Bioconjugafe Chem. 1994,5,631-635
631
Characterization of Protein-Hapten Conjugates. 1. Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Immuno BSA-Hapten Conjugates and Comparison with Other Characterization Methods1 Maciej Adamczyk,* Alex Buko,+Yon-Yih Chen, Jeffrey R. Fishpaugh, J o h n C. Gebler, a n d Donald D. Johnson Division Organic Chemistry Research (D-SNM), Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, Illinois 60064, and Structural Chemistry (D-4181, Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois 60064. Received April 5, 1994@
Several different low molecular weight haptens were conjugated to BSA to produce immunogens useful for antibody development. The extent of BSA modification due to covalent attachment of hapten was estimated by matrix-assisted laser desorption ionization mass spectrometry. The average number of hapten incorporated to immunogen was determined from the difference in the measured molecular weights of the conjugate from nonmodified BSA. The results from mass spectrometry were compared with results obtained from other more traditional methods of immunogen characterization (Wanalysis, trinitrobenzenesulfonic acid titrations, and gel electrophoresis). In each case we were able to calculate the average number of hapten covalently bound to BSA for each synthetically prepared immunogen using matrix-assisted laser desorption ionization mass spectrometry. The other methods presented limitations in certain cases.
INTRODUCTION
Immunogens are structurally complex high molecular weight materials which evoke a n immune response in host animals leading to antibody production. Small molecules (haptens < 1000 Da) generally do not, as such, trigger a n immune response since their low molecular weight and simplicity is not sufficient to trigger a recipient’s immune system. However, it is possible to elicit antibodies with affinity to such haptens by conjugating to a carrier protein forming an immunogen (1). Two very common carrier proteins employed are bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) (2). BSA is well suited as a carrier protein due to its high solubility in various aqueous buffers, moderate molecular weight (-66 kDa), and high content of available primary amines (59 lysines plus the terminal amine) which facilitate easy attachment of hapten. The popularity of BSA is further enhanced by its availability from several different manufacturers in large quantities, reasonable cost, and high purity. Resulting immunogens are generally easy to reproduce and characterize using a variety of methods. Immunogens based on KLH have been frequently used; however, they are difficult to characterize due t o poor solubility, variable mass (4.5 x 105-1.3 x 10’) of the starting protein, and inconsistencies in the purity of the KLH between manufacturers and manufacturing lots. Chemistries used for covalent attachment of haptens to the carrier protein have been well established for many functional groups available on Abstract published in Advance ACS Abstracts, October 1, 1994. Abbreviations: BSA, bovine serum albumin; DMF, dimethylformamide; equiv, molar equivalent; GMP, good manufacturing practice; ISO, International Organization for Standardization; kV, kilovolts; MALDI, matrix-assisted laser desorption ionization mass spectrometry; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; TNBS, trinitrobenzenesulfonic acid. @
1043-1802/94/2905-0631$04.50/0
polypeptides. This process may be accomplished either by direct conjugation between an existing functional group on the hapten and the protein carrier ( 3 )or by more complex methods involving modification of the hapten andlor insertion of linker arms to present a certain molecular feature (4, 5). A fundamental characteristic of an immunogen is the number of hapten covalently attached to the carrier protein which can be determined from the new molecular weight of the modified protein. The optimal number of hapten attached to the carrier protein has been debated concerning relevance t o immunogenicity directed to the newly created epitope (6-8). Several methods have been used to determine the extent of hapten incorporation. The W absorbance spectrum of the synthesized conjugate is commonly used when the hapten or linker has a strong chromophore which differs from the carrier protein (9). If the haptedinker’s chromophore is similar, a differential W spectrum obtained by subtraction of the nonconjugated protein spectra from conjugated protein spectra has been employed (10). When immunogens are obtained by modification of the reactive free amines (lysine) of the polypeptide, the degree of incorporation can be estimated as a difference between the number of reactive groups before and after conjugation by titration with TNBS (11, 12). When available, employment of radiolabeled haptens can permit determination of hapten associated with protein (13). Gel electrophoresis has also been found to have some utility. SDS-PAGE, which separates proteins on the basis of molecular size, has been useful when small proteins (520 000 Da) are conjugated with larger haptens (’800 Da) (14). More recently reported is the use of isoelectric focusing electrophoresis of protein-ligand conjugates where the separation method is based on charge (PI)which will change when amines or carboxylic acids of the polypeptide are modified by conjugation with hapten (9, 15). Matrix-assisted laser desorption ionization mass spectrometry (MALDI) has been effectively utilized in the molecular weight determination of various proteins with 0 1994 American Chemical Society
632 Bioconjugate Chem., Vol. 5,No. 6,1994
a claimed limit of 500 000 Da (16). The practical limit for routine analysis of proteins is approximately 300 000 Da with a sensitivity in the low pmol range and an accuracy of up to f0.1% (17). There are a few published reports employing MALDI for the characterization of protein-hapten conjugates (18-22). Wengatz et al. analyzed various conjugates by MALDI and compared the results to W analysis which revealed discrepancy between the two methods (17). Siege1 et al. measured the amount of attached anticancer drugs and sugars conjugated to human serum albumin and found these values to be in close agreement, in some cases, with data obtained by W spectrometry (22). In light of these findings we were interested in investigating MALDI characterization of immunogens and comparing the results against commonly used methods: colorimetric titration with trinitrobenzenesulfonic acid (TNBS), UV analysis, and gel electrophoresis. MATERIALS AND METHODS
Haptens for desipramine, doxepin, and nortriptyline (1-5) were prepared using previously described methods (4, 5, 9, 23, 24). The remaining haptens, 6-9, were purchased: 6, 3-methoxy-4-hydroxyphenylglycol,and 8,
17P-estradiol 6-(O-carboxymethyl) oxime (Sigma Chemical Go., St. Louis, MO); 7, homovanillic acid (Aldrich, Milwaukee WI); and 9, 3a-hydroxy-5-androsten-17-one hemisuccinate (Steraloids, Wilton, NH). All other chemicals were of the purest grades commercially available. All solutions were prepared using water from a Millipore water system (Millipore, Marlborough, MA). Dialysis tubing (MW cutoff 15 000 Da) was obtained from Spectrum Medical Industries, Inc. (Los Angeles, CA). Hapten was conjugated to BSA by activation of the carboxylic group of the hapten to a reactive ester using the following general procedure (4, 5,25). Each hapten was dissolved in DMF (1.0 mg/mL), 1.1 equiv of Nhydroxysuccinimide and 1.1 equiv of 1,3-dicyclohexylcarbodiimide were added, and the solution was stirred for 24 h. The reaction mixture was filtered through a glass pipette equipped with a cotton plug, and the resulting filtrate was combined with 0.026 equiv of a BSA solution (46 mg/mL in 4:l pH 7.8, 100 mM phosphate buffer:DMF) and stirred overnight. The protein solution was placed into cellulose dialysis tubing and dialyzed against 2 L of 0.1 M sodium phosphate (pH 7.8) for 4 h and then against water (2 L) with changes every 2 h for a total of seven changes. The resulting dialyzate from the dialysis tubing was lyophilized and the conjugate was sealed in amber vials under argon and stored a t -20 "C (conjugation of hapten 3 required additional protection/ deprotection steps which are described in detail in ref 5). TNBS titrations were carried out by adding to 0.5 mL of a BSA o r conjugate solution (1.0 mg/mL) in a glass vial, 1.0 mL of 4% sodium bicarbonate, and 1.0 mL of a 0.1% TNBS solution. The vials were incubated for 2 h a t 37 "C in a water bath, and then 1.0 mL of a 10% SDS solution was added followed by 0.5 mL of 1M HC1. UV absorbence were measured a t 342 nm (a sample which contained 0.5 mL water instead of protein was used as a blank). Each sample was tested in triplicate, and the results were averaged. The calculated percent of hapten bound to BSA was derived from the extent of BSA free amine modification (11). Gel electrophoresis employed a Pharmacia PhastSystem (Piscataway, NJ) utilizing 12.5%polyacrylamide SDS-PAGE precast gels (PhastGel). Conjugates were dissolved in SDS-PAGE sample buffer (10 mM TrisMCl,
Adamczyk et al.
1 mM EDTA, 2.5% SDS, and 5.0% P-mercaptoethanol, pH 8.0) to 1mg/mL and heated to 100 "C for 5 min. Gels were loaded with 2-3 p L of sample and run under denaturing conditions using the manufacturer's protocol. Upon completion, the gels were stained with Coomassie Blue using a standard developing routine from the manufacturer. UV absorption spectra were recorded on a Beckman 640 UV spectrophotometer. Conjugates and nonmodified BSA (0.1-0.5 mg/mL in deionized water) were scanned from 200 t o 500 nm. The number of hapten attached to BSA was calculated from the intensity of a difference spectra obtained by subtracting nonmodified BSA from the conjugates (10). Molar extinction coefficients for hapten were taken from the literature for 1-6 (26) and were determined for homovanillic acid (7),estradiol carboxymethyl oxime (81, and 3a-hydroxy-5-androsten17-one hemisuccinate (9) by W titration of the hapten in water. Analysis by matrix-assisted laser desorption ionization mass spectrometry employed a Bruker Reflex (Bruker Instruments, Billerica, MA) time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm). The crystal matrix, sinapinic acid (Aldrich Chemical Co., Milwaukee, WI), was prepared a t a concentration of 15 mg/mL in acetonitrile. Protein samples were typically 10-50 pmoL'pL in 2:l water:acetonitrile solution. Sample and matrix solutions were mixed in equal volumes (typically 1.5pL each) directly on the stainless steel probe tip (target) and allowed to dry (-10 min) in a fume hood a t room temperature. The crystallized analyte-matrix sample was then rinsed with 0.1% TFA solution by placing approximately 2 pL of the solution on the probe sample a t room temperature, allowing it to stand for about 5 s, and then gently drying the crystals with a stream of nitrogen. Spectra were recorded a t a threshold laser irradiance for 50-150 shots in the linear mode a t 30 kV. The resulting data were analyzed using XMASS, ver. 2.0.0, the post acquisition software supplied with the spectrometer. RESULTS AND DISCUSSION
Immunogens were prepared by covalent attachment of haptens 1-9, containing a terminal carboxylic group, to the free-amino groups (lysine residues and N-terminal amine) of BSA. In each case the carboxylic group of the hapten was activated by formation of the N-hydroxysuccinimidate ester using 1,3-dicyclohexylcarbodiimide (see Materials and Methods). Resulting conjugates were freed of unbound hapten by extensive dialysis in aqueous buffer and then lyophilized. Conjugates in this study were characterized by the following four methods: MALDI, differential UV analysis, titration with TNBS, and gel electrophoresis. In addition, the immunogens prepared were used for the immunization of animals and, in each case, resulted in the production of antibodies with affinity to the specific hapten. Table 1 lists the results of conjugate characterization by MALDI, UV, and TNBS. All of the results are reported as percent modified free amines (60 total, 59 lysines plus N-terminal amine) in BSA by the hapten. Molecular weight determination of proteins by MALDI is becoming a routine procedure. Typically, 10-50 pmol of a sample is cocrystallized with sinapinic acid and then irradiated with a high intensity pulsed laser beam to generate, intact, gas-phase molecular ions of the intact protein. A molecular weight of 66 521 Da was observed for nonmodified BSA which is within 0.2% of the molecular mass (66 430 Da) based on the complete amino acid sequence (27). The observed value for BSA was used
Characterization of Protein-Hapten
Bioconjugate Chem., Vol. 5, No. 6,1994 633
Table 1
Hapten Incorporation2 Hapten
Hapten Mol. Wt.'
MS
uv
TNBS
324.42
28%
2 1%
31%
I,
(71,808)
430.51
9% (68,704)
4%
32%
400.49
19%
21%
13%
I
(70,999)
365.47
45% (76,038) 100% (89,893)
25%
64%
321.41
42%
22%
40%
ND
20%
HO-
N/
I
5
(74,365)
6
242.22
CW&H
O V H
0
'
16% (68,646) 100% (81,995)
MPmoH 182.17
18% (68,923)
ND
37%
359.42
13%
34%
11%
HO
8 HO
@
(69,305)
N ' O v0 o H
388.50
9
38% (75,048)
>loo%
48%
0
1. Hapten mol wt. decreases by 17 mass units upon conjugation to BSA.
2. All substitution values are based on the number of primary amines (lysine and N-terminus) which have been modified.
634 Bioconjugate Chem., Vol. 5, No. 6,1994
e
1
20,000
Adamczyk et al.
- 681704(M*'
'1
-M'z
40,000
"'
80,000
60,000
Figure 1. Mass spectrum of immunogen 2.
- 68,646 M i
I
40,000
. -
I
60,000
I
I
.
80,000
,
.
,
.
,
100,000
.
,
.
,
120,000
Figure 2. Mass spectrum of immunogen 6.
in the following equation to determine the percent of modified BSA free amino groups. % modification = (conjugate mol wt) - (BSA mol wt) (loO)(haptenmol wt) (total BSA free amines)
We successfully obtained spectra for all immunogens discussed in this paper (Figure 1 illustrates a n representative example). Each spectrum contained a t least two peaks which represent singly (M+) and doubly (Mf2) charged states. The molecular weight of each conjugate was calculated from the peak centroid using the software supplied with the instrument. Experimentation with different concentrations of the conjugate were found to be an important factor for successful generation of a MS spectrum (see Materials and Methods). In addition, a n enhancement of the ion generated signal was observed by washing the sample-matrix crystals with a weak solution of TFA just prior to analysis. It is presumed that washing removes residual salts from the sample which had precipitated out during crystallization of the matrix. It was very interesting to observe additional peaks of low ion abundance for conjugates 4 and 6 (Figure 2) which represent the maximum number of hapten that could be introduced onto the free amines of BSA. These peaks represent different populations of hapten incorporation. Circumstances behind distinct populations could be a phenomenon of folding/unfolding of BSA during conjugation, hapten type and size, conjugation conditions, and/or coupling reagents used. Distinct populations of hapten within immunogens would effect immunological response. It has been reported that immunogens containing high incorporation of hapten generally result in poor immunogenicity or antibodies of the IgM type with low binding affinity to the antigen (7, 8). On the other hand, some evidence has been provided that low incorporation of hapten triggers a thymus dependent immuno response giving rise to high affinity antibodies of the IgG type (7). UV analysis was limited to those conjugates which were water soluble and possessed a usable chromophore.
Conjugates of tricyclics antidepressant drugs 1-5 were soluble, yet 4 and 5 produced slightly turbid appearance in water, the solvent used for analysis. Evaluation was not performed on the conjugates 7 and 9 which lacked solubility in water. The remaining conjugates 6 and 8 exhibited good solubility. Analysis was performed using conjugate/water solutions (0.5 mg/mL) by measuring the absorbence a t a known chromophore and subtracting out the contribution from BSA. The ratio of hapten to free amines in BSA was determined from the ratio of calculated hapten molarity to the molar concentration of lysine amines in nonmodified BSA (0.46 mM, a t 0.5 mg/mL). The range of estimated hapten incorporation was 4% for 2 to 100% for 9 with only three conjugates (1-3)having values similar to the MALDI analysis. Wengatz et al. observed higher values from UV analysis when compared with MALDI (18). They proposed that noncovalently bound hapten was associated with their conjugates resulting in the higher values. We observed higher UV values for immunogens 8 and 9,and it is possible that these conjugates have noncovalently bound hapten which was not completely removed by dialysis. On the other hand, we observed low UV values (when compared with MALDI) for immunogens 4 and 5 and attribute the results to limited solubility of these conjugates. Analysis of immunogens by W has a number of routinely applied assumptions such as the following: all noncovalently bound hapten (including salts and buffers) are removed, the immunogen is completely soluble in the matrix used for analysis, and the chromophores of BSA and hapten do not change upon conjugation. In reality these assumptions outline the deficiencies and dificulties associated with W analysis of immunogens since the perfect situation is virtually impossible to achieve. Titrations of immunogens using TNBS is a popular colorimetric method used to determine the number of free amino groups in proteins and peptides (11).This method is specific for primary amines producing trinitrophenyl derivatives which are easily quantified using a W - v i s spectrophotometer (28). As with the W analysis, solubility of the conjugates is critical for obtaining useful results. Conjugates 1-9 were all solubilized a t room temperature (1mg/mL) by the addition of 10% SDS (11). TNBS results were in close agreement with MALDI estimates except for conjugates 2 and 7 where the titration greatly overestimated the number of hapten. As with UV analysis, similar problems are associated with this method which are difficult to control. It is presupposed that the TNBS reagent reacts with all non-modified free amines of a given protein. However, folding/unfolding and aggregation between proteins could hinder some nonmodified amines from reacting with the reagent and result in overestimation of amine modification. Gel electrophoresis of our immunogens yielded limited information about their molecular weight. We encountered difficulties of immunogen solubility in the SDSPAGE buffer gel along with precipitation of the conjugate on the gel during electrophoresis. In addition, bands on the gel associated to the immunogens were often smeared, which suggested heterogeneous distribution of hapten but prevented accurate assessment of the immunogen molecular weight. The accuracy of SDS-PAGE is poor (>5%)when compared with MS techniques (29). We needed valid methods for characterization of hapten-protein conjugates prepared in our labs to meet the high demands of quality policies (GMP and ISO) being implemented by governments worldwide (30, 31 1. Each procedure presented here has its own advantages. We found analysis using MALDI to be very useful, it being the only method which directly measures the conjugate's
Characterization of Protein-Hapten
molecular weight. Unlike the other methods assessed, MALDI is not limited by rigid solubility constraints, has a high tolerance for impurities such as saltlbuffers used in immunogen preparation, and is not dependent on the physicalhhemical composition of the hapten. TNBS titrations were surprisingly close to the MALDI estimates. UV analysis had the most limitations in our hands giving results which did not correlate well with the other methods. Electrophoresis was found to have limited utility in this study. MALDI is also not without its own demands. Our initial attempts to obtain spectra of the immunogens were challenging. We found it necessary to test different conditions of sample concentration and sample preparation. An improvement of the ion generated signal was obtained by treating the sample-matrix crystals with a TFA solution. Such optimization might be necessary with any immunogen. Our work was limited to small haptens conjugated to BSA under very similar conditions. It may be necessary to investigate other variables such as crystal matrixes to obtain useful spectra for other hapten-protein conjugates. Further work is needed to determine the scope and limitations of MALDI in this area. In summary, we have demonstrated that MALDI can be very useful for the analyses of synthetically prepared immunogens to determine the number of covalently bound hapten. This tool is particularly useful due to the discovery of multiple populations of hapten incorporation observed for some immunogens. Such information is advantageous in determining the possible impact an immunogen may have on antibody development. Furthermore, the results of this work illustrate the power of physicalkhemical methods of characterization of organic compounds which for many years was reserved only for small molecular weight haptens. LITERATURE CITED (1) Benjamini, E., and Leskowitz, S. (1991) Immunology: A short course, 2nd ed., Chapter 3, Wiley-Liss, New York. (2) Liddell, J. E., and Cryer, A. (1991) A practical guide to monoclonal antibodies, Chapter 3, John Wiley & Sons, Ltd., New York. (3) Erlanger, B. F. (1980)The preparation of antigenic haptencarrier conjugates: A survey. Methods Enzymol. 70, 85. (4) Adamczyk, M., Fishpaugh, J., Harrington, C., Hartter, D., Johnson, D., and Vanderbilt, A. (1993) Immunoassay reagents for psychoactive drugs I. The method for the development of antibodies specific to amitriptyline and nortriptyline. J . Immunol. Methods 162, 47. (5) Adamczyk, M., Fishpaugh, J., Harrington, C., Johnson, D., and Vanderbilt, A. (1993) Immunoassay reagents for psychoactive drugs 11. The method for the development of antibodies specific to imipramine and desipramine. J. Immunol. Methods 163, 187. (6) Niswender, G. D., and Midgley A. R., J r . (1970) Zmmunological methods in steroid determination, Chapter 8, Appleton, New York. (7) Klaus, G. G. B., and Cross, H. M. (1974) The influence of epitope density on the immunological properties of haptenprotein conjugates. Cell. Immunol. 14, 226. (8) Malaitsev, V. V., and Azhipa, 0. Y. (1993) Influence of Epitope Density on Immunogenic Properties of HaptenProtein Conjugates. Bull. Exp. Biol. Med. 115, 726. (9) Barbarakis, M. S., and Bachas, L. G. (1991) Isoelectric focusing electrophoresis of protein-ligand conjugates: Effect of the degree of substitution. Clin. Chem. 37, 87. (10) Metelitza, D. I., Eryomin, A. N., Karasyova, E. I., and Markina, V. L. (1991) Catalytic activity and thermostability of dehydrogenase conjugates with cortisol and progesterone. Bioconjugate Chem. 2 , 309. (11) Shinoda, T., and Tsuzukida, Y. (1974) Identification of rapidly trinitrophenylating amino groups of human Bence-
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