Communications to the Editor Protein Chromatography in Neat Organic Solvents

Nancy Chang and Alexander M. Klibanov* Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received June 17, 1991/Accepted September 13, 1991

Pure formarnide and ethylene glycol are used instead of wa- ter as processing media for protein chromatography. A num- ber of common proteins are freely soluble in these solvents and most do not undergo irreversible inactivation in them. Batch adsorption studies reveal that proteins readily adsorb to various ion-exchangers in formamide and ethylene glycol and subsequently can be completely desorbed by adding in- organic salts (LiCI and NH,NO,) to these solvents. The idea of protein separations in formamide and ethylene glycol is illustrated by column chromatography and preparative sep- aration of mixtures of ( i ) oxidized A and B chains of insulin and (i i ) lysozyme and ribonuclease on the anion-exchanger triethylaminoethylcellulose and the cation-exchanger phos- phocellulose, respectively. Key words: protein separations formamide ethylene glycol downstream protein processing chromatography ion-exchange resins

INTRODUCTION

The growing demand for novel therapeutic and diagnos- tic proteins (derived from recombinant DNA processes) necessitates further innovations in downstream protein processing. Indeed, over the last few years the develop- ment of new, potentially scaleable approaches to protein purification has attracted much a t t e n t i ~ n . ~ ~ ~ ~ ~ ” ~ ” ~ ~ ~ ’ Among other unit operations, chromatographic tech- niques appear to be particularly promi~ing.~,~

Virtually all protein separations to date have em- ployed water as the processing medium. Although proteins are insoluble in the vast majority of organic sol- vents, a few nonaqueous solvents, namely dimethyl sul- foxide, formamide, and ethylene glycol, dissolve most common proteins.” Consequently, we recently proposed to carry out protein separations and purifications in pure dimethyl sulfoxide and validated this idea by means of ion-exchange chromatography on carboxy- methylcellulose and of fractional precipitation with ethyl acetate in that solvent.’ In the present study, pro- tein separations in nonaqueous solvents have been extended to formamide and ethylene glycol. Relevant protein behavior and batch adsorption/desorption char- acteristics as well as chromatographic separations on various anion-exchangers and cation-exchangers have been investigated.

* To whom all correspondence should be addressed.

MATERIALS AND METHODS

All enzymes and other proteins were purchased from Sigma Chemical Company: hen egg-white lysozyme (EC 3.2.1.17) and ovalbumin; bovine pancreatic a- chymotrypsin (EC 3.4.21.1), trypsin (EC 3.4.21.4), ribo- nuclease (EC 3.1.27.5), Zn-insulin, and oxidized A and B chains of insulin (oxidation of cysteine resi- dues to Cys[S03H] was carried out according to a modified Sanger’s procedure”); horseradish peroxidase (EC 1.11.1.7); and bovine serum albumin (prepared from fraction V, crystallized and lyophilized). Zinc-free insulin was prepared from the commercial sample using the method of Sluyterman.’’ Formamide and ethylene glycol (both 99+% pure) were obtained from Aldrich Chemical Company and contained below 0.03% and 0.05% of water, respectively; they were used without further purification or drying. All resins were pur- chased from Sigma: carboxymethylcellulose and diethylaminoethylcellulose (CM-cellulose and DEAE- cellulose, respectively; both microgranular), triethyl- aminoethylcellulose (TEAE-cellulose, fibrous), and phosphocellulose (fibrous). All other chemicals used were obtained commercially and were of analytical grade or purer.

Protein determinations were carried out according to the Lowry method’’ following at least a 10-fold dilution of a protein solution in formamide or ethylene glycol with water (10 mM phosphate buffer, pH 7.0); for lyso- zyme, the protein concentration was also assayed by measuring the absorbance at 280 nm directly in the nonaqueous solvent. Calibration curves were obtained beforehand for each protein under each set of con- ditions. Catalytic activities of enzymes were assayed according to the literature procedures: ref. 17 for lyso- zyme, ref. 7 for both a-chymotrypsin and trypsin, ref. 9 for ribonuclease, and ref. 20 for peroxidase.

In order to determine their solubility in formamide and ethylene glycol, proteins were dissolved in distilled water at 4 mg/mL, the pH was adjusted to the desired value, and the solutions were lyophilized. Each solid protein sample was then added to formamide or ethyl- ene glycol and stirred at 30°C. Following complete dis- solution, more protein was added, and the procedure

Biotechnology and Bioengineering, Vol. 39, Pp. 575-578 (1992) 0 1992 John Wiley & Sons, Inc. CCC 0006-3592/92/050575-04$04.00

was repeated until no more protein dissolved. The un- dissolved protein was subsequently removed by centrifu- gation, and the protein concentration in the supernatant was measured by the Lowry method, as outlined above.

RESULTS AND DISCUSSION

There are at least three reasons for considering neat (also called straight or pure) organic solvents as process- ing media for protein separations. First, a significant hurdle in downstream protein processing, degradation of proteins by endogenous proteases, will be eliminated since proteolytic (and other) enzymes are catalytically inactive in protein-dissolving organic solvents such as dimethyl sulfoxide, formamide, and ethylene glyc01.8~'~~'~ Second, protein conformations and other characteris- tics in protein-dissolving organic solvents should be dif- ferent from those in water," thus augmenting separation opportunities beyond those existing in water. Third, pro- tein separations in organic solvents may be particularly attractive for membrane and other hydrophobic proteins where this approach may obviate the currently unavoid- able use of detergent^.'^ Having established the feasibil- ity of protein processing in dimethyl sulfoxide? in this work we have explored chromatographic separation of proteins in two other protein-dissolving neat organic solvents, formamide and ethylene glycol.

At the outset, it was essential to characterize the solu- bility of proteins in the two solvents. We selected hen egg-white lysozyme as the initial model. The enzyme was dissolved in distilled water, pH was adjusted to 6.0, and then the solution was lyophilized. The resultant protein was found to have a very high solubility in both formamide and ethylene glycol-45 and 28 mg/mL, respectively (at 30°C). Then we established that reason- able solubility in these solvents is a rather general phe- nomenon. In addition to lysozyme, eight out of nine proteins tested- trypsin, ribonuclease, peroxidase, oval- bumin, a-chymotrypsin, insulin, and oxidized A and B chains of insulin-were readily soluble in formamide at 1 mg/mL, and only bovine serum albumin was not (all the proteins were lyophilized from aqueous solution ad- justed to pH 6.0, except for insulin and its oxidized chains, which were lyophilized from pH 8.0). Likewise, the first four of these proteins were soluble in ethylene glycol at 1 mg/mL, while a-chymotrypsin and bovine serum albumin were not.

Next, we examined enzyme stability in formamide and ethylene glycol. Note that from the bioseparation standpoint, the parameter of interest is not whether en- zymes are active in the solvents but whether the enzy- matic activity is retained following subsequent dilution with water or removal of the solvent. To this end, we dissolved lysozyme, trypsin, and a-chymotrypsin in formamide (all three at 0.5 mg/mL), then immediately diluted them 100-fold with the aqueous buffers later used for the assays, and measured the catalytic activity.

In all three cases the latter was, within the experimental error, indistinguishable from that prior to the disso- lution in formamide. However, 24h incubation in formamide at 30°C did result in irreversible decline of enzymatic activity: approximately half for lysozyme and trypsin and 90% for a-chymotrypsin. This irre- versible inactivation was substantially reduced when the temperature was lowered to 4"C-to 0%, 20%, and 80%, respectively. When the same experiments were performed for lysozyme and ribonuclease in ethylene glycol, neither enzyme underwent appreciable irre- versible inactivation following either immediate dilu- tion or 24h incubation in the solvent at 30°C. Thus in most, although not all, of the instances examined en- zyme stability is sufficient for protein processing in the two solvents.

In order to establish the feasibility of protein chroma- tography in formamide and ethylene glycol, we investi- gated batch adsorption of proteins to ion-exchangers in these solvents as well as salt-induced desorption under these conditions. Figures 1A and 1B depict the adsorp- tion isotherms for lysozyme onto CM-cellulose in for- mamide and ethylene glycol; for comparison, adsorption isotherms in dimethyl sulfoxide and distilled water (pH 6.0) were also determined and are presented in Figures 1C and 1D. In all cases both the enzyme and

12 I 26

1 3 1 0 0 16 32

50 I

0 25 50

Total amount of lysozyme added, mg

Figure 1. Adsorption of lysozyme to CM-cellulose in forma- mide (A), ethylene glycol (B), dimethyl sulfoxide (C), and distilled water at pH 6.0 (D). The resin (lyophilized from distilled water at pH 6.0; 25 mg) was suspended in 1 m L of one of the solvents, and then various amounts of lysozyme (also lyophilized from pH 6.0) dissolved in 9 mL of the same solvent were added. The resultant mixtures were stirred at 30°C for 1 h and then centrifuged to re- move the resin. The protein content of the supernatant was deter- mined by the Lowry assay following at least a 10-fold dilution with a buffered aqueous solution. Each sloped straight line is drawn with the theoretically predicted tangent of 1, and its intercept with the abscissa axis affords the binding capacity of the resin under the specified conditions.

576 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 39, NO. 5, MARCH 5, 1992

the resin were lyophilized from aqueous solutions at pH 6.0. The points in Figure 1 correspond to the experi- mentally observed amounts of lysozyme in the superna- tant at given total amounts of enzyme present, and the sloped line has the theoretical tangent of unity. The data in Figures 1A-D afford the binding capacities of the cation-exchanger for lysozyme, which were calcu- lated to be 900, 280, 200, and 1500 mg/g resin, respec- tively. Thus the binding capacities in the nonaqueous solvents are both substantial and dependent on the na- ture of the solvent.

We also established protein binding to the anion- exchangers DEAE-cellulose and TEAE-cellulose in formamide and ethylene glycol. For example, at 30°C at least 20 mg protein was readily and completely adsorbed by 1 g resin (in all cases lyophilized from pH 6.0) in the following combinations: (i) ovalbumin (lyophilized from pH 6.0) and both ion-exchangers in both solvents, (ii) in- sulin (lyophilized from pH 8.0) and both ion-exchangers in formamide, and (iii) oxidized A and B chains of in- sulin (lyophilized from pH 8.0) and TEAE-cellulose in formamide.

Ion-exchange chromatography in water is greatly af- fected by the pH of the aqueous solution." We found that in nonaqueous solvents the analogous variable was the pH of the aqueous solution from which the resin had been lyophilized. One would expect that the higher the pH, the greater the net negative charge of a cation- exchanger, such as CM-cellulose, and hence the higher the affinity of the latter to a protein with a net positive charge. Indeed, while the binding affinity for lysozyme (lyophilized from pH 6.0) of CM-cellulose lyophilized from pH 6.0 in ethylene glycol is 280 mg/g (Fig. lB), when the resin was instead lyophilized from pH 2.0 or pH 10.0, the binding capacity under the same conditions dropped to zero or jumped to 430 mg/g, respectively. In principle, the pH of the aqueous solution of the protein prior to lyophilization should influence the adsorption behavior as well. This phenomenon, however, should be much less predictable than the situation with the resin because changing the pH, in addition to altering the net charge of the protein, will also affect the protein's con- formation and, in turn, adsorption characteristics. In agreement with this reasoning, when the pH from which lysozyme was lyophilized was lowered from 6.0 to 4.0 to 2.0 (thereby increasing its net positive charge and thus anticipated affinity to cation-exchangers), the binding capacity of CM-cellulose (lyophilized from pH 6.0) for the protein in ethylene glycol actually decreased from 280 to 150 to 110 mg/g resin, respectively.

Since many common inorganic salts are freely soluble in formamide and ethylene glycol, we decided to use them in order to desorb proteins from ion-exchangers in these solvents. Two representative salts, LiCl and NH4N03, easily dissolved in both solvents at 1M. At this concentration, lithium chloride and ammonium ni- trate completely desorbed lysozyme from CM-cellulose

in formamide and oxidized A and B chains of insulin from TEAE-cellulose in formamide; in addition, ribo- nuclease was completely desorbed from CM-cellulose by 1M NH4N03 in ethylene glycol.

Having established the feasibility of protein adsorp- tion onto and desorption from ion-exchangers in for- mamide and ethylene glycol in batch systems, we then proceeded to column chromatography of proteins in these solvents. Figure 2A depicts chromatographic sepa- ration of oxidized A and B chains of insulin on TEAE- cellulose in formamide. A 1 : 1 (w/w) mixture of the two peptides in formamide was loaded onto the column preequilibrated with that solvent and then washed with it. Selective desorption was achieved with a NH4N03 gradient (0.1-0.4M) in formamide. As seen in Fig- ure 2A, a satisfactory separation of the peaks for A and B chains was attained; subsequent high-performance liq- uid chromatography (HPLC) analysis revealed that the A chain fraction contained no B chain and vice versa.

E J 125 250

C a 0050 0 S 0

CI

B 0 C Q, .- c

2 0025 n

0 000

0 500 1000

Volume, ml Figure 2. Chromatographic separation of (A) oxidized A (0) and B (0) chains of insulin on TEAE-cellulose in formamide and (B) lysozyme (0 ) and ribonuclease (m) on phosphocellulose in ethylene glycol. A 13 x 2-cm glass column was packed with 6 g of one of the resins (both lyophilized from distilled water at pH 6.0). Following equilibration at 30°C with the respective solvent, 10 mg of a 1 : l (w/w) protein mixture (lyophilized from pH 8.0 in A and from 6.0 in B) dissolved in the same solvent was loaded, and then the column was washed with that solvent. In both cases, 2.5-mL fractions were collected and assayed for protein (A) or both for protein and lysozyme and ribonuclease activities (B). In A the proteins were eluted with a NH4N0, gradient (from 0.1 to 0.4M); the flow rate was 0.5 mL/min. In B the proteins were eluted with LiCl (0.45M and l M , see text); the flow rate was 0.3 mL/min.

COMMUNICATIONS TO THE EDITOR 577

The recoveries were 96% for the A chain and 98% for the B chain.

Figure 2B illustrates the protein separation of lyso- zyme from ribonuclease in ethylene glycol on the cation-exchanger phosphocellulose. A 1 : 1 (w/w) mix- ture of the two enzymes in ethylene glycol was loaded onto the column preequilibrated with that solvent and then washed with it. We found that 0.45M LiCl selec- tively desorbed lysozyme without eluting ribonuclease. Subsequent increase in the salt concentration to 1M afforded complete desorption of the second enzyme (Fig. 2B). Protein recoveries for lysozyme and ribonu- clease were 74% and 82%, respectively; recoveries of enzymatic activity were 70% and 78%, respectively.

This work demonstrates the ion-exchange chromatog- raphy of proteins in neat formamide and ethylene glycol. Combined with our previous study on protein separation and purification in neat dimethyl sulfoxide,* the results obtained validate the idea of downstream protein pro- cessing in these three protein-dissolving organic sol- vents. The immediate issue to be addressed in order to broaden the scope of our methodology is that of the mechanism of time-dependent enzyme inactivation in these media. If elucidated, this mechanism may be con- ducive to approaches to prevent this process, as in our recent investigation of subtilisin inactivation in several nonaqueous solvents.'6 In addition, we are currently applying our bioseparation methodology to the purifica- tion of membrane-bound proteins.

This research was supported by the National Science Foun- dation Biotechnology Process Engineering Center at the Massachusetts Institute of Technology. We thank Lucia Gardossi for the HPLC tests.

References

1 . Arnold, F. H . 1991. Metal-affinity separations: a new dimen- sion in protein processing. Bio/Technol. 9: 151-156.

2. Chang, N., Hen, S. J., Klibanov, A.M. 1991. Protein separation and purification in neat dimethyl sulfoxide (DMSO). Biochem. Biophys. Res. Commun. 176: 1462-1468.

3. Chisti, Y., Moo-Young, M. 1991. Large scale protein separa- tions: engineering aspects of chromatography. Biotechnol. Adv. 8: 699-708.

4. Cramer, S. M., Subramanian, G. 1990. Recent advances in the theory and practice of displacement chromatography. Separ. Purif. Meth. 19: 31-91.

5. Gordon, N. F., Moore, C. M.V., Cooney, C. L. 1990. An over- view of continuous protein purification processes. Biotechnol. Adv. 8: 741-762.

6 . Huddleston, J. G. , Lyddiatt, A. 1990. Aqueous two-phase systems in biochemical recovery. Appl. Biochern. Biotechnol. 26: 249-279.

7. Hummel, B. C.W. 1959. A modified spectrophotometric deter- mination of chymotrypsin, trypsin, and thrombin. Can. J. Bio- chem. Physiol. 37: 1393-1399.

8. Klyosov, A. A,, Van Viet, N., Berezin, I.V. 1975. The reactions of a-chymotrypsin and related proteins with ester substrates in non-aqueous solvents. Eur. J. Biochem. 59: 3-7.

9. Kunitz, M. 1956. A spectrophotometric method for the mea- surement of ribonuclease activity. J. Biol. Chem. 164: 563-568.

10. Ladisch, M.R., Willson, R.C., Painton, C.C. , Builder, S.E . (eds.). 1990. Protein purification: from molecular mechanisms to large-scale processes. ACS Symp. Series No. 427. American Chemical Society, Washington, DC.

11. Lillehoj, E. P., Malik, V. S. 1989. Protein purification. Adv. Biochem. Eng./Biotechnol. 40: 19-71.

12. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.

13. Michel, H. 1991. Crystallization of membrane proteins. CRC Press, Boca Raton, FL.

14. Rees, E. D., Singer, S. J. 1956. A preliminary study of the prop- erties of proteins in some nonaqueous solvents. Arch. Biochem. Biophys. 63: 144-159.

15. Sanger, F. 1949. Fractionation of oxidized insulin. Biochem. J. 44: 126-128.

16. Schulze, B., Klibanov, A.M. 1991. Inactivation and stabiliza- tion of subtilisins in neat organic solvents. Biotechnol. Bioeng. 38: 1001 -1006.

17. Shugar, D. 1952. Measurement of lysozyme activity and the ultraviolet inactivation of lysozyme. Biochim. Biophys. Acta 8:

18. Singer, S. J. 1962. The properties of proteins in non-aqueous solvents. Adv. Protein Chem. 17: 1-68.

19. Sluyterman, L. A. 1955. Electrophoretic behavior in filter paper and molecular weight of insulin. Biochim. Biophys. Acta 17: 169-176.

20. Trinder, P. 1969. Determination of glucose in blood using glu- cose oxidase with an alternative oxygen acceptor. Ann. Clin. Biochem. 6: 24-27.

21. Walter, H., Brooks, D. E., Fisher, D. (eds.). 1985. Partitioning in aqueous two-phase systems: theory, methods, uses, and ap- plications to biotechnology. Academic, New York.

302-309.

578 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 39, NO. 5, MARCH 5, 1992