1. Field of the Invention
It is well known that the information required for the 3-dimensional organization of polypeptides and proteins (herein referred to collectively as "polypeptides"), such as, without limitation, hormones, enzymes, antibodies and virus coats into biologically active structures is present within the amino acid sequence of the polypeptide and if the latter can be placed in a suitable environment the biologically active structures will generally form spontaneously by folding of the amino acid chain(s). The resulting structures are often stabilized by one or more disulfide cross-links formed from sulfhydryl (thio, mercapto) residues in some of the sulfur-bearing amino acids. Unfortunately some biosyntheses, otherwise generally advantageous, result in the production of difficulty soluble aggregates. This is the case, for example, in the production of so-called refractile bodies during biosyntheses by modified E. coli of foot-and-mouth-disease (FMD) virus coat, insulin, some human interferons, human-interleukin 2, porcine-growth-hormone (pGH), urokinase, bovine-growth-hormone (bGH), hepatitus-B-surface-antigen (HBsAg), tissue plasminogen activator (TPA), human-growth-hormone (hGH) and prorennin. Such polypeptide aggregates are often solubilized in aqueous denaturant (chaotropic, lyotropic) solutions (most popularly 4 to 9 molar aqueous solutions of guanidine hydrochloride (guanidinium chloride, "Gu.HCl"), less popularly in solutions of sodium thiocyanate or urea (typically 4 to 9 molar) or in sodium dodecyl sulfate (typically 0.1 to 2.0 percent by weight)) containing typically small amounts of reducing agents, for example sulfhydryl compounds such as beta-mercapto-ethanol ("BME"), dithiothreitol ("DTT") or glutathione ("GSH") to create oxidation-reduction ("redox") potentials sufficient to reduce disulfide links to sulfhydryl residues. In such solutions the polypeptides apparently have little of the structural organization present in the biologically active polymer although the amino acid sequences remain intact.
Biological activity may also be lost from polypeptides (having the correct amino acid sequence for activity) as a result of folding errors introduced during biosynthesis or as a result of the recovery methods used. In such cases it may be desirable to unfold the polypeptides, for example by dissolving them in denaturant solutions generally containing a reducing agent for disulfide links.
In any of the above cases the polypeptide is allowed spontaneously to refold by decreasing the denaturant power (chaotropism, chaotropy) of the chaotropic agent, generally while maintaining conditions under which disulfide links between sulfur bearing amino acids either do not form or (preferably in the case of renatured polypeptides which are stabilized by disulfide links) are in dynamic equilibrium with the precursor amino acid sulfhydryl residues. It is known that the formation of permanent disulfide linkages during the process of refolding may also be inhibited by converting a substantial fraction of the amino acid sulfhydryl and/or disulfide moieties to S-sulfonate groups by reaction of the polypeptide dissolved in chaotropic solution with a sulfite salt and a mild oxidizing agent such as a tetrathionate salt. (The preferred redox potentials for maintaining predominantly S-sulfonate groups are of course different from those potentials which will maintain predominantly sulfhydryl groups.) Following at least partial reduction of the chaotropy and substantial refolding the S-sulfonate groups are removed by reaction with a soluble sulfhydryl compound, preferably in the presence of small amounts of disulfide compound, air, oxygen and/or a mild oxidizing agent.
Decreasing the denaturant power or activity of chaotropic solutions is commonly accomplished by dilution, dialysis or buffer exchange (for example by dialysis against a concentrated solution of a suitable buffer, by diafiltration or by gel permeation chromatography). It is found however that such processes, known in the prior art, used to renature unfolded polypeptide typically result in substantial precipitation of the polypeptide or incorrect refolding thereby reducing substantially the yield of biologically active materials.
It is therefore an objective of this invention to provide processes which result in increased yield of biologically active (renatured) polypeptide from solution in denaturants. This and other objectives will become clear from the brief description of the drawings and description of preferred embodiments herewith.
2. Description of the Prior Art
Electrodialysis ("ED") has become an accepted process and apparatus for transferring ions from one solution to another. The state of the art is well described in pages 726 through 738, Volume 8, Kirk-Othmer Encyclopedia of Chemical Technology, 3d Edition, Wiley, N.Y. 1979. Typically one to several hundred repeating groups of spaced electrolytically conducting membranes are positioned between a single pair of electrodes. Typically in each group of membranes at least one membrane has an electrolytic (Hittorf) transport number for ions of one charge sign substantially different from the transport number of such ions in the solutions to which the ED apparatus is applied. Solutions to be processed are introduced into narrow spaces (compartments, chambers, cells) between the membranes and between the electrodes and the membranes adjacent thereto. A direct electric current (which may have some alternating current component) is applied between the electrodes causing ions to be transferred through the membranes. In the case of ED apparatus operating in a diluting/concentrating mode in which the repeating group comprises two membranes, electrolyte enriched solution is withdrawn from every other compartment (enriching, concentrating, brine, receiving or rinsing compartment) and electrolyte depleted solution from the intervening compartments (depletion, dilution, diluate, dilute, donating, demineralizing, desalting compartments). In the case of ED apparatus operating in a metathesis mode in which the repeating group of membranes comprises three (single decomposition) or four (double decomposition) compartments ions are interchanged among the compartments but the total concentration of electrolyte (expressed in electrical equivalents per unit volume) in each compartment typically remains substantially unaltered.
Ions passing through the above described membranes are accompanied by (electroosmotic) transport of water, typically 4 to 10 moles of water through each membrane per electrical equivalent of ions transferred. Such (specific) water transport through a membrane depends upon the structure of the membrane, on the concentration of electrolyte in the solutions bathing the membrane and upon the current density applied. If the solutions bathing each side of the membrane are both very concentrated then it may be that the ratio of ions to water passing through the membrane is about the same as the ratio in the solution in the compartment donating said ion. In such case ED can result in substantial transfer of solution from the ion donating compartment(s) to the ion receiving compartment(s) without substantial reduction in the concentration of said ions in the nominal donating compartment(s). For example, during ED in desalting mode of a solution originally having about 7 gram-equivalents of Gu.HCl per liter to about 6 gram-equivalents per liter against a rinsing solution having a log-mean concentration of about 0.6 normal, about 70 percent of the volume of the dilution solution was electrolytically transferred to the rinsing stream and about 76 percent of the Gu.HCl, i.e. the solution transferred had a concentration of about 7.3 gram-equivalents per liter. In contrast, during the electrodialysis desalting of a solution originally having about 2 gram-equivalents of Gu.HCl per liter to about 0.33 gram-equivalents per liter (i.e. about 76 percent removal of Gu.HCl) against a rinsing stream having a log-mean concentration of about 0.3, about 50 percent of the volume of the dilution stream was transferred and the solution transferred had a concentration of about 2.7 gram-equivalents per liter.
As mentioned above, ED can also be used in "metathesis" modes in which the ionic composition of a solution can be altered without substantially changing the ionic strength of a solution. For example if a solution of NaCl is introduced to an ED chamber bounded by cation selective ED membranes, Na.sup.+ ions can be replaced in part or substantially completely by other cations, the concentration of Cl.sup.- anions being substantially unaltered. On the other hand if such NaCl solution is introduced to an ED chamber bounded by anion selective ED membranes Cl.sup.- ions can be replaced by other anions without substantially altering the concentration of cations.
It is known in the prior art to decrease the concentration of a solution of a strong denaturing agent and a potentially biologically active polypeptide by adding either water or a buffer to the solution, effectively diluting both denaturant and polypeptides. The method typically results in substantial loss of active polypeptide through precipitation and/or incorrect refolding.
It is also known to decrease the concentration of a strong low molecular weight denaturant in a solution thereof containing potentially active polypeptide by dialysis in which the denaturant diffuses through a semipermeable membrane into water or a buffer. The transfer of polypeptide is inhibited by the membrane. This method also typically results in unsatisfactory yield of biologically active polypeptide.
Diafiltration with either water or a buffer also typically results in unsatisfactory yield of active polypeptide.
USPTO Backgrounds
USPTO Backgrounds