Bioconjugate Chem. 1995, 6, 313-31 5
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TECHNICAL NOTES Electrophoretic Method for the Quantitative Determination of a Benzyl-DTPA Ligand in DTPA Monoclonal Antibody Conjugates Diep T. Pham, Frans M. Kaspersen, a n d Ebo S. Bos* N.V. Organon, P.O. Box 20, 5340 BH OSS, The Netherlands. Received December 12, 1994@ ~~
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A simple electrophoretic IEF procedure was developed for the quantitation of bifunctional DTPA ligand molecules in DTPA-protein conjugates. From a calibration plot of PI versus substitution ratios of reference conjugates, the concentrations of DTPA conjugated to protein were determined. Molar ratios of DTPA to protein agreed satisfactorily with the ratios obtained by a spectrophotometric technique using a colored yttrium(II1) complex of arsenazo 111. The IEF method was successfully applied on preparations of benzyl-DTPA to mAbs MOPC-21, SC-20 (aCEA), and human serum albumin.
INTRODUCTION
For over a decade, radiolabeled monoclonal antibodies have been studied as a tool in the diagnosis and therapy of cancer (1-3). The most interesting isotopes employed in radioimmunodiagnosis and -therapy, e.g., “‘In and are metal ions, which can be bound to the targeting moiety only when captured in a chelation structure either directly to the antibody or via carrier molecules like serum albumin or polylysine. Determination of the number of ligands introduced is a n essential part in the quality control of chelator-conjugate preparations for preclinical purposes and clinical use. The number of metal binding sites in ligand mAb conjugates can be quantitated by several analytical methods, e.g., lllIn or 57C0binding assays (4, 5 ) or the spectrophotometric titration with a yttrium-arsenazo I11 complex (6). A drawback of the two former procedures is that radioactive isotopes are employed as analytical reagents and the procedure has to be carried out in restricted areas, whereas in the latter method the use of the suspectedly carcinogenic arsenazo I11 compound requires special precautions. Moreover, the reproducibility of the isotope binding assays is poor a t least in our hands in contrast to that of the spectrophotometric assay. The introduction of metal-binding ligands from the amino polycarboxylate class (e.g., DTPA, DOTA, LILOl) will add to the total negative charge of a protein causing a n acidic shift in PI of the conjugate compared to the unmodified protein. As a consequence, these ligandmAb conjugates will show an anodal shift in IEF dependent on the number of ligands introduced. The objective of this study was to develop a qualitative and quantitative determination of the chelator content
of DTPA-protein conjugates by IEF and to compare this electrophoretic method with the spectrophotometric procedure using the yttrium-arsenazo complex. EXPERIMENTAL PROCEDURES
Materials. MOPC-21 and SC-20 were a kind gift of Dr. B. Butman (PerImmune Inc., Rockville MD). HSA was purchased from the Central Blood Bank, Amsterdam, the Netherlands. Arsenazo I11 complex was obtained from Sigma, St. Louis, MO. All chemicals used were of analytical grade from Baker Deventer, The Netherlands, or Merck, Darmstadt, FRG. Preparation of @-Isothiocyanatobenzyl) -DTPA. (p-Isothiocyanatobenzy1)-DTPA was synthesized according to the procedure of Brechbiel et al. (7) except that tert-butyl groups were used for protection of the carboxylic acid groups. (p-Nitrobenzy1)diethylenetriaminewas reacted with excess (15 equiv) tert-butyl bromoactate in refluxing ethanol in the presence of triethylamine (15 equiv) for 20 h. After removal of the salts by extraction, the product was purified by chromatography on & 0 3 (n-hexanelethyl acetate, 85/15 v/v), yielding pure tert-butyl (p-nitrobenzy1)diethylenetriaminepentaacetate as an oil in 45% yield; FAB-MS mlz = 809 ((M HI+). Subsequently, tert-butyl (p-nitrodibenzy1)ethylenetriaminepentaacetate was reduced for 4 h in ethanol using Hz (3 atm of pressure) with PdC (10%)as catalyst. After removal of the catalyst, the product was purified by chromatography on A 1 2 0 3 (n-hexanelethyl acetate; 8:2 vlv), yielding pure tert-butyl p-aminobenzyldiethylenetriamine pentaacetate a s a n oil in 45% yield. This was further reacted in dichloromethane with thiophosgene for 4 h a t room temperature. The product, tert-butyl (p-isothiocyanatobenzy1)diethylenetriaminepentaacetate, was isolated as a n oil in 50% yield by Abstract published in Advance ACS Abstracts, April 1,1995. chromatography on A1203(n-hexanelethyl acetate, 85/15 ‘Abbreviations: CBB, Coomassie brilliant blue; CEA, carcivlv). IR (liquid): 2090 cm-I (N=C=S); 1744 cm-I (COOnoembryonic antigen; DOTA, 1,4,7,10-tetraazacyclododecane tBu). lH-NMR (CDC13): 1.40 ppm (tBu); 7.08 and 7.25 NPJTJV-tetraacetic acid; DTPA, diethylenetriaminepenppm (phenyl). FAB-MS: mlz = 821 ((M + H)-) and mlz taacetic acid; HSA, human serum albumin; IEF, isoelectric focusing; LILO, 1,3-bis[N-[N-(2-aminoethyl)-2-aminoethyll-2- = 843 ((M + Na)+). Preparation of Benzyl-DTPA Conjugates. The aminoacetamidol-2-~4-isothiocyanatobenzyl)propane-N,N,N’, proteins to be conjugated were transferred t o PBS by W,N””’JV’JY”’JV’’’’-octaacetic acid; PBS, phosphate-buffered chromatography on PD10. The PBS used in this procesaline; TFA, trifluoroacetic acid.
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Pham et al.
314 Bioconjugate Chem., Vol. 6, No. 3, 1995
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Figure 1. IEF analysis of antibody- and HSA-DTPA conjugates. (A) HSA-DTPA M, pZ-markers; (1)HSA; (2) HSA-DTPA (0.4 moUmol); (3)HSA-DTPA (1.0 mol/mol); (4)HSA-DTPA (1.7 mollmol); (5) HSA-DTPA (3.3mollmol). (B) MOPC-DTPA M, pZmarkers; (1)MOPC; (2) MOPC-DTPA (2.0 mol/mol); (3)MOPC-DTPA (2.2 mol/mol); (4)MOPC-DTPA (4.4mol/mol); ( 5 ) MOPCDTPA (7.3 mol/mol). (C) SC-20-DTPA M, pl-markers; (1)SC-20-DTPA (1.8mol/mol); (2) SC-20-DTPA (2.2 mol/mol); (3)SC-20DTPA (3.1mol/mol); (4) SC-20-DTPA (3.8mol/mol); ( 5 ) SC-20-DTPA (5.0 mol/mol); (6) SC-20. DTPA / PROTEIN (mol / mol)
DTPA / PROTEIN (mol / mol)
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Table 1. Determination of Chelator Content of DTPA Conjugates with IEF and Spectrophotometric Method MOPC-DTPA
HSA-DTPA SC-20-DTPA
1 2 3 4 1 2 1 2 3
IEF 3.1 6.4 5.3 2.8 3.0 2.7 2.82 f 0.48 4.56 f 0.50 5.55 f 0.27
yttrium-arsenazo 3.0 6.8 5.4 2.6 3.1 2.8 2.22 f 0.45 4.51 f 0.62 5.21 f 2.1
dure was chromatographed on Chelex-100 in order to remove unwanted metal ion contaminants. (tert-Butylbenzyl)-DTPA was deprotected by incubation for 4 h in TFA (1g/L) at ambient temperature. TFA was removed by a gentle stream of nitrogen. The residue was dissolved in 1 mL of dichloromethane and again evaporated to dryness; this procedure was repeated three times leaving the unprotected chelator as a white powder. FAB-MS: mlz = 539 ((M - H)-) and mlz = 577 ((M + K - 2H)-). The unprotected chelator was dissolved in PBS to a final concentration of approximately 5 g/L. Usually, deprotected (p-isothiocyanatobenzy1)-DTPAwas added to the protein solutions in PBS a t a molar proteidchelator ratio of 1:lO. The pH of the reaction mixture was adjusted to 8.9 with 0.1 m o m of triethylamine. Incubation was performed for 2 h at 37 "C. Excess reagent was removed by gel filtration on PDlO equilibrated in Chelex-
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2 3 4 5 6 DTPA / PROTEIN (mol / mol)
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Figure 3. Correlation curve between IEF and spectrophotometric analysis. Abscissa: electrophoretic method. Ordinate: spectrophotometric method.
treated PBS. The substitution efficiency was approximately 30%. Determination of Chelator Content of DTPA Conjugates. Spectrophotometric Method. This method was carried out according to Pippin et al. (6). To 500 pL of yttrium-arsenazo solution was added 10 pL of conjugate solution in PBS, and after mixing the absorbance at 652 nm was measured in a Pye-Unicam 8700 spectrophotometer. A calibration curve was prepared using 10 pL of DTPA solutions in a concentration range of 20100 nmom. Electrophoretic Method. IEF of DTPA conjugates was carried out in pH 3-9 gels in a Phast Electrophoresis System (Pharmacia) essentially according to the manufacturers' instructions. Gels were stained with CBB G250 as described by Neuhoff (8) and scanned in a computing densitometer 300 A of Molecular Dynamics using Imagequant 3.3. The median p l values of the protein and conjugate peaks were estimated using the prestained Electran p l calibration kit pH 4.7-10.6 (BDH) as a reference. RESULTS AND DISCUSSION
In Figure 1, the IEF analysis of MOPC-DTPA, SC20-DTPA, and HSA-DTPA conjugates of different, known substitution ratios (determined by the spectrophotometric method) is presented. The antibody- and albumin-DTPA conjugates showed the predicted anodal
Technical Notes
Bioconjugate Chem., Vol. 6,No. 3, 1995 315
PI - 4,85
- 5,65 - 6,45 - 7,3 - 8,3 M 1 2 3 4 M Figure 4. Qualitative IEF analysis of MOPC- and HSAchelator conjugates: M, pl-marker; (1)HSA-chelator; (2)H S A (3) MOPC-chelator; (4) MOPC. The chelator was a carboxylic acid-containing chelator, analogous to DOTA.
shift compared to the unmodified proteins. Measurement of PI changes by computer imaging resulted in the calibration curves presented in Figure 2. From these curves, the chelator substitution ratio of various antibody and HSA-conjugates was derived and compared with the values obtained by the spectrophotometric analysis. As can be concluded from Table 1, a good correlation was found between the chelator content determined by the electrophoretic and spectrophotometric method, the correlation coefficient being 0.985 (Figure 3). Except a t high substitution ratios, the interassay variation of both methods (as determined in three consecutive experiments) was comparable and in the same order as published by Pippin et al. (6). The reason we found a high variation a t elevated DTPA contents may be the inaccuracy a t low absorbance values of the spectrophotometer used. Since the electrophoretic method is only dependent on the number of carboxylic acid groups introduced a t conjugation of the chelator, no corrections have to be made for the intrinsic metal-binding capacity of the protein itself, thus providing a direct value for the total chelator content of the conjugates rather than the total available binding sites for metal ions. In particular, the intrinsic metal binding capacity of HSA is substantial, i.e., 3 moVmol protein, whereas for mAbs usually a
value under 0.2 moVmol was found. Moreover, IEF allows a convenient and an at least qualitative control on the attachment of carboxylic acid-containing chelators that can bind the (radioactive) metal ions only a t elevated temperatures as is shown in Figure 4. For the DOTAlike structure used in this experiment (trans)chelation occurred only above 70 "C. Thus, a t temperatures compatible with the protein carrier, no reaction of the mAb-chelator conjugate with the yttrium-arsenazo complex was observed, whereas a substantial anodal shift was found with IEF. In conclusion, IEF offers an easy, qualitative and quantitative method for the determination of the chelator content of conjugates between proteins and DTPA or probably other amino polycarboxylate chelators. LITERATURE CITED (1) Waldman, T. A. (1991).Monoclonal Antibodies in diagnosis and therapy. Science 252, 1657- 1662. (2) Goldenberg, D. M. (1993). Monoclonal Antibodies in cancer detection and therapy. Am. J . Med. 94, 297-312. (3) Fritzberg, A. R., Beaumier, P. L., Bottino, B. J., and Reno, J. M. (1994).Approaches to improved antibody- and peptidemediated targeting for imaging and therapy of cancer. J. Controlled. Rel. 28, 167-173. (4) Paik, C. H., Murphy, P. R., Eckelman, W. A., Volkert, W. A., and Reba, R. C. (1983). Optimization of the DTPA mixed anhydride reaction with antibodies a t low concentrations. J. Nucl. Med. 24, 932-936. (5)Meares, C. F., McCall, M. J., Reardon, D. T., Goodwin, D. A., Diamanti, C. I., and McTigue, M. (1984). Conjugation of antibodies with bifunctional chelating agents: Isothiocyanate and bromoacetamide reagents, Methods of analysis and subsequent addition of metal ions. (6) Pippin, C. G., Parker, T. A., McMurry, T. J., and Brechbiel, M. W. (1992). Spectrophotometric method for the determination of a bifunctional DTPA ligand in DTPA-monoclonal antibody conjugates. Bioconjugate Chem. 3, 342-345. (7) Brechbiel, M. W., Gansow, 0. A., Atcher, R. W., Schlom, J., Esteban, J., Simpson, D. E., and Colcher, D. (1986).Synthesis of 1-(p-isothiocyanatobenzyl)derivatives of DTPA and EDTA. Antibody labeling and tumor imaging studies. Znorg. Chem. 25,2772-2781. (8) Neuhoff, V., Stamm, R., and Eibl, H. (1985). Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: a systematic analysis. Electrophoresis 6, 427-448.
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