Development of Cross-Linked Antibody Fab Fragment Crystals for

An antibody Fab fragment capable of enantiospecific separation of a chiral drug was crystallized. Simple glutaraldehyde cross-linking of the crystals ...
0 downloads 0 Views 198KB Size
Download PDF
Development of Cross-Linked Antibody Fab Fragment Crystals for Enantioselective Separation of a Drug Enantiomer Antti Vuolanto,*,† Kristiina Kiviharju,† Tarja K. Nevanen,# Matti Leisola,† and Jouni Jokela†,‡

CRYSTAL GROWTH & DESIGN 2003 VOL. 3, NO. 5 777-782

Laboratory of Bioprocess Engineering, Helsinki University of Technology, P.O. Box 6100, FIN-02015 HUT, Finland, and VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Finland Received February 10, 2003;

Revised Manuscript Received May 28, 2003

ABSTRACT: A novel immunoaffinity separation material, cross-linked antibody crystals (CLAC), was developed by crystallization and cross-linking of a recombinant antibody Fab fragment ENA5His capable of enantiospecific separation of a chiral drug, finrozole. Crystallization conditions of the antibody fragment were screened by hanging drop vapor diffusion experiments. The small-scale vapor diffusion crystallization was easily scaled up to 10-mL batch crystallization with a 70% yield. Also, the low affinity mutant of ENA5His having one single amino acid mutation crystallized at the same conditions. Simple glutaraldehyde cross-linking of the crystals yielded CLAC, which were insoluble in water, 5-100% methanol, and 2% DMSO in PBS buffer. Batch experiments showed that the CLAC material was functional as it specifically bound the desired enantiomer from the drug racemate. Introduction In the development of a protein-containing separation material, proteins are normally immobilized on the surface of an inert matrix such as silica, polysaccharides, or synthetic polymers. However, the use of solid support leads to a low overall specific activity of the matrix, because typically the solid support accounts for >95% of the total mass of the immobilized protein matrix. To produce an immobilized protein matrix with maximal protein content, cross-linked protein crystal (CLPC) technology can be used. A crystalline protein is in its most compact active form, and, thus, the volumetric activity of the protein is maximal. Chemical cross-linking of protein crystals maintains the crystalline state of proteins outside the crystallization conditions and also reinforces the crystal structure. CLPCs form an active, insoluble, mechanically robust,1 and microporous2 protein matrix. CLPCs have great potential in separation of various compounds2,3 and in enzymatic production processes.1,4-6 The stability of proteins is often improved by crystallization and cross-linking. Improved thermostability,7,8 stability against organic solvents,5-7 and improved stability against proteolysis7 have been reported. Cross-linked protein crystal technology has been recently reviewed by, for example, Margolin and Navia,9 Govardhan,10 and Ha¨ring and Schreier.11 Antibodies are binding proteins produced by mammalian immune system to protect the animal by neutralizing invading viruses, parasites, and toxins. Antibodies against haptens, small organic molecules such as drugs and hormones, can be created by immunization of mice with hapten-protein conjugate. By the modern * Corresponding author: Tel., +358-9-451 2560; Fax, +358-9-462 373; e-mail, [email protected]. † Helsinki University of Technology. # VTT Biotechnology. ‡ Current address: Department of Applied Chemistry and Microbiology, University of Helsinki, P.O. Box 56, FIN-00014 University of Helsinki, Finland.

gene technology, it is possible to clone only the antigenbinding domain of the antibody as an antibody fragment, Fab, or a single chain antibody, and produce the fragment in large amounts heterologously in bacteria. Antibody fragments, like intact antibodies, are capable of recognizing even minor differences in small molecules such as free amino acids,12 drugs,13 and hormones.14 Nevanen et al.15 demonstrated that immunoaffinity chromatography can be used for enantiospecific fractionation of the enantiomers of a chiral drug molecule finrozole. This molecule has two chiral carbon atoms, and therefore the synthesis product is a mixture of four stereoisomers. The most potent stereoisomer as a drug is difficult to separate from its enantiomer with conventional methods but was successfully separated with carrier-immobilized Fab fragments in a small scale. However, the 60% methanol needed for the elution of the tightly bound drug enantiomer caused about 30% reduction of the enantiomer binding capacity of the carrier-immobilized high affinity Fab fragments per each binding-elution cycle.15 Due to the many excellent properties of the CLPCs, we decided to study the applicability of CLPC technology to immunoaffinity chromatography in which separation is based on highly specific antibody-antigen interaction. In this study, we show the crystallization and crosslinking conditions for the Fab fragment and that the glutaraldehyde cross-linked antibody crystals (CLAC) specifically separates one drug enantiomer from its racemic mixture. The results also show that the stability of the Fab fragment is improved by crystallization and cross-linking. Experimental Section Drug Racemate. Finrozole, 4-[3-(4-fluorophenyl)-2-hydroxy1-(1,2,4-triazol-1-yl)-propyl]benzonitrile (C18H17FN4O, 323 g/mol), was provided by Hormos Medical corp. (Finland). The drug crystallizes as R,S- and S,R-racemate and the crystal structure of the drug has been published previously.28 The two enantiomers studied in this paper are designated here as a and d.

10.1021/cg034021r CCC: $25.00 © 2003 American Chemical Society Published on Web 07/18/2003

778

Crystal Growth & Design, Vol. 3, No. 5, 2003

There is not a certainty about the absolute configuration of the enantiomers. Antibody Fragments. The recombinant antibody Fab fragments ENA5His (molecular mass of 50.1 kDa based on the amino acid sequence) and its low affinity single mutant ENA5His(Tyr97Val) were provided by VTT Biotechnology (Finland). They were cloned and produced by Escherichia coli bacteria as described elsewhere.15,16 The culture supernatant containing the Fab fragment was centrifuged to remove cell debris. The liberated DNA was removed by treating the solution for 1-2 h with 2 mg/mL DNAse at 22 °C. The solution was clarified by filtration through a Pellicon cross-flow filtration unit (Millipore, USA) with a molecular weight cutoff (MWCO) of 1000 kDa to remove any particles left and to facilitate the concentration step. The clarified solution containing the Fab fragment was concentrated with a Mini-Tan filtration unit (Millipore, USA) with a MWCO of 5 kDa. For metal affinity chromatography, the buffer was changed to PBS buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4) by ultrafiltration with a MWCO of 3 kDa (Amicon, Ireland). A column (10 × 1.6 cm) packed with Ni-NTA Superflow (Qiagen, USA) metal affinity resin was washed with 10 column volumes of PBS buffer by a Bio-Pilot liquid handling system (Pharmacia, Sweden) equipped with a UV detector (280 nm). The protein sample (20 mL) was introduced to the column followed by washing with PBS buffer containing 0.05 M imidazole until the baseline was achieved. The Fab fragment was eluted with 0.5 M imidazole in PBS buffer. After a buffer change to 0.1 M sodium acetate buffer at pH 5.0, the purified Fab fragment was concentrated to 8 mg/mL by ultrafiltration (MWCO 3 kDa, Amicon, Ireland). The purity of the Fab fragment was confirmed by SDS-PAGE. Crystallization. Vapor diffusion crystallization experiments were set up in hanging drops in 24-well tissue culture plates (Hampton Research, USA). An equal volume (5 µL) of protein solution and crystallization mother liquor were mixed on a circular siliconized glass slide. The concentration of ENA5His and ENA5His(Tyr97Val) was 8 and 4 mg/mL, respectively, in 0.1 M sodium acetate buffer at pH 5.0. The slide was placed on a chamber and sealed airtight with grease. The chamber contained 1 mL of crystallization mother liquor. The experiments were conducted at two different temperatures (20 and 5 °C), as especially antibody crystallization has been shown to be temperature sensitive.17 The screening of crystallization conditions for ENA5His consisted of Crystal Screen and PEG/ionic screen (Hampton Research, USA), ammonium sulfate, mixed NaH2PO4 and K2HPO4, 2-propanol, MPD (2,4methyl-pentanediol), and PEGs (poly(ethylene glycol)) molecular weights 600, 3350, 4000, 6000, 8000, and 12000. Crystallization of ENA5His(Tyr97Val) was studied in the conditions producing ENA5His crystals. Batch crystallization was first set up in 0.2 mL scale in 0.5 mL Eppendorf tubes and then at 10 mL scale in plastic test tubes of 15 mL at conditions derived from the hanging drop experiments. The ENA5His solution (8 mg/mL in 0.1 M sodium acetate buffer, pH 5.0) and precipitant stock solutions (50% PEG 3350 in H2O and 4 M NaH2PO4 or 1.5 M K2HPO4 solutions) were mixed in a tube, and thereafter the tube was incubated at 20 °C with or without gentle rocker mixing (Barnstead/Thermolyne, UK). The crystallization was monitored by removing 2 µL samples and viewing them under microscope. The crystal yield on antibody fragment was determined by measuring the absorbance of the liquid phase at 280 nm. Preparation of CLAC. The ENA5His crystals produced in the batch crystallization were resuspended in protein-free mother liquor. 25% glutaraldehyde solution in water was added to a final concentration of 0.5% (w/v) to cross-link the crystals. The crystals were allowed to cross-link for 3 h at 20 °C with gentle roller mixing. The cross-linked ENA5His crystals (CLAC) were washed three times with 0.1 M sodium acetate buffer at pH 5.0 and stored in the same buffer at 5 °C. The solubility of CLAC was studied in water, PBS buffer, 5-80% methanol in water, 100% methanol, and 2% DMSO in PBS buffer. About 0.5 mg of CLAC was incubated in 50 µL of

Vuolanto et al.

Figure 1. Diagram from the hanging drop vapor diffusion experiments showing the crystallization conditions of ENA5His at 20 °C. each solution for 2 h at 20 °C. After the incubation, the soluble protein concentration in the liquid phase was determined by measuring the absorbance at 280 nm. Drug Racemate Binding Test. A total of 18.4 mg of CLAC (dry weight measured by vacuum-drying after the binding test) was suspended into 1 mL of PBS buffer containing 5% DMSO in an Eppendorf tube. To the crystal slurry was introduced 30 µL of the drug racemate dissolved in DMSO (4 mg/mL). The mixture was incubated with gentle rocking mixing at 20 °C. The binding of the drug enantiomers to the CLAC matrix was monitored by removing 50 µL samples intermittently from the solution phase of the reaction mixture and analyzing the samples by HPLC. When the binding was complete, the crystals were separated from the reaction mixture and washed two times with 1 mL aliquot of 2% DMSO in PBS buffer. The bound drug was released by adding 1 mL of 60% methanol in PBS buffer on the crystal slurry and incubating the mixture with rocking mixing. The release was monitored by removing 50 µL samples intermittently and analyzing them by HPLC. HPLC Analysis of Drug Enantiomers. The finrozole enantiomers a and d were analyzed by HPLC equipped with UV detector. The 4.6 × 150 mm Ultron ES-OVM column (Hewlett-Packard, USA) was eluted at 0.25 mL/min with 0.02 M ammonium phosphate (pH 5.0) containing 15% methanol at 30 °C. The finrozole enantiomers were detected at 230 nm.

Results Crystallization of ENA5His. Antibody Fab fragment ENA5His crystals were initially obtained from 20% PEG 3350 with 0.2 M KH2PO4 at pH 4.7 and 20 °C in hanging drop. Figure 1 shows the conditions where crystals were obtained in more precise screening of the conditions. Good crystallization behavior was obtained from 20 to 28% PEG 3350 with 0.1-0.6 M KH2PO4 or NaH2PO4 at 5 and 20 °C. Figure 2 shows that the resulting ENA5His crystals were rods, rod aggregates, rectangular, or wedge-like. Rod was the only crystal form seen when high PEG 3350 concentration (24-30%) was used with high phosphate concentration (0.4 or 0.6 M). At lower concentrations of PEG 3350 and phosphate clear relationship between the crystal shape and the crystallization conditions was not seen. Other precipitants, such as ammonium sulfate, 2-propanol, MPD, and PEGs of molecular weights 600, 4000, 6000, 8000, and 12000 did not crystallize ENA5His at the concentrations tested. Batch crystallization was studied in the conditions found in the vapor diffusion experiments. When the ENA5His solution was mixed with the precipitant stock solutions to a final concentration of 3.5 mg/mL protein,

Cross-Linked Antibody Fab Fragment Crystals

Crystal Growth & Design, Vol. 3, No. 5, 2003 779

Figure 2. Crystals of ENA5His obtained by the hanging drop vapor diffusion method. Crystallization conditions: (a) 20% PEG 3350, 0.2 M KH2PO4, pH 4.7, 5 °C; (b) 20% PEG 3350, 0.4 M KH2PO4, pH 4.7, 5 °C; (c) 24% PEG 3350, 0.4 M NaH2PO4, pH 4.5, 20 °C; (d) 26% PEG 3350, 0.2 M KH2PO4, pH 4.7, 20 °C.

Figure 3. Crystals of ENA5His obtained in batch crystallization from 20% PEG 3350, 0.2 M KH2PO4, pH 4.7, 20 °C, and initial protein concentration of 3.5 mg/mL. The crystallization was accomplished (a) with roller mixing or (b) without mixing.

20% PEG 3350, and 0.2 M KH2PO4 at pH 4.7, amorphous precipitate was immediately formed. According to the absorbance at 280 nm, about 65% of the soluble protein precipitated. The batch was incubated overnight at 20 °C with roller mixing, but crystals were not observed. Next the batch was seeded with a few rodshaped crystals from the vapor diffusion experiments, and incubation was continued at 20 °C with roller mixing. After 24 h, the amorphous precipitate had disappeared and the batch contained very small and thin plate crystals as seen in Figure 3a. The crystal yield was about 70% with a final soluble protein concentration of 1.5 mg/mL. To produce bigger crystals, a batch was accomplished and seeded as above but incubated without mixing. Figure 3b shows that the resulting crystals were aggregates of rod-shaped crystals. The crystal yield

was about 70%. KH2PO4 could be replaced with NaH2PO4 without any effect on the crystal form or crystal yield. Large-scale crystallization (50× scale-up in volume and in total protein) of ENA5His was accomplished in 10 mL batch from 20% PEG 3350, 0.2 M NaH2PO4, and with an initial protein concentration of 3.5 mg/mL. The scale-up did not affect the crystal yield (70%) or the geometry of the crystals. Crystallization of Affinity Mutant ENA5His(Tyr97Val). Nevanen et al. have created several mutants of the finrozole binding antibodies one being a low affinity mutant of ENA5His.16 We decided to study how the crystallization behavior is affected by a single amino acid mutation in the binding pocket area fine-tuning the binding properties of the antibody fragment. This would provide additional information about the applicability

780

Crystal Growth & Design, Vol. 3, No. 5, 2003

Vuolanto et al.

Figure 4. Specific recognition of Finrozole drug d-enantiomer from a racemic mixture by CLAC (ENA5His). Panel a shows the binding of drug d-enantiomer to CLAC, and panel b shows the elution of drug d-enantiomer from CLAC by 60% methanol.

of crystallization methods with antibody fragments. Affinity mutant ENA5His(Tyr97Val) crystallization trials were made in the conditions successful for ENA5His. By the vapor diffusion method ENA5His(Tyr97Val) crystals were grown in 22-28% PEG 3350 with 0.10.6 M KH2PO4 at pH 4.7 and 20 °C. The good crystallization conditions were very close to those shown for ENA5His in Figure 1. Batch crystallization of ENA5His(Tyr97Val) was accomplished from 20% PEG 3350 and 0.2 M KH2PO4 with an initial protein concentration of 3 mg/mL, and seeded by a few crystals from the vapor diffusion experiment. The crystallization yield was 60%. The form of ENA5His(Tyr97Val) crystals in the vapor diffusion experiments and batch crystallization was identical to the crystal form of the corresponding ENA5His crystals. These results showed that the single mutation (Tyr97Val) located in the binding pocket of the ENA5His did not affect the ability of mutated ENA5His to crystallize. Cross-Linked Antibody Crystals (CLAC). ENA5His crystals were cross-linked with glutaraldehyde that, as a bifunctional compound, links the free amino groups of lysine residues to each other. After the cross-linking reaction, initially almost colorless crystals were brown probably due to colored byproducts formed in the crosslinking reaction. The solubility of CLAC was studied at 20 °C in water, PBS buffer, 5-80% methanol in water, 100% methanol, and 2% DMSO in PBS buffer. We could not detect any soluble protein by absorbance at 280 nm from these solutions after the incubations. The soluble protein detection limit was about 0.05 mg/mL that would be only 0.5% of the protein present in the form of CLAC. After the incubation of CLAC in the different aqueous solutions and solvents, the crystal structure was undamaged as judged by light microscopy indicating that the crystals did not dissolve in the conditions tested. Chiral Separation by CLAC. The functionality of CLAC was studied by incubating 18.4 mg of CLAC and 120 µg of the drug racemate in 1 mL of PBS buffer at 22 °C. Figure 4a shows the binding of the drug racemate to the CLAC matrix. During the binding, only the amount of the soluble d-enantiomer decreased in the liquid phase, whereas the amount of the soluble aenantiomer remained constant. During washing with 2% DMSO in PBS buffer, a small amount of a-enantiomer was detected in the wash solution. Figure 4b shows the elution profiles of the drug enantiomers from CLAC material by adding 1.0 mL of 60% methanol in PBS buffer on crystal slurry. The bound d-enantiomer eluted from the CLAC matrix completely. The drug

a-enantiomer was not detected in the elution. The d-enantiomer binding capacity of CLAC matrix was about 2 µg of drug d-enantiomer per 1 mg of ENA5His. The theoretical binding capacity is 6 µg of drug denantiomer per 1 mg of ENA5His that is based on 1:1 molar binding ratio. The binding experiment was repeated three times with the same crystals without any effect on the d-enantiomer binding or elution behavior of CLAC. Discussion Harris et al.17 were the first to report crystallization of intact antibodies by PEG 3350 in low ionic strength solutions. Since then, these conditions have been successfully used in numerous crystallization trials of intact monoclonal antibodies and their fragments. Also in this study, the antibody Fab fragment ENA5His crystallized readily from PEG 3350 with sodium or potassium phosphate at a pH of 4.5-5.0. None of the other precipitants, including other PEGs of higher molecular weights, produced crystals. Crystallization conditions found in the hanging drop vapor diffusion experiments were easily adapted to batch crystallization. Batch crystallization is the only crystallization method for the large-scale crystal production that is needed when antibody fragment crystals are applied as immunoaffinity material in practical applications. The key to success was that seed crystals were available from microdiffusion experiments. The results of Lee et al.31 together with our results show that batch crystallization procedure can easily be developed and scaled up based on crystallization conditions found by routinely used microdiffusion methods. Together with the development of high throughput protein crystallization methods,29,30 these results shows a straightforward procedure in the development of new crystalline protein materials. The single amino acid mutant ENA5His(Tyr97Val) crystallized in the same conditions as the wild-type ENA5His. This mutation in the hapten binding site, which decreases the affinity of the antibody fragment 10-fold, is located inside the antibody fragment molecule.16 Apparently, the amino acid 97 is not involved in the interactions between ENA5His molecules in the crystalline state and, therefore, does not affect the crystallization behavior. Valjakka et al.18 obtained similar results with antitestosterone antibody Fab fragments. They crystallized several affinity mutants of the antibody Fab fragment in one crystallization condition. In general, even a single amino acid mutation can change the crystallization behavior of a protein totally.

Cross-Linked Antibody Fab Fragment Crystals

On the basis of our results and the results of Valjakka et al., it seems that the crystallization of antibody Fab fragments is not very sensitive to mutations fine-tuning the hapten binding site. Antibodies (and their fragments) have very conserved structures and, thus, the mutations needed for fine-tuning the binding activity are often located in a certain small area, which apparently is not located in position affecting to the crystallization behavior. According to this assumption, it is possible to fine-tune the binding/release affinity of the antibody fragments by site directed single amino acid mutagenesis and use the same or very similar crystallization conditions as for the wild-type antibody. Glutaraldehyde is the most popular cross-linking agent for protein crystals.9 It reacts irreversibly with the free amino group of lysine residues in the protein. Sometimes, if there are not enough lysine residues present in the protein surface, extra lysine has been used together with glutaraldehyde to cross-link protein crystals.3,19 Despite of the widespread use of glutaraldehyde cross-linking, the reaction mechanism is not fully understood.20 Strongly CLPCs maintain their crystalline state outside the crystallization conditions. CLPCs are as stable or often more stable than soluble proteins. The reason for the stabilization can be a result of crystallinity, chemical modifications formed in crosslinking, or a combination of both.9 ENA5His contains 24 lysine residues, and, therefore, glutaraldehyde alone was a reasonable choice for crosslinking. The cross-linked ENA5His crystals (CLAC) were proven to be totally insoluble in methanol and in different water solutions. The total insolubility of crosslinked crystals is a prerequisite for the use of crystals in practical applications. The d-enantiomer binding experiment was repeated three times without any effect on the d-enantiomer binding activity of CLAC. Nevanen et al.15 reported that the carrier-immobilized ENA5His lost ca. 30% of its activity per elution cycle in immunoaffinity chromatography. Our results show that the crystallization and cross-linking substantially improved the stability of the Fab fragment ENA5His in 60% methanol that was used in the elution of the bound d-enantiomer. In many cases, the activity of cross-linked protein crystals has been reduced compared to native soluble proteins because of mass-transfer limitations. The effect of mass-transfer limitation depends on the size of the pores in the cross-linked protein crystal, the size of the substrate molecule, the kinetic parameters of the reaction, and the size of the crystals. The problem of substrate diffusion limitations in the crystalline proteins has been discussed in several publications and is recently reviewed by Margolin and Navia.9 Crystallization and subsequent cross-linking of protein crystals may also limit the flexibility of the protein molecules due to specific intermolecular interactions in the crystalline state and to covalent bonds formed in crosslinking.21 In some cases, reduced protein flexibility has led to reduced enzyme activity as shown for carboxypeptidase A,22 carboxypeptidase B,23 subtilisin,24 and phosphorylase B.25 In recent reports, Ayala et al.32 and Costes et al.33 report that the specific activity of crosslinked crystals of chloroperoxidase and hydroxynitrile lyase, respectively, decreased with increasing the glu-

Crystal Growth & Design, Vol. 3, No. 5, 2003 781

taraldehyde concentration used in cross-linking. However, maximal stability and total insolubility were only achieved at conditions using high concentration of glutaraldehyde in both cases. The d-enantiomer binding capacity of CLAC was about 33% of the theoretical value that is based on 1:1 molar binding ratio. The reduced binding activity may be a result of reduced flexibility of the antibody molecule caused by crystallization and cross-linking. In many cases, the recognition of the hapten induces a conformational change in the Fab fragment that may be prevented by cross-linking. There are two lysine residues located in the binding pocket area of the Fab fragment.16 In this work, a functional CLAC material was prepared in the absence of the d-enantiomer in the binding pocket and the participation of the binding pocket lysine residues in the cross-linking by glutaraldehyde may weaken the antibody-antigen interaction and reduce the binding activity. However, the d-enantiomer binding remained highly specific. Lower glutaraldehyde concentrations or shorter cross-linking times on the d-enantiomer binding activity of ENA5His were not studied, because the absolute stability of CLAC against high concentrations of polar organic solvents as methanol was more important than the maximal binding activity. It is also possible that the presence or absence of the hapten during crystallization and cross-linking affect the conformation of ENA5His in CLAC and, therefore, the binding activity of CLAC. Groschulski et al.16,17 showed that the properties of cross-linked lipase crystal preparations were affected by the conformation of lipase during crystallization and cross-linking. If lipase was crystallized and cross-linked “lid open”, the activity was three times higher than if crystallized “lid closed”. The lid movement is related to the binding of the substrate to the active site. So far, we have not crystallized ENA5His as a protein-hapten complex to discover the possible differences in the binding properties of such crystals. In this study, we have presented a novel concept for enantiospecific purification of a drug enantiomer from a synthetically prepared racemic mixture by crosslinked antibody crystals. First, we showed that the ENA5His antibody fragment crystallized in the conditions suitable for numerous intact monoclonal antibodies and their fragments, and that these conditions could be used in batch crystallization for the production of tens of milligrams of crystals. This crystallization was not sensitive to a single mutation in the binding pocket. Second, the crystals could be immobilized into a totally insoluble CLAC material by the most common crosslinking reagent, glutaraldehyde. Finally, the prepared CLAC material was functional and it separated pure d-enantiomer from the synthetically prepared drug racemate. This separation ability was preserved unchanged during the repeated separation cycles. According to our experience with xylose isomerase3 and xylanase19 as cross-linked protein crystals as LC stationary phase, the CLAC material is probably suitable for LC stationary phase as the crystals are big enough and the crystal shape is clearly three-dimensional enabling eluent flow in the LC column. The d-enantiomer binding capacity of CLAC is reduced compared to

782

Crystal Growth & Design, Vol. 3, No. 5, 2003

carrier-immobilized ENA5His, but the increased stability of CLAC probably makes CLAC more suitable for purification of drug racemate than carrier immobilized ENA5His is. However, much more work is needed to characterize and optimize the separation properties of CLAC. The long term stability, mass-transfer properties, the properties of CLAC as an LC-column stationary phase in immunoaffinity chromatography and the use of CLAC crystallized with drug enantiomer must be studied in detail. These studies currently underway in our laboratory will show if CLAC material could be used as a specific chiral resolution matrix for the production of pure drug enantiomers in practical applications and as a matrix for quantitative analysis of the drug enantiomer from biological samples. Acknowledgment. Kalevi Visuri (Macrocrystal Oy, Finland) and PhD Jarkko Valjakka (University of Joensuu, Finland) are thanked for helping in the crystallization work and for many fruitful discussions. Marjaana Rytela¨ is thanked for the analysis of drug enantiomers. The funding of The National Technology Agency (Finland) and the Academy of Finland/Graduate School in Chemical Engineering is gratefully acknowledged. References (1) Vaghjiani, J. D.; Lee, T. S.; Lye, G. J.; Turner, M. K. Biocatal. Biotrans. 2000, 18, 151-175. (2) Vilenchik, L. Z.; Griffith, J. P.; St. Clair, N.; Navia, M. A.; Margolin, A. L. J. Am. Chem. Soc. 1998, 120, 4290-4294. (3) Pastinen, O.; Jokela, J.; Eerika¨inen, T.; Schwabe, T.; Leisola, M. Enz. Microb. Technol. 2000, 26, 550-558. (4) Persichetti, R. A.; St. Clair, N. L.; Griffith, J. P.; Navia, M. A.; Margolin, A. L. J. Am. Chem. Soc. 1995, 117, 27322737. (5) Lalonde, J. J.; Govardhan, C.; Khalaf, N.; Martinez, A. G.; Visuri, K.; Margolin, A. L. J. Am. Chem. Soc. 1995, 117, 6845-6852. (6) Khalaf, N.; Govardhan, C. P.; Lalonde, J. J.; Persichetti, R. A.; Wang, Y.-F.; Margolin, A. L. J. Am. Chem. Soc. 1996, 118, 5494-5495. (7) St. Clair, N. L.; Navia, M. A. J. Am. Chem. Soc. 1992, 114, 7314-7316. (8) Visuri, K.; Pastinen, K.; Wu, X.; Ma¨kinen, K.; Leisola, M. Biotechnol. Bioeng. 1999, 64, 377-380. (9) Margolin, A. L.; Navia, M. A. Angew. Chem., Int. Ed. 2001, 40, 2204-2222.

Vuolanto et al. (10) Govardhan, C. P. Curr. Opin. Biotechnol. 1999, 10, 331335. (11) Ha¨ring, D.; Schreier, P. Curr. Opin. Chem. Biol. 1999, 3, 35-38. (12) Hofstetter, O.; Hofstetter, H.; Schurig, V.; Wilchek, M.; Green, B. S. J. Am. Chem. Soc. 1998, 120, 3251-3252. (13) Ezan, E.; Delestre, L.; Legendre, S.; Riviere, R.; Doignon, J.-L.; Grognet, J.-M. J. Pharm. Biomed. Anal. 2001, 25, 123-130. (14) Hemminki, A.; Niemi, S.; Hautoniemi, L.; So¨derlund, H.; Takkinen, K. Immunotechnology 1998, 4, 59-69. (15) Nevanen, T.; So¨derholm, L.; Kukkonen, K.; Suortti, T.; Teerinen, T.; So¨derlund, H.; Teeri, T. T. J. Chromatogr. A 2001, 925, 89-97. (16) Nevanen, T. K.; Hellman, M.-L.; Munck, N.; Wohlfahrt, G.; Koivula, A.; So¨derlund, H. Submitted to Protein Eng. 2003. (17) Harris, L. J.; Skaletsky, E.; McPherson, A. Proteins: Struct., Funct., Genet. 1995, 23, 285-289. (18) Valjakka, J.; Hemminki, A.; Teerinen, T.; Takkinen, K.; Rouvinen, J. Acta Crystallogr. 2000, D56, 218-221. (19) Finell, J.; Jokela, J.; Leisola, M.; Riekkola, M.-L. Biocatal. Biotrans. 2002, 20, 281-290. (20) Wong, S. S. Chemistry of Protein Conjugation and CrossLinking; CRC Press: USA, 1993. (21) Mozzarelli, A.; Rossi, G. L. Annu. Rev. Biophys. Biomol. Struct. 1996, 25, 343-365. (22) Spilburg, C. A.; Bethune, J. L.; Vallee, B. L. Biochemistry 1977, 16, 1142-1150. (23) Alter, G. M.; Leussing, D. L.; Neurath, H.; Vallee, B. L. Biochemistry 1977, 16, 3663-3668. (24) Tuchsen, E.; Ottesen, M. Carlsberg Res. Commun. 1977, 42, 407-420. (25) Kasvinsky, P. J.; Madsen, N. B. J. Biol. Chem. 1976, 251, 6852-6859. (26) Grochulski, P.; Li, Y.; Schrag, J. D.; Bouthillier, F.; Smith, P.; Harrison, D.; Rubin, B.; Cygler, M. J. Biol. Chem. 1993, 268, 12843-12847. (27) Grochulski, P.; Li, Y.; Schrag, J. D.; Cygler, M. Protein Sci. 1994, 3, 82-91. (28) So¨dervall, M.; Mutikainen, I. Z. Kristallogr. NCS 2002, 217, 38-40. (29) Rupp, B.; Segelke, B. W.; Krupka, H. I.; Lekin, T.; Scha¨fer, J.; Zemla, A.; Toppani, D.; Snell, G.; Earnest, T. Acta Crystallogr. 2002, D58, 1514-1518. (30) Karain, W. I.; Bourenkov, G. P.; Blume, H.; Bartunik, H. D. Acta Crystallogr. 2002, D58, 1519-1522. (31) Lee, T. S.; Vaghjiani, J. D.; Lye, G. J.; Turner, M. K. Enzyme Microb. Technol. 2000, 26, 582-592. (32) Ayala, M.; Horjales, E.; Pickard, M. A.; Vazquez-Duhalt, R. Biochem. Biophys. Res. Com. 2002, 295, 828-831. (33) Costes, D.; Whtje, E.; Adlercreutz, P. J. Mol. Catal. B: Enzymol. 2001, 11, 607-612.

CG034021R