Bioconjugate Chem. 2006, 17, 258−260
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A General Approach for Antibody Purification Utilizing [Protein A-Catechol:Fe3+] Macro-Complexes Guy Patchornik†,* and Amnon Albeck§ Affisink Biotechnology Ltd, 11 Hamaccabee St. Kiryat-Ono 55572, Israel, and The Julius Spokojny Bioorganic Chemistry Laboratory, Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel. Received December 23, 2005; Revised Manuscript Received January 27, 2006
A general platform for antibody purification utilizing free nonimmobilized Protein A modified with the strong metal chelator catechol (ProA-CAT) and Fe3+ ions is presented. The mechanism of purification requires formation and precipitation of macro-complexes composed of [ProA-CAT:IgG:Fe3+]. Target IgGs are eluted directly from the precipitates (i.e. pellets) at pH 3 in high yields (71-80%) and high purity (>95%), without dissociating the [ProA-CAT:Fe3+] insoluble macro-complex.
Highly purified antibody preparations represent a major scientific and industrial need. In a previous study (1) we presented a novel purification approach (affinity-sinking, AS1), utilizing free nonimmobilized desthiobiotinylated ligands (e.g., Protein A; Concanavalin A) and free avidin. The nonimmobilized state of the ligand circumvents the need for immobilizing ligands to polymeric supports; hence, polymers are excluded from the process and purification is accomplished without chromatographic columns. This study further demonstrates the implementation of AS on a novel, more challenging platform, the metal:chelator platform. Therefore, Protein A, a 42 kDa factor produced by several stains of Staphylococcus aureus which binds specifically to the Fc region of different classes of immunoglobulins (2), was covalently modified according to ref 3 with a synthesized active ester derivative of the strong metal chelator catechol, catechol-NHS (Figure 1; see synthesis in Supporting Information) (4). The modified Protein A (ProACAT) serves as the nonimmobilized ligand and is used for purification of rabbit and bovine IgGs from E. coli cell lysate. The mechanism of purification according to AS requires three successive steps: 1. Incubation of the modified ligand (ProA-CAT) with the target IgG to initiate specific binding and formation of the [ProA-CAT:IgG] soluble complex (Figure 2A). 2. Precipitation of the [ProA-CAT:IgG] complex upon addition of Fe3+ ions which generate insoluble macro-complexes composed of [ProA-CAT:IgG:Fe3+], whereas impurities are left in the supernatant and are discarded by centrifugation (Figure 2B). 3. Elution of the IgG from the [ProA-CAT:IgG:Fe3+] insoluble macro-complex (i.e. pellet) under conditions which essentially do not dissociate the [ProA-CAT:Fe3+] macro* Corresponding author. E-mail:
[email protected], Tel: 972-89302575, Fax: 972-8-9302565. † Affisink Biotechnology Ltd. § Bar-Ilan University. 1 Abbreviations: AC, affinity chromatography; AS, affinity sinking; Cys, cysteine; desferal, deferoxamine mesylate; EDTA, ethylenediamine tetraacetic acid; Gly, glycine; His, histidine; HPLC, high-pressure liquid chromatography; IgG, immunoglobulin G; ProA-CAT, the modified Protein A catechol derivative.
Figure 1. Synthesis of the catechol-NHS derivative 1 used to modify Protein A.
complex, thus leading to a simple and fast recovery of the target IgG (Figure 2C). In a typical experiment, 0.46 mg/mL of ProA-CAT was added to E. coli cell lysate (first dialyzed to remove possible iron chelators) containing 0.5 mg/mL rabbit IgG, 10 mM NaPi, 400 mM NaCl at pH 7. After 3-5 min of incubation at 4 °C, 3 mM Fe3+ ions were added to initiate precipitation of the [ProA-CAT: IgG] soluble complex (Figure 2B). The short incubation time (3-5 min) reflects the advantage of binding in homogeneous solutions rather than heterogeneous solutions in affinity chromatography. To suppress nonspecific interactions between the generated macro-complexes and impurities possessing weak chelating amino acid residues (e.g., His, Cys), 200 mM imidazole was added as well. After a short spin at 14 000 rpm, the supernatant was analyzed and found to contain primarily impurities whereas ProA-CAT and the IgG were absent (Figure 3A, lane 7). The pellet (containing the complexed IgG) was then washed once with 100 µL of fresh buffer containing 20 mM NaPi pH 7, to remove traces of impurities. Rabbit IgG was eluted from the washed pellet by resuspending it for 3-5 min at 4 °C in 0.4 M Gly and 0.3 M His at pH 3. After a short spin (20 s) at 14 000 rpm, the supernatant was removed, neutralized, and found to contain the target IgG. The average recovery yield was 80% with purity greater than 95% by densitometry (Figure 3A, lane 6). Similar results (yield: 71%; purity >95%) were obtained with bovine IgG, thus demonstrating the applicability of the approach to targets possessing lower affinity toward Protein A (data not shown).
10.1021/bc050361+ CCC: $33.50 © 2006 American Chemical Society Published on Web 02/21/2006
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Bioconjugate Chem., Vol. 17, No. 2, 2006 259
Figure 2. Illustration of antibody purification utilizing a modified Protein A (ProA-CAT) and Fe3+ ions. A. Specific binding of ProA-CAT to the target IgG leads to the formation of the [ProA-CAT:target IgG] soluble complex. B. Precipitation of the latter upon addition of Fe3+ ions, which generate insoluble macro-complexes containing the target IgG. Impurities, left in the supernatant are discarded by centrifugation. C. Target IgG is eluted under acidic conditions without dissociating the [ProA-CAT:Fe3+] macro-complex, kept insoluble in the pellet. D. Regeneration of ProACAT in the presence of strong metal chelators which compete for the complexed Fe3+ ions thereby dissociating the macro-complex (i.e., pellet). The complexed Fe3+ ions and free chelators are excluded by dialysis while the free ProA-CAT can be reused.
Catechol was chosen as the preferred chelator since it (a) exhibits high affinity toward diverse transition metals (5, 6), thus enabling utilization of a variety of transition metals, (b) requires three independent catechol moieties to chelate a single Fe3+ ion, thereby increasing the possibility of interconnecting adjacent [ProA-CAT:IgG] soluble complexes, and (c) was expected to retain its chelating ability even at acidic conditions (pH 3) due to the absence of basic atoms (e.g. nitrogen) required for complex formation. A nitrogen atom (if existed) would be protonated at low pH and presumably not be available for chelating Fe3+ ions. ProA-CAT was regenerated without any chromatographic process at neutral pH in the presence of strong metal chelators such as EDTA and catechol. It was assumed that these chelators will compete with the ProA-CAT on the complexed Fe3+ ions, thereby leading to dissolution of the [ProA-CAT:Fe3+] macrocomplex (Figure 2D). Indeed, a short incubation (5 min) at 4 °C in the presence of 50 mM NaPi pH 7, 100 mM EDTA, 50
mM catechol, and 10% ethylene glycol led to quantitative dissolution of the pellet and regeneration of the ProA-CAT in 75-85% yield (data not shown). The recovery yield may increase in the presence of low concentration of a detergent such as SDS. Free and complexed chelators together with all other reagents could then be dialyzed, enabling the reuse of ProA-CAT. Several independent results imply that Fe3+ ions function as the interconnecting entity: (a) Precipitation of the [ProA-CAT: IgG] complex was abolished in the presence of strong chelators (e.g., EDTA, catechol) as well as in the presence of a specific Fe3+ chelator: desferal (7, 8). (b) Other transition metals (e.g., Cu2+, Zn2+, Mg2+, Ni2+) possessing lower affinity toward catechol (5, 6) did not lead to substantial precipitation under identical precipitating conditions. (c) Regeneration of ProACAT at physiological pH was accomplished only in the presence of strong metal chelators. Generally, greater volumes of buffer are required to remove
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Figure 3. A. Purification of rabbit IgG from E. coli cell lysate utilizing ProA-CAT and Fe3+ ions. Lane 1 rabbit IgG; lane 2 native protein A; lane 3 ProA-CAT; lane 4 E. coli cell lysate; lane 5 rabbit IgG, ProA-CAT and E. coli cell lysate; lane 6 recovered rabbit IgG after elution at pH 3; lane 7 content of supernatant after addition of Fe3+ ions to the mixture shown in lane 5. B. The effect of increased background contamination on the purity of the recovered IgG. Lane 1 rabbit IgG; lane 2 ProA-CAT; lanes 3-5 constant concentration of rabbit IgG and ProA-CAT in the presence of increased E. coli cell lysate concentrations; lanes 3P-5P recovered IgG from pellets generated in lanes 3-5, respectively.
impurities that adsorb nonspecifically to polymeric matrixes in chromatographic columns as the contamination increases. Since no polymeric matrixes exist in AS, we postulated that an increase in the background contamination should not affect the purity of the recovered IgG. To demonstrate such a phenomenon, constant concentration of rabbit IgG and ProA-CAT were added into increasing concentrations of E. coli cell lysate (Figure 3B, lanes 3-5) and all generated pellets were washed only once with a minute volume (100 µL) of buffer regardless their contamination background. Though the recovery yield of the IgG decreased with increased contamination background (from ∼80% to ∼70-75%), the purity (>95%) was similar (Figure 3B, lanes 3P-5P), thus supporting our assumption and emphasizing the advantage of a purification approach lacking a polymeric component. The following are points of emphasis: (a) Ligands other than Protein A may be utilized similarly (e.g. Protein G and L, lectins). (b) The simple (no HPLC) and fast purification process would be advantageous to targets that tend to aggregate or denature rapidly. (c) The purification process can be easily up scaled by the addition of higher concentrations of the modified ligand (being a reagent), whereas affinity columns are limited by the concentration of the immobilized ligand. Moreover, efficient capture of low abundance targets is expected due to the possibility of increasing the modified ligand concentration without significantly diluting the sample, thereby increasing the rate of complex formation (i.e. rate ) k[free ligand][target]). (d) Targets are not diluted within the process (unlike column chromatography) and are eluted into small volumes of elution buffer, resulting in concentrated preparations which may be used for other applications such as crystallization. (e) The core macrocomplex [ProA-CAT:IgG:Fe3+] can be further utilized for isolation of targets other than antibodies. For example, positive (or negative) cell selection can be performed by incubating a cell suspension with a mAb targeted at a specific epitope on the target cell, followed by the addition of ProA-CAT and Fe3+ ions. Immunoprecipitation and virus depletion can be performed accordingly.
In conclusion, the metal:chelator platform, together with the previously described desthiobiotin:avidin platform (1), represents two distinct practical alternatives with characteristic advantages to known purification methodologies utilizing immobilized ligands. Supporting Information Available: Synthetic procedure and spectroscopic characterization of compounds 1 and 2. This material is available free of charge via the Internet at http:// pubs.acs.org/BC.
LITERATURE CITED (1) Patchornik, G., and Albeck, A. (2005) Free nonimmobilized ligands as a tool for purification of proteins. Bioconjugate Chem. 16, 13101315. (2) Nilsson, J., Sta˚hl, S., Lundeberg, J., Uhle´n, M., and Nygren, P. (1997) Affinity Fusion Strategies for Detection, Purification and Immobilization of Recombinant Proteins. Protein Expr. Purif. 11, 1-16. (3) Bayer, E. D., Wilchek, M., and Skutelsky, E. (1976) Affinity cytochemistry: the localization of lectin and antibody receptors on erythrocytes via the avidin-biotin complex. FEBS Lett. 68, 240244. (4) Morpurgo, M., Bayer, E. A., and Wilchek, M. (1999) N-hydroxysuccinimide carbonates and carbamates are useful reactive reagents for coupling ligands to lysines on proteins. J. Biochem. Biophys. Methods 38, 17-28. (5) Martell, A. E., and Smith, R. M. (1974) Critical Stability Constants, Volume 3, pp 303-304, Plenum Press, New York. (6) Binding affinities, expressed as logK values at 25 °C and ionic strength 1.0, of catechol with Cu2+, Ni2+, Co2+, Fe2+, Mn2+, Fe3+ are 13.62, 8.77, 8.32, 7.95, 7.47, 43.8, respectively. (7) Martell, A. E., and Smith, R. M. (1974) Critical Stability Constants, Volume 3, pp 200-202, Plenum Press, New York. (8) Binding affinities, expressed as log K values at 25 °C and ionic strength 0.1, of desferal with Cu2+, Ni2+, Co2+, Fe2+, Zn2+, Fe3+ are 14.12, 10.90, 10.31, 7.20, 10.07, 30.60, respectively. BC050361+
