a major (87%) and minor (13%) peak which differed by a mass of 162 were observed (see

Table 1). This Fab heterogeneity is consistent with either a ragged papain digest (17) or Fab glycosylation (18). Table 2 summarizes the distribution of each acridiniumconjugated fragment for the three antibodies, the average number of acridinium labels in the Fab and Fc regions, and the average number of acridinium labels per antibody. Various factors need to be considered during the calculation of label load by ESI-MS. The determination of the distribution and average labeling value of conjugated species by ESI-MS is independent of drift in the instrument calibration because these values are calculated from the deconvoluted distribution of a single molecular ion envelope measurement. These values are also independent of the absolute concentration of conjugates. However, during the calculation of label-to-fragment ratio, the instrument response for each species in the distribution is presumed to be identical. In practice, the conjugation of an antibody fragment with the acridinium label 1 may alter its ionization efficiency and propensity to form sodium adducts. To check how these factors effect the quantitative nature of the MS results, the total number of labels per antibody as determined by the MS method was compared to the value as determined with UV-vis spectroscopy, a common method to determine label load. When plotted against each other, these values were in good agreement and yielded a slope of 0.95 (Figure 6). Overall, the mass spectrometric data compare favorably with the coupling number obtained by UV-vis absorption spectrophotometry. On the basis of this correlation, we would assume that, on average, factors such as ionization efficiency and sodium adduct formation do not play a significant role in the determination of acridinium label load by ESI-MS. To compare the extent of region-modification between the Fab and Fc region, the number of acridiniums per region were normalized against the MW of the nonglycosylated fragments (Figure 7). For the sake of comparison, the “Fab region” was defined as the F(ab′)2 for antibody F and the sum of two Fabs for antibodies B and T. Fc and Fc region are used synonymously. Normalization eliminates any error associated with the size of a fragment which may skew the apparent level of incorporation per region by virtue of a larger fragment simply having more nucleophilic lysine residues than a smaller fragment. For a hypothetical, completely randomized protein in which every amino acid residue capable of conjugation to the acridinium label 1 has identical reactivity, the ratio of labels per region would be expected to be similar for all reasonably large regions of that protein. For the conjugates studied here, there are clear antibody-dependent differences between the Fab and Fc

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Figure 5. (Left panel) Deconvoluted spectra of Fab of antibody B before and after conjugation with 4, 8, and 16 equivalents of the acridinium active ester 1. The calculated acridinium labels per Fab region are 0, 1.1, 2.1, and 4.4, respectively. (Right panel) Deconvoluted spectra of the Fc heavy chain of antibody B before and after conjugation with 4, 8, and 16 equivalents of the acridinium active ester 1. The calculated number of acridinium labels per Fc are 0, 0.8, 1.8, and 3.6, respectively. Table 2. Summary of Label Distribution and Load of the T, B, and F Antibody Conjugates as Determined by LC/ESI-MS mAb conjugatea

label distribution of Fab or F(ab′)2b

label distribution of Fc heavy chainb

labels per Fab regionc

labels per Fc regionc

labels/antibodyd

T1 T2 T3 B1 B2 B3 F1 F2 F3

0-3 0-4 1-7 0-3 0-3 0-6 0-4 0-5 2-9

0-2 0-2 0-3 0-2 0-3 0-4 0-1 0-1 0-3

1.69 2.80 6.30 1.06 2.10 4.40 1.25 2.15 5.80

1.08 1.28 2.19 0.82 1.75 3.60 0.46 0.65 1.74

2.8 4.1 8.5 1.9 3.9 8.0 1.7 2.8 7.5

a T ) anti-L-T4 mAb; B ) anti-biotin mAb; F ) anti-FITC mAb. The numerals 1, 2, and 3 correspond to reactions in which 4, 8, or 16 equiv of the acridinium active ester 1, respectively, were added. b The values shown indicate the range of conjugate species observed and used in the calculation of label load. For example, seven species were observed for the B3 Fab, the lowest MW of which corresponded to unconjugated Fab and the highest MW of which corresponded to a species with six acridinium labels/Fab. For a detailed mass spectrum of the B3 Fab, see Figure 5. c For the sake of comparison, the “Fab region” is defined as the F(ab′)2 for antibody F and the sum of two Fabs for antibodies B and T (Figure 2). “Fc region” and Fc are synonymous terms. By definition, an antibody Fc is composed of two Fc heavy chains. d Sum of the previous two columns.

regions. For antibodies T and F, there is a preference for conjugation of the Fab region over the Fc region. For antibody B, the trend is clearly reversed. Several simple reasons could account for the observed region-selective labeling. First, and most likely, there could be more lysine residues present on one fragment versus the other. Since the variable region, by definition, does not have a defined sequence of residues, it may give the Fab region a disproportionate number of reactive residues relative to the Fc region. In this scheme, the reactivity of all lysines is assumed to be the same; what differs is the number of lysines. Alternatively, changes in conformation or microenvironment induced by the composition of the variable region could change the reactivity of specific lysine residues. In turn, this effect may create conjugatable sites on the Fab region that are more or less reactive than the “average” lysine residue present in Fc region. Finally, the answer may be a combination of these two proposals.

Mass spectrometry has opened new doors to the analysis of biological macromolecules. For example, antibody microheterogeneity caused by glycosylation and truncated amino acid residues has been observed by MS. In addition, both MALDI and ESI-MS have been applied to confirm the amino acid sequence of monoclonal antibodies (19-26). Such applications have demonstrated the ability of MS to structurally characterize proteins with a high degree of mass accuracy and precision. As a tool for the analysis of protein conjugates, MS can now be routinely applied to a wide variety of systems. The label load and distribution of ligands covalently bound to proteins such as BSA, antibodies, and glucose 6-phosphate dehydrogenase have been readily characterized by MALDI and ESI-MS (9, 27-30). One advantage of MS for the analysis of conjugates is that its use is independent of the spectroscopic nature of the label. While capillary gel electrophoresis has been applied to the analysis of doxorubicin modified antibody heavy and

Region-Selective Labeling of Antibodies by ESI-MS

Figure 6. Plot of number of acridinium labels:antibody as estimated by ESI-MS versus the same values obtained by UVvis spectroscopy. The slope of the best fit line is 0.95 and the correlation coefficient is 0.98.

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Carica papaya) was purchased from BoehringerMannheim (Indianapolis, IN). The syntheses of the acridinium active ester (1, Figure 1) and the acridinium free acid (2, Figure 1) are described elsewhere (32, 33). Cysteine-HCl, EDTA, sodium hydroxide, formic acid, and guanidine-HCl were products of Sigma (St. Louis, MO). Sodium phosphate was purchased from Fisher Scientific (Fair Lawn, NJ). DMF was purchased from Aldrich Chemical Co. (Milwaukee, WI). DMF was purchased anhydrous and degassed prior to use with N2 gas to remove traces of dimethylamine. Microconcentrators (Microcon-10) were purchased from Millipore Corporation (Bedford, MA). Preparation of Antibody-Acridinium-9-carboxamide Conjugates. General Procedure. To each antibody (3.0 mg of mAb in 1.4 mL of 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) was added (a) 4, (b) 8, or (c) 16 equiv of the acridinium-9-carboxamide active ester 1 (Figure 1) in DMF (0.2 mL). Upon addition, the solutions were briefly vortexed then allowed to stand for 18 h in the dark. Each reaction was purified by HPLC using a SEC-250-5 gel permeation chromatography column (Bio-Rad, Hercules, CA) eluting with 100 mM sodium phosphate, 150 mM NaC1, 0.1% CHAPS, pH 6.3 (1.0 mL/min) and subsequently stored at 2-8 °C in the dark. Labeled antibody (Rt ≈ 8.3 min) and unconjugated acridinium-9-carboxamide label (Rt ) 11.1 min) were readily separated under these conditions. The concentration of antibody conjugate was determined by UV-vis spectroscopy using the following formula:

[conjugate] ) [A280 - (A369/4.1)]/280

Figure 7. Region-modification for conjugates of antibodies T, B, and F. Bar graph of labels per region normalized by MW for each antibody conjugate. Each bar represents the average of two runs with the high and low value indicated by the error bar. Notice that the number of labels per Fab region or Fc region varies on a per MW basis. For antibodies T and F there is a preference for conjugation of the Fab region over the Fc region. For antibody B, the trend is clearly reversed.

light chains, the quantitative use of this technique requires a label that is both UV-active and fluorescent (31). No such requirement is needed for the ESI-MS approach shown here. Characterization of antibody conjugates can play an important role in their development as immunoreagents. In the area of small organic molecules, issues of regioselectivity are well understood and manipulated to synthesize molecules with defined properties. In the case of biological macromolecules, this avenue has not been actively explored, perhaps because of the limited number of methods to investigate the phenomenon. The ESI-MS approach demonstrated here will allow for the ready analysis of the average labeling number, distribution, and the region-modification of various labels conjugated to monoclonal antibodies. Such analysis can be applied to study, prepare, and characterize antibody conjugates to ensure the production of high quality immunoreagents. MATERIAL AND METHODS

Materials. The anti-L-T4 (T, IgG2a), anti-biotin (B, IgG2a), and anti-fluorescein isothiocyanate (F1TC) (F, IgGI) monoclonal antibodies were obtained from the Abbott cell culture facility (Abbott Laboratories, Abbott Park, IL). Papain (10 mg/mL crystalline suspension from

where A280 and A369 are the absorbance readings of the acridinium-9-carboxamide-antibody conjugates at the corresponding wavelengths; 4.1 is the A369/A280 of the acid 2, and 280 (210 000 M-1 cm-1) is the extinction coefficient of an IgG antibody at 280 nm (34). The ratio of acridinium label to antibody (r) conjugate was determined by UVvis spectroscopy using the following formula:

r)

A369/369 [A280 - (A369/4.1)]/280

where 369 (14 700 M-1 cm-1) is the extinction coefficient of the acridinium 2 at 369 nm. Papain Digestion of Antibody Conjugates. Papain (10 µL of a 10 mg/mL suspension) was mixed with 90 µL of freshly prepared activation buffer (1 mM EDTA, 10 mM cysteine, and 50 mM sodium phosphate buffer, pH 7.0) and incubated at 37 °C for 10 min. The activated papain solution was added to 100 µL of anti-L-T4 or antibiotin conjugate solution (∼0.5 mg/mL) to give a papain/ antibody ratio of 0.5% (w/w). The resulting solution was incubated at 37 °C for 2 h. The digestion mixture was desalted via ultrafiltration through a Microcon 10 (MWCO ) 10K) and stored at -20 °C for LC/MS analysis. The digestions of the anti-FITC conjugates were performed following the same procedure, except the papain/antibody ratio was adjusted to 1.5% (w/w). LC/ESI-MS Analysis of Fragments. The papain digests in 1N guanidine-HCl solution (2-5 µL) were injected onto a reversed-phase HPLC column (PLRP-S column, polystyrene/divinylbenzene, 1000 Å, 2.1 × 50 mm, Polymer Laboratories Ltd., Amherst, MA) at a flow rate of 0.1 mL/min. The mobile phases A and B contained 0.1% formic acid/H2O and 0.1% formic acid/acetonitrile, respectively. A linear gradient elution was performed

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from 90 to 10% A over 20 min. Typically, salts eluted at the void volume of the column (∼4 min). The column was linked to a electrospray ion source which operated under the following conditions: ion spray voltage ) 4800 V, orifice voltage ) 50 V, nebulizing gas flow ) 1.5 L/min, and TurboProbe jet (350 °C) air flow ) 5 L/min. Mass analysis was carried out on a Sciex 100 mass spectrometer (Perkin-Elmer Sciex, Foster City, CA) equipped with a TurboIon spray source. The samples were scanned over m/z 1000-3000 with a total acquisition time of 5.0 s. Molecular masses and their distributions of the fragments under both HPLC peaks were determined by converting the raw spectra of multiply charged species to the mass domain using the manufacture’s deconvolution software (BioMultiview 1.3.1, Perkin-Elmer Sciex, Foster City, CA). The deconvolution mass window was chosen to be appropriate for the mass ranges of the expected antibody or antibody conjugate fragment. For example, for the digestion products of antibody B conjugates, mass windows of 25-28.5 kDa and 47-52 kDa were chosen to observe derivatives of the Fc heavy chain and Fab, respectively (see Figure 5). The mass domain was then saved as text files and imported into Grams software for further processing (Galactic Industries Co., Salem, NH). The peak intensity (area) corresponding to the respective acridinium conjugated antibody fragments were integrated and the contribution of each component in the distribution (relative intensity) was computed, assuming that the instrumental response is independent of the number of conjugates attached to the antibody. The average labeling number was then calculated from ∑ nx%, where n is the number of acridinium molecules covalently bound to the fragment and x% is the percent relative intensity of the corresponding mass spectrum peaks. For the calculation of total peak area, peak areas from both protonated molecules [M + xH]x+ and sodium adducts [M + xNa]x+ were included. LITERATURE CITED (1) Koppel, G. A. (1990) Recent advances with monoclonal antibody drug targeting for the treatment of human cancer. Bioconjugate Chem. 1, 13-23. (2) Kanellos, J., Pietersz, G. A., and McKenzie, I. G. C. (1985) Studies of Methotrexate-Monoclonal Antibody Conjugates for Immunotherapy. J. Natl. Cancer Inst. 75, 319-329. (3) Abraham, R., Moller, D., Senter, P., Hellstro¨m, I., and Hellstro¨m, K. E. (1991) The influence of periodate oxidation on monoclonal antibody avidity and immunoreactivity. J. Immunol. Methods 144, 77-86. (4) Baldwin, R. W., Durrant, L., Embleton, M. J., Garnett, M., Pimm, M. V., Robins, R. A., Hardcastle, J. D., Armitage, N., and Ballantyne, K. (1985) Design and Therapeutic Evaluation of Monoclonal Antibody 791T/36-Methotrexate Conjugates. Monoclonal Antibodies and Cancer Therapy, pp 215-231, Alan R. Liss, Inc., New York. (5) Sashidhar, R. B., Capoor, A. K., and Ramana, D. (1994) Quantitation of epsilon-amino group using amino acids as reference standards by trinitrobenzene sulfonic acid. A simple spectrophotometric method for the estimation of hapten to carrier protein ratio. J. Immunol. Methods 167, 121-127. (6) Riddles, P. W., Blakeley, R. L., and Zerner, B. (1979) Ellman’s reagent: 5,5′-dithiobis(2-nitrobenzoic acid)sa reexamination. Anal. Biochem. 94, 75-81. (7) Bennett, K. L., Smith, S. V., Lambrecht, R. M., Truscott, R. J. W., and Sheil, M. M. (1996) Rapid characterization of chemically-modified proteins by electrospray mass spectrometry. Bioconjugate Chem. 7, 16-22. (8) Bennett, K. L., Hick, L. A., Truscott, R. J. W., and Sheil, M. M. (1995) Optimum conditions for electrospray mass spectrometry of a monoclonal antibody. J. Mass Spectrom. 30, 769-771.

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