Adsorption of Glucagon and Insulin on an Immobilized Metal Ion Affinity Chromatography Silica Matrix

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1 883 Adsorption of Glucagon and Insulin on an Immobilized Metal Ion Affinity Chromatography Silica Matrix Cristiane S. Farinas 1, Sônia M.A. Bueno 2 and Everson A. Miranda 1 * (1) LEBp: Laboratório de Engenharia de Bioprocessos, Departamento de Processos Biotecnológicos, FEQ, UNICAMP, CP 6066, CEP Campinas, SP, Brazil. (2) LIMBio: Laboratório de Interação Molecular e Bioengenharia, Departamento de Processos Biotecnológicos, FEQ, UNICAMP, CP 6066, CEP Campinas, SP, Brazil. (Received 25 May 2003; accepted 18 July 2003) ABSTRACT: Glucagon is a hormone that increases blood glucose concentrations and is used as a pharmaceutical product mainly in the treatment of hypoglycaemia associated with diabetes. Given both its importance and high current cost, improved purification processes are in demand. By using immobilized metal ion affinity chromatography (IMAC), we conducted tests aimed at the purification of glucagon from complex mixtures. Adsorption studies of glucagon, insulin and a mixture of the two on adsorbents of silica IDA Me 2+ (Cu 2+, Ni 2+ and Zn 2+ ) were carried out. Fixed-bed chromatographic experiments were performed and the adsorption affinity verified for all three metals tested. The most promising condition for glucagon and insulin separation was achieved by using Ni 2+ as the metal ligand and desorption with a ph step gradient. Two industrial insulin-processing fractions (one glucagon-rich and the other insulinrich) were evaluated qualitatively under these conditions, resulting in an increase in glucagon purity for both fractions with a purification factor of five for the latter. INTRODUCTION Glucagon is a hormone found mainly in pancreatic extracts whose main role is to raise the level of glucose in blood, counteracting the action of insulin (Kimball and Murlin 1923). It is used as a pharmaceutical product in the treatment of hypoglycaemia, especially in patients with diabetes. The primary structure of the human glucagon molecule is identical to the bovine and swine molecules, allowing a direct application of purified glucagon from these sources to the treatment of humans. Glucagon consists of a single chain of 29 amino-acid residues (molecular mass = 3.5 kda) including an N-terminal histidine. It has an isoelectric point of 7.0 and can self-associate at high concentrations, forming aggregates and gels at mild temperatures in acid and basic solutions (Joshi et al. 2000). In the crystalline state, glucagon has a helical conformation (Sasaki et al. 1975). Glucagon is produced commercially as a by-product of swine and bovine insulin (Andrade et al. 1997), but production of glucagon by DNA recombinant technology has been reported (Okamoto et al. 1995; Olsen and Diderichsen 2000). The complexity of pancreatic extracts, where a variety of proteins can be found, makes purification of glucagon a difficult task. Processes used to purify glucagon for therapeutic applications are in most cases based on a sequence of chromatographic steps (Andrade et al. 1997; Maskalick and Anderson 1986; Carrea et al. 1985). Andrade et al. described a process employing three chromatographic steps, viz. ion exchange, gel filtration and *Author to whom all correspondence should be addressed. everson@feq.unicamp.br.

2 884 C.S. Farinas et al./adsorption Science & Technology Vol. 21 No another ion-exchange chromatography step, to purify glucagon from a pancreatic source at the pilot-scale level. A different process, comprising hydrophobic adsorption chromatography, anionexchange chromatography and crystallization, has been described as a way to recover glucagon from a pancreatic source, yielding a product considered to be of high purity (Maskalick and Anderson 1986). Sub-unit exchange chromatography, based on the self-association of glucagon molecules, was also used to purify this polypeptide (Carrea et al. 1985). Although the purity of the glucagon obtained from the latter process was ca. 90%, the product also contained trace amounts of insulin. Recombinant glucagon has been purified through isoelectric precipitation (Okamoto et al. 1995) with a level of purity over 99.5% being achieved. However, this process seemed to depend on the primary structure of the fusion protein and was apparently limited to the purification of the specifically designed recombinant molecule. The presence of a histidine as the N-terminal residue in the amino-acid sequence of the glucagon molecule is an indication that immobilized metal ion affinity chromatography (IMAC) may be a suitable adsorption technique to be investigated as a basis for a glucagon purification process. However, no report of such a study has appeared in the literature. IMAC is an adsorption method based on the affinity interaction between certain amino-acid residues on the surface of proteins (tryptophan, cysteine and mainly histidine) and metal ions immobilized on a solid support by a chelating agent. Porath et al. (1975) first proposed this technique and since then it has been widely used for the purification of natural and recombinant proteins and peptides. The main advantages of IMAC include high protein-binding capacities and ligand stabilities, matrix versatility and the low cost of ligands (Arnold 1991). It thus combines selectivity and low cost, making IMAC suitable for large-scale applications. The purpose of the present work was to study the affinity adsorption of glucagon and insulin on an IMAC adsorbent using Cu 2+, Ni 2+ and Zn 2+ ions immobilized on silica IDA (iminodiacetic acid). Experimental data were gathered for use as a basis for the development of a process for glucagon purification from native or recombinant sources. Since insulin is the main impurity present in extracts from natural sources of glucagon (animal pancreas), the interaction of glucagon in the presence of insulin with these IMAC adsorbents was evaluated. Insulin is a small globular protein of molecular mass 5.8 kda comprised of two polypeptide chains joined by two disulphide crosslinks (Blundell et al. 1972). Moreover, the insulin molecule has two histidine residues and to a certain extent it can therefore be considered to be a model of impurity for glucagon purification by IMAC. MATERIALS AND METHODS Materials High-purity hormones (swine glucagon, purity of 94%; swine insulin, purity of 97%) and two industrial insulin-processing fractions (one insulin-rich, ca. 70% insulin, and another glucagonrich, ca. 70% glucagon) were generously donated by Biobrás (Brazil). The glucagon-rich fraction was collected after a precipitation step with zinc acetate during the commercial production of insulin. The insulin-rich fraction was collected in a previous step of the same process after an isoelectric precipitation step. The IMAC adsorbent, Prosep -Chelating I, referred to here as PCI, was obtained from BioProcessing (England). This affinity adsorbent consists of a metal-binding IDA immobilized on a porous silica matrix. All buffers were prepared with deionized water (Milli-Q System, Millipore, USA) and degassed under vacuum. All other chemicals used were of at least

3 Adsorption of Glucagon and Insulin 885 analytical grade. Unless otherwise stated, the equilibration buffer used in all experiments was 20 mm sodium phosphate buffer (ph 7.0) containing 1.0 M NaCl. Methods Determination of protein concentration The total protein concentration was determined by measuring the absorption of solutions at 280 nm using a DU 650 spectrophotometer (Beckman, USA). Samples collected from IMAC chromatographies of mixtures of glucagon and insulin and of industrial insulin-processing fractions were analyzed for glucagon and insulin concentrations in a HPLC system (Shimadzu, Japan) using a reverse-phase column C18 Asahipack ODS-H series STR (Shimadzu, Japan). A discontinuous gradient was developed over 30 min using 0.1 M sodium phosphate (ph 3.1) as buffer A and 50% (v/v) acetonitrile in buffer A as buffer B. The UV detector was set at 220 nm. Preparation of protein solution Glucagon solutions were prepared by dissolving glucagon in 20 mm sodium phosphate buffer containing 1.0 M NaCl at ph 10.0 and diluting this solution to the desired concentration with this same buffer at ph 7.0 (equilibration buffer). The ph value was then adjusted to 7.0 with phosphoric acid. Due to solubility limitations, the maximum concentration of glucagon in all experiments was 0.56 mg/ml. Insulin solutions were prepared following the same glucagon procedure and the maximum concentration of insulin in all experiments was 0.90 mg/ml. These two hormones were mixed by joining the two previously prepared solutions. Since insulin solubility in the buffer was higher than glucagon solubility, mixtures of these two hormones were not equimolar due to glucagon precipitation after mixing. This precipitate was removed before use by centrifugation. Chromatographic procedures The chromatographic system consisted of a Miniplus 3 peristaltic pump (Gilson, France), an Econo UV Monitor detector, a fraction collector, a chart recorder (all three by Bio Rad, USA) and a C5/5 chromatographic column (Pharmacia, Sweden) (5 mm i.d. 5 cm length). This column was packed with 1 ml of the adsorbent PCI, prepared by following the protocol suggested by the manufacturer of the adsorbent. Briefly, the adsorbent-packed bed was washed with at least five volumes of water and percolated with 20 mm copper, zinc or nickel sulphate solutions. Unbound metal ion was washed out of the column with five volumes of the elution buffer. After that, the column was equilibrated with the equilibration buffer. Protein solution in this buffer (5 or 10 ml) was fed into the column, followed by a washing step with this same equilibration buffer. Elution was undertaken with a ph step gradient from ph 6.0 to 2.1, using 20 mm sodium phosphate solution containing 1.0 M NaCl. The column was regenerated with 50 mm EDTA at ph 8.0. The flow rate during all procedures was 0.35 ml/min and the 1.0 ml fractions collected were analyzed for total protein, glucagon and insulin concentration. Adsorption isotherm experiments Glucagon and insulin adsorption experiments for isotherm determination at 25 C were undertaken using 3-ml syringes as the stirred tank. These syringes had a filter at their tip allowing separation

4 886 C.S. Farinas et al./adsorption Science & Technology Vol. 21 No of the solution from the adsorbent when liquid was ejected from them. Volumes of glucagon- or insulin-containing solutions (1 ml) at different concentrations (from 0.10 to 0.55 mg/ml) were fed into a series of syringes containing 5 mg of PCI Ni 2+ previously equilibrated with equilibration buffer. In order to minimize mass-transfer resistance, syringes containing adsorbent and protein solution were agitated end-over-end for 30 min at 25 C. After this period, considered sufficient for equilibrium to be achieved (data not shown), the protein concentration in the liquid phase was measured spectrophotometrically at 280 nm. The amount of protein adsorbed was equal to the difference between the initial protein concentration and the concentration of protein remaining in solution at equilibrium. Adsorption isotherms were then constructed by plotting glucagon or insulin adsorbed by the IMAC matrix (adsorption capacity as mass of protein per volume of adsorbent) as a function of the concentration of unbound protein. RESULTS AND DISCUSSION Chromatography with high-purity hormones Chromatographic column experiments were carried out in order to evaluate how glucagon and insulin (separately and in a mixture) were adsorbed on PCI Me 2+ under dynamic conditions. The three transition metals chosen, i.e. Cu 2+, Ni 2+ and Zn 2+, are among the most widely used in IMAC. Recovery was calculated on the basis of the protein concentration determined spectrophotometrically at 280 nm and HPLC analysis of the binary mixture. Once a potential condition for glucagon and insulin separation was identified, this condition was evaluated using chromatographic runs of two different samples obtained from the industrial insulin production process. Chromatography with high-purity glucagon The chromatograms obtained when the IMAC column was fed with glucagon dissolved in the equilibration buffer (Figure 1 and Table 1) showed that glucagon adsorbed on all the metal-charged adsorbents tested. Since glucagon has only one histidine residue in its structure, it was expected that its adsorption on an IMAC matrix would only occur if Cu 2+ ions were used as the ligand; at least two histidyl residues are essential for adsorption on immobilized IDA Ni 2+ and two histidyl residues in the vicinity are required for adsorption on IDA Zn 2+ (Sulkowski 1985). However, glucagon can self-associate in solution and form trimers, resulting in a structure with three histidine residues positioned superficially (Sasaki et al. 1975). In this type of conformation, glucagon can be expected to adsorb on immobilized Ni 2+ and Zn 2+ ions. The percentage of glucagon washed out of the column was only ca. 13% in the chromatography using PCI Cu 2+, indicating a strong interaction between this biomolecule and Cu 2+ ions. Elution took place only when a buffer at ph 2.1 was fed into the column. When glucagon solution was fed into a PCI Ni 2+ column, the fraction not retained was ca. 16%; however, elution took place at a higher ph, viz. at ph 4.0. This increasing elution ph trend was also observed using PCI Zn 2+ since ca. 16% of the glucagon did not adsorb and elution took place when a buffer at ph 6.0 was fed into the column. Hence, the strength of glucagon binding to the immobilized metal ions tested decreased in the order Cu 2+ > Ni 2+ > Zn 2+, i.e. according to Sulkowski s rule (Sulkowski 1985).

5 Adsorption of Glucagon and Insulin 887 Figure 1. IMAC chromatograms for glucagon solution with Cu 2+, Ni 2+ or Zn 2+ ions immobilized on PCI. Feeding step: mg glucagon in the equilibration buffer [20 mm phosphate buffer (ph 7.0) containing 1.0 M NaCl]. W = washing step with the equilibration buffer. Elution was carried out using a ph step gradient with 20 mm phosphate buffer containing 1.0 M NaCl at the indicated ph. Bed volume, 1 ml. Feed volume and glucagon concentration for the different metal ions used as ligands were 10 ml at 0.56 mg/ml for Cu 2+ ions, 10.0 ml at 0.57 mg/ml for Ni 2+ ions and 5.0 ml at 0.50 mg/ml for Zn 2+ ions.

6 888 C.S. Farinas et al./adsorption Science & Technology Vol. 21 No TABLE 1. Glucagon and Insulin Mass Balance for Separate Chromatograms of these Proteins on PCI Me 2+ Cu 2+ ion Ni 2+ ion Zn 2+ ion Glucagon Insulin Glucagon Insulin Glucagon Insulin mg % mg % mg % mg % mg % mg % Protein injection Protein recovery on (1) Washing (2) Elution at ph value (3) Regeneration Total protein recovery Chromatography with high-purity insulin The chromatograms for insulin (Figure 2 and Table 1) demonstrated that insulin bound tightly to all three transition metals, since little or no protein was detected in the washed-out fractions. This was expected given that insulin has two histidine residues. In the PCI Cu 2+ experiment, insulin was bound strongly in a similar manner to glucagon, with elution only taking place at ph 2.1. No insulin was detected in the through flow for chromatograms with PCI Cu 2+ and PCI Ni 2+. However, the strength of insulin binding on PCI Ni 2+ appeared lower than that on PCI Cu 2+ since almost complete elution (82%) from PCI Ni 2+ was possible at ph 5.0. The profile obtained for insulin on PCI Zn 2+ showed that a small amount of insulin (8.9%) was not retained. This was probably due to the presence of the Zn 2+ ion in the protein solution (the insulin used in these experiments had ca. 2% Zn 2+ ion added to it during the crystallization process). These Zn 2+ ions would probably compete for the metal-binding site of the protein with the immobilized Zn 2+ ions in the matrix. However, in other respects, insulin showed a similar chromatographic profile for PCI Ni 2+ and PCI Zn 2+ : a major peak during elution with buffer at ph 5.0 followed by a small peak during elution with buffer at ph 4.0. Chromatography with mixtures of high-purity glucagon and insulin When the adsorption strengths of glucagon and insulin on PCI Cu 2+ are compared (Figures 1 and 2), a high-resolution separation of these proteins would not be expected with the Cu 2+ ion as ligand due to the strong adsorption of both proteins. However, Ni 2+ or Zn 2+ ions would be capable of carrying out this task since the ph required for insulin elution (ph 5.0 for both metal ions) was different from that required for glucagon elution (ph 4.0 and 6.0, respectively). Nevertheless, chromatograms of a mixture of these two proteins were compared with those obtained for each

7 Adsorption of Glucagon and Insulin 889 Figure 2. IMAC chromatograms for insulin solution with Cu 2+, Ni 2+ or Zn 2+ ions immobilized on PCI. Feeding step: mg insulin in the equilibration buffer [20 mm phosphate buffer (ph 7.0) containing 1.0 M NaCl]. W = washing step with the equilibration buffer. Elution was carried out using a ph step gradient with 20 mm phosphate buffer containing 1.0 M NaCl at the indicated ph. Bed volume, 1 ml. Feed volume and insulin concentration for the different metal ions used as ligands were 10 ml at 0.81 mg/ml for Cu 2+ ions, 10.0 ml at 0.50 mg/ml for Ni 2+ ions and 5.0 ml at 0.90 mg/ml for Zn 2+ ions.

8 890 C.S. Farinas et al./adsorption Science & Technology Vol. 21 No protein using all three metal ions (Figure 3). Different results were found with these mixtures due to competition between the proteins for the adsorption-binding site. All the adsorbents, i.e. PCI Cu 2+, PCI Ni 2+ and PCI Zn 2+, showed a different degree of separation of glucagon from insulin under the conditions tested. The protein content of the solution fed into the column packed with PCI Cu 2+ was 35% glucagon and 65% insulin. HPLC analysis of the fraction collected after elution with 55 ml mobile phase showed that the major polypeptide mass fraction (78%) corresponded to glucagon. Insulin was only eluted during the regeneration step with EDTA. Given that the retention of both glucagon and insulin on PCI Cu 2+ adsorbent was extremely strong and thereby required drastic elution conditions, this metal ion was not studied further. On the other hand, mild conditions were required for the elution of the two peaks observed in the chromatogram of PCI Ni 2+ (elution ph not lower than 4.0). The amount of insulin fed into the column packed with PCI Ni 2+ was larger than the amount of glucagon (81% insulin and 19% glucagon). HPLC analysis revealed that insulin eluted at ph 5.0, but that glucagon also started to elute at this peak tail. The fraction collected at the maximum of the first peak (after the passage of 29 ml mobile phase) contained mainly insulin (97%), but this percentage decreased to 63% at the beginning of the second peak (after the passage of 48 ml mobile phase). Probably due to kinetic effects during desorption, protein elution attained a plateau above the original baseline and exhibited a broad elution peak. When buffer at ph 4.0 was fed into the column, glucagon was eluted together with some insulin not eluted at ph 5.0. A difference in affinity existed between these two proteins for PCI Ni 2+, since the elution of insulin occurred at a different ph from the elution of glucagon (ph values of 5.0 and 4.0, respectively). The conditions tested for the experiment using PCI Zn 2+ did not allow the separation of glucagon and insulin, indicating that some further operational adjustments are still required. HPLC analysis of the feeding solution indicated 46% glucagon and 54% insulin, while analysis of the pool of fractions collected during elution with buffer at ph 6.0 indicated that the total protein contained 70% glucagon whereas the pool collected during elution at ph 5.0 contained 88% insulin. Of the various conditions tested, the use of PCI Ni 2+ as the adsorbent and elution by a decreasing ph step gradient were selected for the remainder of the experiments. Adsorption isotherm experiments In order to identify a condition for better selectivity during the adsorption step, adsorption isotherms of glucagon and insulin on PCI Ni 2+ at 25 C and different ph values were determined via batch experiments using stirred tanks (Figure 4). The distribution coefficient, K, was calculated from the slope of the linear portion of each isotherm (Table 2). Despite a low correlation factor for the determination of K for glucagon at ph 6.0, comparison of the values for the separation coefficient a [the ratio of K for insulin (Ki) and K for glucagon (Kg) at different adsorption ph values] indicated that a increased as the ph increased, thereby suggesting that a selective adsorption could be achieved at ph 7.0 or possibly higher. Given that the ph value commonly used for IMAC adsorption is ca. 7.0 [the ph at which the electron-donor group, in this case the imidazole group of histidine, is partially unprotonated, thus favouring protein adsorption], this ph was tested for the recovery of glucagon from industrial insulin-processing fractions via adsorption on PCI Ni 2+. The insulin and glucagon adsorption isotherms were also analyzed using three models frequently employed to describe the adsorption behaviour of proteins, i.e. the Langmuir, Freundlich and Langmuir Freundlich models (Sharma and Agarwal 2001). All model parameters were evaluated

9 Adsorption of Glucagon and Insulin 891 Figure 3. IMAC chromatograms for a mixture of glucagon and insulin solution with Cu 2+, Ni 2+ or Zn 2+ ions immobilized on PCI. Feeding step: mixture in the equilibration buffer [20 mm phosphate buffer (ph 7.0) containing 1.0 M NaCl]. W = washing step with the equilibration buffer. Elution was carried out using a ph step gradient with 20 mm phosphate buffer containing 1.0 M NaCl at the indicated ph. Feed volume for the different metal ions used as ligands were 10.0 ml for Cu 2+ ions, 10 ml for Ni 2+ ions and 5.0 ml for Zn 2+ ions. The glucagon/insulin mass ratios fed into the PCI Cu 2+, PCI Ni 2+ and PCI Zn 2+ columns were 35:65, 19:81 and 46:57, respectively.

10 892 C.S. Farinas et al./adsorption Science & Technology Vol. 21 No Figure 4. Adsorption isotherms for glucagon ( ) and insulin ( ) on PCI Ni 2+ at 25 C. Adsorption buffer: 20 mm phosphate buffer containing 1.0 M NaCl at the indicated ph.

11 Adsorption of Glucagon and Insulin 893 TABLE 2. Comparison between Adsorption Isotherms for Glucagon and Insulin at Different Adsorption ph Values ph Protein Linear section Distribution Correlation Separation C (mg/ml) a coefficient (K) factor coefficient a b (Ki/Kg) insulin glucagon insulin glucagon insulin glucagon a C is the protein equilibrium concentration in solution. b Ki and Kg are the distribution coefficients for insulin and glucagon, respectively. using non-linear regression analysis using the Levenberg Marquardt method (Table 3). This analysis indicated that only the Freundlich model was suitable for describing all the adsorption data for glucagon and insulin at the different ph values tested. However, a theoretical drawback of the Freundlich isotherm is that the amount of adsorbed protein increases indefinitely with the protein concentration in solution, resulting in unrealistic parameters values (Sharma and Agarwal 2001). The Langmuir Freundlich model fitted the insulin adsorption data at ph 5.0 and 7.0 better, while at ph 6.0 and also at ph 7.0 the Langmuir model was adjudged to be best. In contrast, the Langmuir Freundlich model only fitted the glucagon adsorption data at ph 5.0. A fit of the glucagon adsorption data by the Langmuir model was not possible at any ph value, probably because of insufficient experimental data (due to glucagon solubility limitations). Hence, it was not possible to use the parameter K d (dissociation coefficient) that represents the affinity between the solute and the adsorbents to compare the affinity interaction of glucagon and insulin with the adsorbent. Chromatography with two industrial insulin-processing fractions The conditions selected previously for the separation of glucagon from insulin, i.e. PCI Ni 2+ as the adsorbent and elution via a decreasing ph step gradient, were tested on two industrial fractions with the aim of obtaining a qualitative evaluation of the operational settings chosen. The chromatogram for the glucagon-rich fraction [Figure 5(a)] showed that 44% of the total protein was eluted during the feed and washing steps. Glucagon was eluted only when a buffer at ph 3.0 was percolated through the column. The amount of insulin detected at this peak was below the detection limit of the equipment, indicating that the glucagon recovered under these conditions was of relatively high purity. Three main peaks were obtained when an insulin-rich fraction was fed into the column packed with PCI Ni 2+ [Figure 5(b)]. HPLC analysis of the first two peaks revealed unknown impurities. However, analysis of the fraction eluted at ph 2.3 indicated that glucagon was the major protein component. Insulin elution took place mainly during column regeneration

12 894 C.S. Farinas et al./adsorption Science & Technology Vol. 21 No TABLE 3. Adsorption Isotherm Parameters for Glucagon and Insulin at Different Adsorption ph Values Hormone ph Isotherm model Langmuir Langmuir Freundlich Freundlich a Q m ± K ± K d 0.22 ± 0.18 h 0.97 ± 0.06 n 1.61 ± 0.28 R = 0.952; s 2 = R = 0.966; s 2 = Q m ± K ± 4.00 Insulin 6.0 K d 0.45 ± 0.09 a h 0.64 ± 0.05 R = 0.983; s 2 = R = 0.975; s 2 = Q m ± 2.86 Q m ± K ± K d 0.14 ± K d 0.82 ± 0.49 h 0.48 ± 0.02 R = 0.977; s 2 = n 0.65 ± 0.07 R = 0.984; s 2 = R = 0.989; s 2 = Q m ± 4.21 K ± a K d ± h 1.49 ± 0.11 n 3.29 ± 0.48 R = 0.927; s 2 = R = 0.970; s 2 = K ± Glucagon 6.0 a a h 2.22 ± 0.18 R = 0.912; s 2 = K ± a a h 1.01 ± 0.10 R = 0.876; s 2 = a Fitting was not possible (see Discussion section). Q m is the maximum protein binding capacity in mg/ml. n is the Langmuir Freundlich coefficient. K d is the apparent dissociation constant in mg/ml. K and h are the Freundlich equilibrium constants. Equations: Langmuir: Q = Q m C/(K d + C); Freundlich: Q = KC n ; Langmuir Freundlich: Q = Q m C n /(K d + C n ). with EDTA. A purification factor of five for glucagon was achieved in this chromatography. This provides an indication of the potential of IMAC as a technique for purifying glucagon present in a complex medium. The ph required for glucagon elution from PCI Ni 2+ decreased in line with the complexity of the feed, since the ph required varied in accordance with the sample added to the column. Glucagon added separately or mixed with insulin (Figures 1 and 3) was eluted at ph 4.0, glucagon present in the glucagon-rich fraction was eluted upon decreasing the ph down to ph 3.0 (Figure 5) and glucagon present in the insulin-rich fraction was eluted at an even lower ph value, viz. 2.3 (Figure 5).

13 Adsorption of Glucagon and Insulin 895 Figure 5. IMAC chromatograms for (a) glucagon-rich and (b) insulin-rich industrial fractions on PCI Ni 2+. Feeding step: industrial fraction in the equilibration buffer [20 mm phosphate buffer (ph 7.0) containing 1.0 M NaCl]. W = washing step with the equilibration buffer. Elution was carried out using a ph step gradient with 20 mm phosphate buffer containing 1.0 M NaCl at the ph indicated. CONCLUSIONS Interaction of glucagon and insulin with IMAC adsorbents was studied with the aim of purifying glucagon in a complex medium. Despite the fact that glucagon and insulin exhibited an adsorption affinity towards all the metal ions tested (Cu 2+, Ni 2+ and Zn 2+ ), selectivity towards glucagon was achieved by using Ni 2+ as the metal ion ligand with desorption involving a ph step gradient. Studies with an insulin-rich industrial fraction showed a fivefold increase in glucagon purity. Since the ph required for glucagon elution varied according to the complexity of the feed, the feasibility of using IMAC as a purification tool for glucagon will depend on an appropriate adjustment of the elution protocol based on requirements. Thus the results presented in this paper could serve as guidelines for the purification of glucagon using the IMAC adsorption technique.

14 896 C.S. Farinas et al./adsorption Science & Technology Vol. 21 No ACKNOWLEDGMENTS The authors would like to thank Luciano Vilela (formerly at Biobrás, Brazil, and currently at Biomm, Brazil) for his donation of the hormones, CAPES (Brazil) for the financial support provided to C.S. Farinas and FAPESP (Brazil) for its financial support. REFERENCES Andrade, A.S.R., Vilela, L. and Tunes, H. (1997) Braz. J. Med. Biol. Res. 30, Arnold, F.H. (1991) Bio/Technology 9, 151. Blundell, T., Dodson, G., Hodgkin, D. and Mercola, D. (1972) Adv. Protein Chem. 26, 279. Carrea, G., Pasta, P. and Antonini, E. (1985) Biotechnol. Bioeng. 27, 704. Joshi, A.B., Rus, E. and Kirsch, L.E. (2000) Int. J. Pharm. 203, 115. Kimball, C.P. and Murlin, J.R. (1923) J. Biol. Chem. 58, 337. Maskalick, D.G. and Anderson, M.T. (1986) US Pat Okamoto, H., Iwamoto, H., Tsuzuki, H., Teraoka, H. and Yoshida, N. (1995) J. Protein Chem. 14, 521. Olsen, T. and Diderichsen, B. (2000) J. Biotechnol. 79, 185. Porath, J., Carlsson, J., Olsson, I. and Belfrage, G. (1975) Nature (London) 258, 598. Sasaki, K., Dockerill, S., Adamiak, D.A., Tickle, I.J. and Blundell, T. (1975) Nature (London) 257, 751. Sharma, S. and Agarwal, G.P. (2001) Anal. Biochem. 288, 126. Sulkowski, E. (1985) Trends Biotechnol. 3, 1.