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R. Kuhn et al.

Electrophoresis 1990, 1 1 , 942-941

Reinhard Kuhn* Sabrina Hoffstetter-Kuhn** Horst Wagner

Free-flowelectrophoresis for the purification of proteins: 11. Isoelectric focusing and field step electrophoresis

Fachrichtung Anorganische Analytik und Radiochemie, Universitat des Saarlandes, Saarbrucken

Two modes of continuous isoelectric focusing are described. The development of a natural pH gradient, consisting of a mixture ofthree buffer solutions, and the focusing behavior of human serum albumin is investigated. The advantages of isoelectric focusing in an artificial pH gradient of three buffer solutions are demonstrated on the purification of a-amylase from an E . coliprotein extract. Furthermore the principleof field step electrophoresis is presented. The most important factors influencing the efficiency: (i) residence time, (ii) conductivity ofthe sample and (iii) sample zone width, are discussed. The use of a larger sized device to allow simultaneous multiple injections of the sample demonstrates the feasibility of scaling-up field step electrophoresis. This approach permits a throughput of about 20 mL sample solution per minute.

1 Introduction

2 Materials and methods

Free-flow electrophoresis (FFE) is a continuous run operation, without the need for supporting materials, and an interesting alternative to preparative chromatographic techniques. Therefore FFE is a labor-saving method and well suited for the purification of labile biopolymers, since - in contrast to chromatography - there are no disturbing interactions with stationary phases, often inducing a loss in biological activity. Over the last few years the versatility of F F E has been increased by the development of new techniques which have not only extended its application range but also allow a concentration of sample compounds during their separation. In part I of this series [ I1 we reported the principles and some applications of zone electrophoresis (ZE) and isotachophoresis (ITP). This part introduces isoelectric focusing (IEF) in a stepwise pH gradient and field step electrophoresis (FSE).

2.1 Chemicals

Bier 121 developed a preparative IEF apparatus using carrier ampholytes to generate a continuous pH gradient. The establishment of the pH gradient during electrophoresis is a slow process; therefore, the sample has to be recycled several times through the separation chamber to ensure that sample compounds are focused. We were engaged to develop techniques for a continuous mode of operation without carrier ampholytes [31. Simple amino acids or Good buffers are used to set up a stepwise pH gradient, which is either preformed prior to electrophoresis [41 or generated electrophoretically within an acceptable time [51. While IEF is limited to amphoteric compounds, FSE is also suitable for the purification of small ions [6,71 in addition to proteins [8-101, viruses [ l I1 or particles [121. Depending on the width of the admitted sample zone, individual compounds can be concentrated more than 10-fold I 131 over the original concentration.

Correspondence: Dr. R. Kuhn, Analytical Research and Development, Sandoz Pharma Ltd., CH-4002 Basel, Switzerland Abbreviations: ADH, alcohol dehydrogenase; Ala, L-alanine; AMPD, 2-amino-2-methyl-l,3-propanediol; Arg, L-arginine: Asn, L-asparagine; Bicine, N,N-bis(hydroxyethy1)glycine; cSer, cycloserine; EACA, t-aminocaproic acid; FFE, free flow electrophoresis: FSE, field step electrophoresis; Glu, L-glutamic acid: GlyGly, glycylglycine; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonicacid; HSA, human serum albumin IEF, isoelectric focusing; ITP, isotachophoresis; MES, 2-(N-morpho1holino)ethane sulfonic acid; Tris, tris(hydroxymethy1)-aminomethane; ZE, zone electrophoresis 0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

All chemicals used were of analytical grade unless otherwise stated. L-Alanine (Ala), p-alanine, alcohol dehydrogenase (ADH) from baker’s yeast, a-amylase from Aspergillus oryzae, 2-amino-2-methyl- 1,3-propanediol (AMPD), L-arginine (Arg), L-asparagine (Asn), N,N-bis(hydroxyethy1)glycine (Bicine), cycloserine (cSer), 6-aminocaproic acid (EACA), Bromophenol Blue (sodium salt), chicken egg albumin, Lglutamic acid (Glu), glycine (Gly), N-2-hydroxyethylpiperazine-N’-2-ethanesulfonicacid (HEPES), human serum albumin (HSA), methionine, 2-(N-morpholino)ethane sulfonic acid (MES), polyethyleneimine (pract.), starch according to Zulkowsky and tris(hydroxymethy1)-aminomethane (Tris) were obtained from Serva (Heidelberg, FRG). All other substances were purchased from Merck Darmstadt, FRG). 2.2 Analytical methods The enzymatic activity of a-amylase (EC 3.2.1.1) was measured according to Rick et al. [14] using the absorbance at 546 nm. Alcohol dehydrogenase (EC 1.1.1.1) was determined by the methodofBergmeyer [ 151. The total protein concentration was measured at 280 nm using chicken egg albumin as standard. All photometric measurements were performed using a Hitachi “Model1 100-60” photometer. For the measurements of pH and conductivity the pH meter “WTW 530” and conductometer “WTW L F 52 1” from WTW (Lauda, FRG), were employed. 2.3 Sample preparation The preparation of the crude sample containing ADH is described in [ 11. The a-amylase extract was prepared as given in t51. 2.4 Electrophoretic apparatus Most of the experiments were performed with the free-flow instrument Elphor VaP 22 (Bender & Hobein, Munich, FRG) in a vertical, rectangular separation chamber, 50 x 10 x 0.05

* **

Present address: Analytical Research and Development, Sandoz Pharma Ltd., Basel, Switzerland. Present address: FO 3 Analytical Research, Ciba-Geigy Ltd., Basel, Switzerland 0113-0835/90/1111-0942 $3.50+.25/0

Isoelectric focusing and field step electrophoresis

Eleclrophoresis 199O,Il, 942-947

cm. A description of the apparatus and its application is given in detail elsewhere 151. Cellulose acetate membranes were used in all experiments. A prototype manufactured by the same company was used for the experiment with multisample dosing (see Fig. 11). The only difference to the Elphor VaP 22 is the bigger separation chamber, measuring 100 x 15 x 0.05 cm.

3 Results and discussion 3.1 IEF There are two different basic modes of performing continuous I E F in free solution. The first is to set up a pH gradient during electrophoresis by admitting a mixture of selected buffer substances with low electrophoretic mobilities, e. g., amino acids or Good buffers, into the separation chamber 151.These simple buffers then migrate electrophoretically to produce a stepwise p H gradient according to their individual pK or p l values in a manner analogous to the formationof acontinuous pH gradient with carrier ampholytes. The number of pH steps is determined by the number of different buffer compounds. The dynamics of the development of a three-step pH gradient of L-glutamic acid (Glu), cycloserine (cSer) and Arg is illustrated in Fig. l. The development of the p H steps with increasing residence time is clearly visible. Without recycling the solution. a residence time of at least 40 min is necessary for a well-shaped p H profile. The emerging p H profile is accompanied by conductivity changes indicative of the moving boundaries of the buffer compounds. Finally, after a residence time of 40 min, three conductivity zones demonstrate the completeness of the amino acid separation. The focusing behavior of HSA added to the buffer mixture is illustrated in Fig. 2. As the pH profile evolves, the protein molecules migrate within the changing gradient from both sides of the chamber until the steady state is achieved - after a residence time of about 40 min. Intermediate focusing peaks of the protein

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counterfeiting different sample compounds (e. g., three at a 20-min residence time) are the result of concentration at the transient conductivity steps. In the second mode, an artificial stepwise pH gradient is established by admitting the individual buffer solutions into the separation chamber as parallel zones (illustrated diagrammatically in Fig. 3) [4]. The sample can be dissolved in, e. g., buffer B and introduced through two inlet tubes. Other methods of introducing a narrow band of sample can also be employed. In our example the stepwise p H profile is formed by the four buffer solutions A, B, C and D. When the voltage is applied, the compound to be purified migrates under the influence of its charge until it reaches the pH step at which it loses its charge and is focused. This experimental design is best suited to the purification of one or two components of a complex mixture. Since the p H gradient is preformed, residence times can be much shorter in this mode (typically 5- 10rnin). The optimization ofthe residence time for the purification ofaamylase from a crude E. coli protein extract is illustrated in Fig. 4. When the residence time is 2 min, there is a tendency for the enzyme to focus at the acidic p H step (fractions 35-37, Fig. 4a); this process is completed after a residence time of 6 min (Fig. 4c). Since most other E. coli cell proteins possess p l values of 4-6, they migrate slowly at pH 4.7 and therefore d o not focus at the pH step during the chosen residence time.

3.2 Field step electrophoresis 3.2.1 Principle The low solubility of proteins near their isoelectric points inhibits high concentration factors and therefore high throughputs in all continuous I E F methods. This drawback can be avoided by using a discontinuous conductivity profile instead of a discontinuous p H profile. The fact that the migration velocity of charged compounds can be drastically reduced by

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Figure 1. Dynamics of IEF in a buffer system composed of 10 mM each of glutamic acid, cSer and arginine. The pH gradient is established directly in the cell. The distributions of (a) pH and (b) conductivity (b) at different residence times are shown. 0.1 M phosphoric acid and 0.05 M NaOH served as anolyte and catholyte, respectively. The voltage applied was 500 V.

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increasing the conductivity of the medium means that a concentration of migrating compounds can occur in a conductivity step just as it can in a pH step. FSE takes advantage of this principle. The sample is dissolved in a low conductivity buffer and introduced as a more or less broad zone between the anolyte and catholyte, which act as flanking zones ofhigh conductivity (Fig. 5). The field strength in the sample zone is much higher than that in the flanking zones (Fig. 6) since it is inversely proportional to the conductivities. Hence charged sample compounds migrate with high velocity towards the oppositely charged electrode. On reaching the anolyte or catholyte boundary they decelerate and are concentrated.

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Figure 2. IEF behavior of 0.05 mg/mL HSA in a Glu/cSer/Arg buffer system. See Fig. 1 for more details.

Figure 3.Schematic representation of IEF in an artificial pH gradient. Top panel shows the inlet system; middle panel, focusing of three ampholytes in pH profile given below. For explanation see text.

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Figure 4 . Stepwise IEF of 0.35 mg/mL E. coli cell proteins containing 0.05 mg/mL a-amylase; influence of the residence time on purification and focusing of a-amylase. The arrows indicate sampleinlet zone;black area, the enzyme activity (as E5J; and blank area, the total protein concentration (as EZ8J.The protein mixture was dissolved in MES/GlyGly, each 0.03 mol/L. Asp (0.03 mol/L) and Arg (0.03 mol/L) were introduced through 2 inlet ports, the sample through one inlet. The residence time was (a) 2, (b) 4 and (c) 6 min. A voltage of 800 V was applied in all runs. The anode is to the left.

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Isoelectric focusing and field step electrophoresis

Electrophoresis 1990, 1I , 942-941

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Figure 7. FSE of a-amylase (0.3 g/L) and E. coli cell proteins (1.0 g/L); effect of residence time on enzyme focusing. Black area, a-amylase activity measured at 546 nm; blank area, total protein concentration at 280nm. The high conductivity electrolyte was HEPES/Bicine (each 0.03 mol/L) adjusted to a conductivity of 3 rnS/cm with KC1. The proteins were dissolved in HEPES/Bicine(each0.03 mol/L),pH 5.3, conductivity0.07 mS/cm. The arrows indicate the sample inlet zone (width 2 cm). The residence time was (a) 1, (b) 2 and (c) 4 min. The voltage was 500 V.

Figure 5. Schematic representation of FSE. The top panel shows the inlet system, lower panel illustrates the purification of 2 components.

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Figure 8 . FSE of alcohol dehydrogenase; effect of conductivity of sample solution on protein migration. The arrows indicate the sample inlet zone. K,SO, with aconductivityof 3 mS/cm served as high conductivitysolution. 0.1 g/L ADH was dissolved in EACA/Ala, each 0.05 mol/L, pH 7.0. The conductivities of the sample solutions were adjusted to (a) 0.1 mS/cm, (b) 0.2, (c) 0.4, (d) 0.6, and (e) 0.78 mS/cm with potassium sulfate. Broken line represents the conductivity profile. Voltage, 400 V; residence time, 2 min; sample zone width, 2 cm.

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Figure 6. Conductivity and field strength profile of FSE.

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3.2.2 Residence time The high electrophoretic mobilities of the sample compounds mean that only short residence times are required for complete focusing in the conductivity steps. Since, in contrast to IEF, a steady state is attained, the optimization of residence time is of utmost importance for successful focusing. If too short a residence time is chosen, as depicted in Fig. 7a, tailing of the sample component will result, indicating incomplete concentration. In the case of too long a residence time (Fig. 7c), the throughput is decreased and the sample component migrates into the high conductivity zone, reducing the concentration factor. A sixfold concentration was achieved by optimizing the residence time (Fig. 7b).

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Figure 9. Current/potential curve at different conductivities of sample zone. Sample solution as in Fig. 8, adjusted to the conductivities in the figure.

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Electrophoresis 1990,II, 942-941

3.2.3 Conductivity

to remove the heat if separations are to be made with samples of high conductivity. Whenever possible, samples with high salt concentrations should be dialyzed to reduce the conductivity and to improve the efficiency.

The concentration of the sample component is not only affected by the applied voltage, residence time and pH, but also by the conductivity of the sample zone. As described above, increasing the conductivity of the sample solution causes a decrease in the field strength and in the migration velocity. Figure 8 shows the migration of ADH at different sample conductivities. Even a small increase in conductivity from 0.1 to 0.2 mS/cm (Fig. 8a, b) decreases the concentration factor, and conductivities greater than 0.4 mS/cm (Fig. 8d, e) dramatically lowered the migration velocity. Increasing the conductivity also increases the electric current. The dependence of the current on applied voltage at different sample conductivities is illustrated in Fig. 9. Ifthe sample conductivity is low (e.g., 0.06 mS/cm) compared to the conductivity of the flanking zones (3 mS/cm), the resulting current is determined by the sample conductivity alone and there is a nonlinear increase with voltage. At higher voltages the sharp conductivity profile across the chamber deteriorates, allowing higher current flows. If the sample conductivity becomes similar (e.g., 1 mS/cm) to the conductivity of the flanking zones, a dependence of the current on the applied voltage becomes more linear. Finally, when the conductivities ofthe sample and the flanking zones are the same, the current increase is strictly linear with respect to the voltage in obedience to Ohm’s law, just as in ZE. Since the Joule heat generated within the separation chamber is P x R,an efficient cooling system is necessary

3.2.4 Width of the sample zone The width ofthe sample zone also influences the separation efficiency, especially with respectto samplethroughput and concentration factor. Sincethe electrophoreticmobilityofproteins is low, optimal purification demands that the sample has to be admitted as a narrow band. Figure 1Odisplaystheinfluence of the sample application width upon the separation of aamylase from E. colicell proteins. The voltage was adjustedin such a way that the same field strength was applied within the sample zone in every experiment. Despite the use of the same field strength, the enzyme was only completely focused when sample addition was restricted to a width of 2 cm. Pronounced peak tailing was observed when the sample was introduced into the separation chamber in a broad zone, thus demonstrating that enzyme velocity does not increase linearly with voltage. If longer residence times were employed to overcome this problem, the tailing was only slightly reduced because of the deterioration this caused in the conductivity profile. The theoretical concentration factor of about 8 for a zone width of 2 cm was almost achieved in Fig. 10a. The throughput cannot be increased by using broader sample zones; it can, however, be increased by simultaneously in-

Figure 10. FSE of 0.6 g/L E. coli proteins containing 0.3 g/L a-amylase; effect of sample zone width on enzyme concentration. The arrows indicate sample inlet zones; black area, enzyme activity (as E5J; blank area, total protein concentration (as Ezs0).The proteins were dissolved in HEPES/Bicine, each 0.03 mol/L, pH 5.3. HEPES/Bicine (each 0.03 mol/L)/K,SO, with a conductivity of 3 mS/cm served as high conductivity solution. The sample zone widths were (a) 2, (b) 4 and (c) 6 cm. The voltage was (a) 400, (b) 800 and (c) 1200 V at a residence time of 2 min.

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Fig. 11. FSE of ADH and cytochrome c, each 0.1 g/L, with 4-fold sample dosing, The arrows indicate sample zone; black area, the ADH concentration (as Ezs0); blank area, thecytochromecconcentration (as E&). The proteins were dissolved in EACA/Ala, 0.05 mol/L each, pH 7.0; voltage, 1200 V; residence time, 3 min; sample zone width, 4 x 2 cm. A potassium sulfate solution, adjusted to 1 mS/cm, served as high conductivity electrolyte.

Electrophoreris 1990, Ii, 947-952

Stability of borate-glycerol pH gradients in RIEF

troducing the sample into the separation chamber in several narrow zones, as illustrated in Fig. 1 I for the separation of ADH from cytochrome c. The injection of 4 sample streams resulted in a maximum throughput of about 20 mL sample solution per minute at a concentration factor of nearly 6, compared to the initial concentration. Purification was carried out in a prototype electrophoresis chamber after optimization with the Elphor VaP 22. All experimental parameters were the same as in the optimization experiments with the exception of voltage, which had to be multiplied by 1.5 to take into account the different chamber widths.

This work was supported by the Bundesminister f u r Forschung und TechnoIogie, FR G,Forderkennzeichen 03 18809 Al. Received May 23, 1990

4 References [ I ] Hoffstetter-Kuhn, S., Kuhn, R. and Wagner, H., Electrophoresis 1 9 9 0 , I I , 304-309. 121 Bier, M., in: Asenjo, J. A. and Hong, J. (Eds.), American Chemical Society Symposium Series 314, Washington, 1986, pp. 185-192.

Paul Todd William Elsasser Bioprocessing and Pharmaceutical Research Center, Philadelphia, PA

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131 Manzoni, R.,Ph. D. Thesis. Universitat des Saarlandes, Saarbrucken 1986. 141 Kuhn, R., Wagner, H., Mosher, R. A. and Thorrnann, W., Electrophoresis 1987,8,503-508. L5l Mosher, R. A., Thormann, W., Kuhn, R. and Wagner. H., J . Chromatogr. 1989,, 39--49. 161 Kessler, R., Manz, H. J. and Szkkely, G.,J. Chromatogr. 1989,469, 444-447. [71 Wagner, H. and Neupert, D.,J. Chromatogr. 1978,156, 219-224. [Sl Barth, F., Gruetter, M., Kessler, R. and Manz, H. J., Electrophoreyis 1986, 7,372-375. 191 Kessler, R., Manz, H. J. and Walliser, H. P.. in:3. EngineeringFoundation Conference on Recovery of Bioproducts, Uppsala 1986. l l O l Kuhn, R. and Wagner, H.,J. Chromatogr. 1989,481,343-351. I 1 11 Heydt, A., Wagner, H. and Miiller, P.,J. Virolog. Methods 1988,19, 13-22. 1121 Dickenscheid-Simon, R., Ph. D. Thesis, Universitat des Saarlandes Saarbrucken 1988. [ 131 Wagner, H., Kuhn, R. and Hoffstetter, S., in: Wagner, H. and Blasius, E. (Eds.), Praxis der elektrophoretischen Trennmethoden, Springer, Berlin, 1989, pp. 223-278. I141 Rick, W. and Stegbauer, H. P., in: Bergmeyer, H. U. (Ed.), Methoden der enzymatischen Analyse, Verlag Chemie, Weinheim, 1962, pp. 918-923. [I51 Bergmeyer, H. U., in: Bergmeyer, H. U. (Ed.), Methoden der enzymatischenAna[yse,VerlagChemie, Weinheim, 1962,pp. 392-393.

Nonamphoteric isoelectric focusing: 11. Stability of borate-glycerol pH gradients in recycling isoelectric focusing By complexing polyols with borate in recycling isoelectric focusing and by varying the ratio ofpolyol to borateovertheusefulpH range of4.0-6.0, it is possibleto control pH. Twelve solutions of 0.1 M boric acid and varying glycerol concentration were used to vary pH in a twelve-compartment commercial recycling isoelectric focusing (RIEF) system. Various concentrations of boric acid were tested as anolyte, and various Tris(hydroxymethy1amino)methane-borate buffer systems were tested as catholyte. Electroosmosis, hydrogen ion flow, and fluid balancing were characterized in two glycerol gradients; one was maintained at 0.06 pH/fraction and the other at 0.12 pH/fraction. In the latter case, ovalbumin (~14.70)migrated to the pH 4.6 1 and 4.72 compartments. It is concluded that the borate-glycerol system can be adequately stabilized in RIEF for isoelectric purification of certain proteins.

1 Introduction 1.1 Reyciing isoelectric focusing With the advent of pharmaceutical and diagnostics manufacture based on proteins from tissues, cells, or genetically engineered organisms, the need for large-scale, free-fluid purification methods has intensified. Recycling isoelectric focusing (RIEF) was introduced by Bier etal. 11-31 as a means of scaling up protein purification in free fluid. In this Correspondence: Dr. Paul Todd, National Institute of Standards and Technology, 325 Broadway 583.10, Boulder, CO 80303, USA Abbreviation: RIEF, recycling isoelectric focusing 0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

method isoelectric focusing takes place in a small multicompartment chamber in which each compartment corresponds to a single value of pH along the pH gradient and is (typically) separated from adjacent compartments by nylon screens. Synchronized pumps circulate the focused material from each compartment through a separate heat exchanger, and separands migrate from compartment to compartment until they reach the compartment that contains their isoelectric pH. Separands are then drained from their corresponding individual heat exchangers. Several grams of protein can be processed in a fraction of an hour by this method. Kyhse-Andersen [41 used a 32-compartment chamber (with mixing flow but not recycling) and claimed the fractionation of 10 g ofprotein in 660 ml of ampholyte solution [51. 0173-0835/90/11 11-0947 %3.50+.25/0