Bioconjugate Chem. 2001, 12, 421−427
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A Robust Method for the Preparation and Purification of Antibody/ Streptavidin Conjugates Mark D. Hylarides,† Robert W. Mallett,† and Damon L. Meyer‡ NeoRx Corporation, 410 W. Harrison Street, Seattle, Washington 98119, and Seattle Genetics, Inc., 22215 26th Avenue SE, Bothell, Washington 98021 . Received October 26, 2000; Revised Manuscript Received January 26, 2001
The many uses of antibody-protein conjugates, especially antibody-streptavidin conjugates, give rise to the need for a reliable conjugation method offering reasonable yields and reproducible quality. We describe a method for preparing antibody-streptavidin conjugates that has consistently produced conjugates of quality and in sufficient quantity to be used in the clinical development and evaluation of the Pretarget delivery system. In this method antibody disulfides are reduced to generate reactive thiols, and maleimides are linked to streptavidin with the heterobifunctional cross-linking agent, SMCC. The two activated proteins are then mixed briefly before the conjugation is terminated with an oxidizing agent that reforms disulfides from unreacted thiols. The preponderance of the conjugate produced is 1:1 and 1:2 Ab:SA conjugate. This fraction is isolated from unconjugated proteins and high molecular weight byproduct by iminobiotin affinity and ion-exchange chromatography. The resulting conjugate is at least 90% 1:1 + 1:2 Ab:SA conjugate, contains no SA or Ab, and is produced reproducibly in 37% yield.
INTRODUCTION
Protein conjugates of monoclonal and polyclonal antibodies are used in many in vitro and in vivo applications. Researchers at NeoRx Corporation have investigated an approach to the treatment of solid and hematologic malignancies, “Pretarget” technology, which makes use of antibody-streptavidin conjugates to localize therapeutic radionuclides to tumors (1). The antibody causes the conjugate to localize at antigen bearing disease sites; the streptavidin captures subsequently administered biotinylated radionuclide chelates. This strategy has been shown (1-12) to circumvent the dose-limiting toxicity associated with covalent delivery of radionuclides by labeled antibodies (13-18). Recent literature (9, 11, 12, 19, Pierce Catalog) offers a plethora of methods for the covalent conjugation of proteins. These methods produce conjugates with a wide range of yields and purity and are often incompletely characterized with respect to reproducibility and scalability. The conjugation method described in this report was used throughout the development and evaluation of the Pretarget delivery system and has been compared with a number of other conjugation methods either in the laboratory or in the literature. It meets the key criteria for the development of Pretarget: it produces a high yield (relative to other available methods) of 1:1 + 1:2 Ab:SA conjugate; it succeeds on scales from a few milligrams to tens of grams; and it succeeds, with some optimization, for many different antibodies. The ideal targeting vehicles for the Pretarget method and many other applications are fusion proteins, which are homogeneous, and which, given a suitable expression system, may be prepared in large quantities at minimal cost. However, synthetic conjugates may be preferable during the investigation of a combined-function protein † ‡
NeoRx Corporation. Seattle Genetics, Inc.
because development of a suitable expression system for a fusion protein can be a lengthy and uncertain process. Furthermore, synthetic conjugation methods offer a relatively straightforward process for generating conjugates with a range of properties such as specificity, molecular weight, and binding valency to find out which are most suitable, and to aid the design of an optimal fusion protein. Hence, antibody-protein conjugation methods, and especially antibody-streptavidin conjugation methods can be helpful in the evaluation of new therapeutic modalities. Streptavidin is a 52 kD tetrameric protein produced by Streptomyces avidinii that is commercially available in high purity and shows good in vivo stability (20, 21). The strong interaction and rapid association between streptavidin and biotin (Kd ) 10-15 M) are ideal for capture of the biotinylated radionuclide chelate (90Y DOTA biotin) from circulation at the low concentrations reaching the disease site following systemic administration (20). Streptavidin was chosen in preference to avidin as a receptor because it is not glycosylated and has a reported lower retention in normal tissue (22). The murine NR-LU-10 antibody recognizes a noninternalizing 40 kD glycoprotein antigen abundantly expressed on carcinomas of the lung, colon, breast, prostate, and ovary (23, 24). This antibody has been studied both in animals and in human clinical trials (25, 26). The conjugation method takes advantage of thiols generated by reducing disulfides in IgG’s. These thiols react with maleimides placed on the surface of streptavidin at an appropriate loading by reaction with SMCC. The reduced IgG and the maleimide-functionalized streptavidin are combined at an equimolar ratio, and the progress of the reaction is monitored by size exclusion chromatography. At an optimal reaction time, when the modified proteins are maximally consumed, but before too much high molecular weight byproduct has formed, the reaction is terminated by addition of the oxidizing
10.1021/bc0001286 CCC: $20.00 © 2001 American Chemical Society Published on Web 04/27/2001
422 Bioconjugate Chem., Vol. 12, No. 3, 2001
agent sodium tetrathionate, which reforms disulfides from unreacted thiols. Thus, the proteins are conjugated through thioether and amide linkages. While the conditions described represent an optimum incorporation of reactants into usable conjugate, the reactants are not entirely consumed; some streptavidin remains underivatized with SMCC and, hence, unreactive. Consequently, the final conjugation reaction mixture contains, in addition to usable conjugate, unreacted streptavidin, unreacted antibody, and cross-linked byproduct. The purification procedure separates the three impurities in two steps. Unreacted antibody is removed by iminobiotin affinity chromatography. Unreacted streptavidin is removed by ion-exchange chromatography under conditions at which conjugate binds to the matrix but streptavidin does not. Cross-linked byproduct is separated by careful elution of the desired 1:1 + 1:2 conjugate from the ion-exchange matrix (after the streptavidin has flowed through) at a salt concentration at which the cross-linked byproduct remains immobilized on the matrix. Conjugates produced using this process were evaluated in LS-180 and SW1222 nude mouse xenograft models. The conjugates show favorable pharmacokinetic properties and biodistribution. MATERIALS AND METHODS DL-Dithiothreitol (DTT), 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), 2-iminobiotin N-hydroxysuccinimide, and sodium tetrathionate dihydrate were purchased from Sigma (St. Louis, MO). Succinimmidyl 4-(N-maleimidomethyl)cyclohexane 1-carboxylate (SMCC) was purchased from Pierce (Rockford, IL). Recombinant core streptavidin (SA) was purchased from Boehringer Mannheim (Mannheim, Germany). The monoclonal antibody, NR-LU-10, was prepared by Boehnringer Ingelheim for exclusive use by NeoRx Corporation. Coarse G-25 Sephadex chromatography matrix was purchased from Pharmacia LKB (Pisctaway, NJ). Epoxy-activated Marcroprep chromotography support 45-90 um was purchased from BioRad (Hercules, Ca) and supplied as a dry powder. N-(3Aminopropyl)-1,3-propanediamine was purchased from Aldrich (Milwaukee, WI). Fractogel EMD TMAE-650 (M) was purchased from EM Separations (Wakefield, RI). Calcium- and magnesium-free phosphate-buffered saline (PBS) was from BioWhittaker (Walkersville, MD). Preparative chromatography was done using XK series columns on a Pharmacia FPLC system. LS-180 cells were obtained from ATCC (Rockville, MD). Size Exclusion Chromatography. Analytical size exclusion chromatography (SEC) was performed on a Beckman 114M HPLC system fitted with a 9.4 mm × 250 mm Zorbax GF-250 column (MacMod Analytical, Inc., Chadds Ford, PA) using Borwin data processing software. The mobile phase consisted of 20 mM phosphate, pH 6.8 plus 0.5M NaCl at a flow rate of 1.0 mL/min. The effluent was monitored at 254 nm. Immunoreactivity. Immunoreactivity of the conjugate was determined using a competitive ELISA (27). Briefly, a solution of crude LS-180 cell extract was allowed to dry onto 96 well microtiter plates as the target. Peroxidase-labeled NR-LU-10 was used as the competitor and reporting agent at a concentration that gave OD values of 0.5 at 490 nm. NR-LU-10 and NR-LU-10/SA were serially diluted into aliquots of optimized NR-LU10/HRP conjugate and reacted with the antigen-coated microtiter plate for 1 h at room temperature. The microtiter plates were washed, and a substrate solution
Hylarides et al.
of ABTS was added, incubated for 30 min, and then read at 490 nm on a Molecular Devices Spectra Max Plus spectrophotometer. Polyacrylamide Gel Electrophoresis. PAGE analyses were done using the Mini-PROTEAN II system from Bio-Rad Laboratories (Hercules, CA). All electrophoresis was done using the procedure reported by Laemmli (28) using 5% acrylamide with sodium dodecyl sulfate (SDS). Samples were not boiled prior to electrophoresis, to maintain the tetrameric form of streptavidin. IEF gels were run on a PHAST system from Pharmacia. Precast IEF gels resolving proteins with isoelectric points between pH 3 and 9 were used in all evaluations, under the running conditions recommended by Pharmacia. Molecular Weight Determination. Molecular weights of NR-LU-10/SA conjugates were determined using a Zorbax GF-250 column connected in series with a Varian Star 9040 refractive index detector and a MiniDawn light scattering instrument (Wyatt Technologies, Santa Barbara, CA). This method involves no assumptions regarding protein shape. Molecular weights were determined as described by Wyatt (29) using a dn/dc value of 0.185 for a protein in an aqueous buffer solution. Iminobiotin Affinity Matrix Preparation. Immobilized iminobiotin affinity matrix was prepared by suspending a known amount of the dry Macroprep epoxyactivated matrix in dry methanol. N-(3-Aminopropyl)-1,3propanediamine was added to the slurry at 10 mmol/g of dry matrix. An excess of triethylamine was added, and the slurry was continuously rotated on a Buchi RE 111 rotary evaporator. After 16 h, an equal volume of 1.0 M Tris pH 10.6 buffer was added, and the slurry was rotated for an additional 2 h. The slurry was filtered, washed with several volumes of H2O, resuspended in 0.1 M H2SO4, and rotated for an additional 16 h to hydrolyze any residual epoxide residues. After filtration and H2O wash, the matrix was resuspended in 0.5 M sodium borate, pH 8.0 buffer, and 50 µmol of 2-iminobiotin N-hydroxysuccinimide hydrobromide was added per gram of dry gel. The reaction mixture was rotated overnight, filtered, and washed with alternate cycles of 1.0 M acetic acid and 1.0 M NaOH. The affinity matrix was capable of binding 3.0 mg of SA/mL of packed bed volume. Iminobiotin resin, available from Pierce, is suitable for this purification, but its expense precluded use on large scale. Affinity Purification of Streptavidin. Recombinant streptavidin (SA), pI ) 7.3; A2801% ) 34, was supplied as a lyophilized powder. Removal of excipients, endotoxin, and residual proteases was accomplished by affinity purification using immobolized iminobiotin. Lyophilized SA (500 mg) was reconstituted in 50 mM glycine buffer pH 9.0, 0.5 M NaCl, and applied to a sanitized 5.0 cm × 10 cm (196 mL) iminobiotin affinity column which was preequilibrated in the SA reconstitution buffer. After the 280 nm absorbance returned to baseline, the column was washed with five additional column volumes of buffer. SA was eluted with 0.1 M acetate buffer, pH 3.0, 0.1 M NaCl, buffer-exchanged into PBS, and concentrated using an Amicon 1 ft2 10 kDa molecular weight cut off CH2 spiral ultrafiltration system. Following concentration, 370 mg of purified SA was recovered at 23.0 mg/mL. Preparation of SMCC-Derivatized Streptavidin. Streptavidin, 368 mg (7.08 µmol), was diluted to 20 mg/ mL in PBS, and 1.84 mL (10% v/v) sodium borate buffer, 0.5 M pH 8.0, was added to increase the pH to 8.0. SMCC (7.10 mg, 21.24 µmol, or 3× over SA mol/mol) in 1.17 mL of DMSO was added to the gently stirring SA solution and reacted for 30-60 min at room temperature. The
Antibody−Streptavidin Conjugation Method
solution was then loaded onto a 2.6 cm × 42 cm Sephadex G-25 size exclusion column equilibrated with PBS at 8.0 mL/min, and the effluent was monitored at 254 nm. SMCC-derivatized SA (368 mg) was collected at the void volume in 71 mL of PBS. The extent of maleimide derivatization (MSR) was determined using cysteine followed by DTNB (30). Briefly, a known quantity of SMCC-SA was reacted with excess cysteine for 15 min. The amount of cysteine consumed by maleimide was determined by treating the solution with DTNB and subtracting the sample OD412 at pH 8.0 from that of a cysteine control. DTT-Reduced NR-LU-10. NR-LU-10 at 23.4 mg/mL (819 mg), pI ) 6.2; A2801% ) 14, was reduced by treatment with 25 mM DTT. After incubation for 30 min, residual DTT was removed by gel filtration as described above using a 2.6 × 42 cm G-25 column with helium-sparged PBS, containing 1 mM DTPA at 8.0 mL/min. A quantitative yield of reduced NR-LU-10 was recovered from the column in 795 mL. The thiol content of the antibody was measured using DTNB at pH 8.0 ( ) 13 600 cm-1M-1). NR-LU-10/SA Conjugate. Equimolar amounts of DTT-reduced NR-LU-10 (824 mg) and SMCC-SA (286 mg) were diluted to 110 mL each (24 µM) with Hesparged PBS. The two components were combined simultaneously over a 2 min period into a sterile 250 mL container using a P-1 peristaltic pump (Pharmacia LKB) to obtain a final protein concentration of 5.0 mg/mL. The stirred protein solution was monitored by HPLC SEC to evaluate the extent of the conjugation reaction. After 45 min, the reaction was quenched, and the remaining reduced disulfides were reoxidized, by the addition of solid sodium tetrathionate dihydrate to a final concentration of 5 mM. NR-LU-10/SA Conjugate Purification. For conjugate purification, glycine was added to the crude reaction mixture to obtain a concentration of 50 mM, NaCl was added to bring the total NaCl concentration to about 500 mM, and the pH was adjusted to 9.0 by addition of 1.0 M NaOH while stirring. The extinction coefficient for a 0.1% solution of an equimolar mixture of NR-LU-10 and SA is 2.0, which also corresponds to the extinction coefficient of a 1:1 NR-LU-10/SA conjugate. For simplicity, this value was used in all purification steps to estimate process yield. A 5.0 cm × 7.5 cm (147 mL) iminobiotin affinity column was prepared and equilibrated in 50 mM glycine buffer pH 9.0 containing 0.5 M NaCl. Crude conjugate solution (255 mL), pH 9.0, with a conductance of 34 mS/cm was loaded onto the iminobiotin column at 8.0 mL/min. Conjugate products and residual streptavidin bound to the matrix while unreacted NR-LU-10 and tetrathionate were collected in the flow-through. After the column was washed with five additional column volumes of equilibration buffer, 715 mg of bound material was eluted in 550 mL at 1.3 mg/mL with 0.2 M sodium acetate buffer, pH 4.0 containing 0.4 M NaCl. The pH of the eluted material was adjusted to 5.0 with 0.5 M borate buffer, pH 8.0 for storage. Following iminobiotin affinity purification, the product was buffer exchanged into 50 mM NaPi buffer pH 9.0 using a CH2 spiral diafiltration cartridge with a 30 kDa cutoff yielding 609 mg in 420 mL of buffer. The material (
