IE84354B1 - Purified, biologically active, bacterially produced recombinant human CSF-1 - Google Patents

Description

PATENTS ACT, 1992 /0213 PURIFIED, BIOLOGICALLY ACTIVE, BACTERIALLY PRODUCED RECOMBINANT HUMAN CSF-1 CHIRON CORPORATION Technical Field The purification recombinant proteins in activity. In procedures which possible the production of biologically active, forms of CSF-1 bacterial hosts expressing genes encoding the monomer. relates to refolding of invention processes for and bacterially produced forms having high particular, it specific biological concerns make dimeric from Background Art Colony stimulating factor-1 (CSF-l) is one of several proteins which are capable of stimulating colony formation by bone marrow cells plated in semisolid culture medium. CSF-1 is distinguished from other colony stimulating factors by virtue of its ability to stimulate these cells to become predominantly macrophage colonies. Other CSFs the colonies which consist of neutrophilic granulocytes and macrophages; predominantly neutrophilic granulocytes; or neutrophilic and eosinophilic granulocytes and A review of these CSFs has been published stimulate production of macrophages.

The characteristics of native human CSF-1 are complex, and in fact it is not yet clear what form of CSP-1 is active in the human body. Soluble forms of naturally-produced CSF-1 have been purified to various degrees from human urine, mouse L-cells, cultured human (MIA Paca) cells, and also from various human and mouse lung cell conditioned media, T—1ymphoblast cells, and from human placental-conditioned mediwn. Many, if not all of the isolated native CSF-1 proteins appear to be glycosylated There the molecular weights pancreatic carcinoma from human The existence of "native-like" CSF-1 reference variety in the monomeric isolated from human urine is of proteins is important because these proteins provide standards against which to compare the quality and biological activity of refolded recombinant forms of CSF-l. For relied upon the soluble CSF-l produced by the Mia Paca cell line as well this purpose, we have eprotein of as properties of other highly purified CSF-l molecules been described in the literature. The these purified which have specific activity of "native-like" reference proteins has typically fallen in the range of 4 to 10 x 107 units per mg (as measured by in vitro mouse bone marrow colony-forming assays).

CSF-l has also been produced from recombinant DNA using two apparently related CDNA clones: (1) a “short” which when translated, produces a monomeric protein of 224 amino amino acids, also preceded by the The long form has been cloned and expressed, by two groups, as disclosed in Ladner, M.B., et al, The EMBO J (1987) 6(9):2693-2698, -amino acid signal sequence. and Wong, G., et al, Science (1987) 235: 1504-1509. (The DNA and amino acid sequences for both "short" and "long" forms are shown in Figures 5 and 6, respectively; however, the 32 amino acid signal sequence is incomplete as illustrated in Figure 6.) The long and short forms of the CSF—l-encoding DNA appear to arise from a variable splice junction at the upstream portion of exon 6 of the CSF-l-encoding DNA. when CSF-1 is expressed in certain eucaryotic cells from either the long or short cDNA appears to be variably processed. at the C-terminus and/or variably glycosylated. genomic forms, it Consequently, CSF-l proteins of varying molecular weights are found when the reduced monomeric form is analyzed by Western analysis. ‘essential for full CSF-l activity.

The amino acid sequences of the long and short forms, as predicted from the DNA sequence of the isolated clones and by their relationship to the genomic sequence, are identical with respect to the first 149 amino acids at the N-terminus of the mature protein, and diverge thereafter by virtue of the inclusion in the longer clone of an additional 894 bp insert encoding 298 Both allow additional amino acids following glutamine 149. forms of the with the shorter and‘ longer gene expression of proteins sequences identical regions at the C—terminus, as well as at the Biologically CSF-l has recovered when CDNA encoding through the first 150 or containing N-terminus. active been 158 amino acids of the short form, or through the first 221. amino acids of the longer form, is expressed in eucaryotic cells. if not all, of the native secreted CSF-l molecules are glycosylated and dimeric, Since most, significant posttranslational processing apparently occurs in vivo. Given the complexity of the" native CSF-l molecule, express the CSF-1 gene organisms. It seemed unlikely that active protein would be obtained when the convenient bacterial hosts, it has been considered expedient to in cells derived from higher gene was expressed in more such as E. coli. Bacterial hosts do not have the capacity to glycosylate proteins, nor are their intracellular conditions conducive to the refolding, disulfide bond formation, and disulfide—stabilized dimerization‘ which Thus, production of recombinant CSF-l in E. coli has, prior to is apparently experimental invention, resulted in this protein of activity, although its identification as monomeric CSF-l very low ’ difficult had been readily confirmed by immunoassay, N-terminal sequencing, and amino acid analysis.

It is by now accepted that inactive forms of recombinant foreign proteins produced in bacteria may require further them useful for the intended. in order to render for which "refolding" steps purposes they are As a dimeric protein containing a large and disulfide bonds, CSF-l represents a particularly challenge for production bacterial Often, recombinant produced in including CSF-l so produced, are in the form of insoluble number of cysteines which are required for activity, from systems. proteins E. coli, highly referred to as intracellular protein precipitates inclusion bodies or refractile bodies. inclusions can readily be separated from the soluble bacterial proteins, but then must be solubilized under conditions which result These in essentially complete denaturation of the protein. Even secreted proteins from bacterial sources, while not necessarily presenting the same solubility problems, may require considerable activity. Each different refolding protocol in order to achieve full biological activity. manipulation in order to restore different protein may require a A number of papers have appeared which report refolding attempts for individual proteins produced in bacterial hosts, or which are otherwise in denatured or non-native form. A representative sample follows.

Reformation of an oligomeric enzyme after denaturation by sodium dodecyl sulfate (SDS) was reported by Weber, K., et al, J Biol Chem (1971) :4504—4509. problem created by the binding of proteins to SDS, and This procedure was considered to solve a the process employed removal of the denatured protein from SDS in the presence of 6 M urea, along with anion exchange to remove the SDS, followed by dilution from urea, all in the presence of which aspartate reducing agents. The least refolded B-galactosidase, proteins were - at partially included: transcarbamylase, rabbit bacteriophage R-17.

Light, A., in Biotechnigues (1985) 3:298-306, describes a variety of attempts to refold a large number muscle aldolase, and coat protein from of proteins. It is apparent from the description in this reference that the techniques which are applicable individual to the concerned. In are highly particular protein fact, in some cases, refolding significant amounts of particular proteins has not been possible and the results are quite unpredictable. In addition, refolding procedures for recombinant urokinase material was dissolved in 8 M urea or 5 M guanidine hydrochloride, and the rearrangement of disulfides was facilitated by use of a buffer containing a glutathione redox system. Recombinant human immune interferon, refolded to chaotropic which has no disulfide bonds, has been generate a more active preparation using absence of thiol-disulfide (PCT application WO 86/06385). In example, bacterially synthesized granulocyte macrophage agents in the exchange reagents another colony-stimulating factor (GM-CSF), a member of the CSF group, was also produced in E. coli and refolded after This CSF CSF-l, since GM-CSF has a distinct amino acid sequence solubilization in 6.M urea. is unrelated to and is also monomeric.

Use of refolding procedures to obtain reconstitution of activity in multimeric proteins has also been described by Herman, R.H., et al, Biochemistry for immunoglobulins. An employ denaturation and the use of appropriate oxidizing and reducing agents or sulfitolysis reagents. A related approach employs the catalyst thioredoxin, and is disclosed by Pigiet, V.P., Certain aspects of solubilization, purification, and refolding of certain recombinant proteins produced as refractile bodies in bacteria are also disclosed in U.S. 4,511,562; 4,511,503; 4,512,912; 4,518,526 and EPO publication 114,506 (Genentech).

The foregoing references are merely representative of a large body of literature which, when taken‘ together, shows individual steps in protocols which may be modified and combined in various sequences to obtain tailored individually procedures for particular subject proteins produced in accordance with that retailoring of the overall procedures to fit a specific particular expression systems. It is evident case is a requirement for producing refolded. product with full biological activity in useful amounts. published procedures describe a step for successful refolding of For example, a number of the the recombinantly produced protein. It is not clear from these references, but is known in the art, that the starting material for refolding may exist in a variety of forms, depending on the nature of the expression system used. In the case of bacterial expression, it is, however, clear that the product is not glycosylated, and that, in addition, production of an intracellular disulfide-bonded dimeric product reducing environment in bacterial cells. is prevented by the Currently the most common form of recombinant starting material for refolding is an insoluble protein which protein intracellular, is produced by expression of a gene for mature or bacterial fusion protein, lacking a functional signal sequence, under the control of standard bacterial promoters such as trp or PL. Because recombinantly produced products in bacteria are produced in high reducing environment, and because typically the constructs do not enable the bacteria to secrete the recombinant protein, concentrations in a these often observed to form insoluble inclusion bodies. signal sequences which function in foreign proteins are However, including the E. coli penicillinase U.S. Patents bacteria are known, sequence disclosed by Gilbert et al, 4,411,994 and 4,338,397, the B. licheniformis sequences disclosed by Chang in U.S. Patent 4,711,843 and 4,711,844, and the phosphatase A signal sequence (phgg) disclosed by Chang, et al, pen? Nos. in European Patent Publication No. 196,864, published 8 October 1986. Secretion can be effected in some strains. However, if Gram-negative hosts are used, complete secretion may not and the protein may reside in the periplasmic space. Nevertheless, it is likely that proteins expressed under control of promoters and signal occur, much more sequences such as phgg will be produced in soluble form if they are capable of refolding and forming required disulfide bonds disclosed hereinbelov are expected to be of in the extracellular environment. The methods value for both intracellular and secreted products where refolding is required.

Nowhere in literature is a specific process described for the preparation of biologically active dimeric CSF-1 from bacteria. The present invention describes several refolding procedures involving CSF-1 proteins of various primary structures. The resulting refolded CSF-1 proteins are fully active and soluble, and the various molecules differ sufficiently in physical properties that they may be expected to exhibit a variety of pharmacokinetic and/or pharmacological properties when used therapeutically in_t/i_\1_o.

Disclosure of the Invention Accordingly the present invention provides an isolated and purified, recombinant, unglycosylated and dimeric CSF-1, said dimeric CSF-1 being biologically active and essentially endotoxin and pyrogen~free, said dimeric CSF- 1 consisting of two monomeric human CSF-1 subunits, said two monomeric subunits being the same or different, with the proviso that when said two monomeric subunits are the same, said monomeric subunits are an NV2 or an NV3 deletion mutein of human mature CSF-1.

The subunits may be different.

Alternatively, the two monomeric human CSF-1 subunits may be the same and are an NV2 or an NV3 deletion mutein of human mature CSF-1.

One or both of said monomeric human CSF-1 subunits may comprise a human LCSF or an NV2 or an NV3 truncated mutein thereof, and optionally a two monomeric human CSF-1 tyrsg, serm, S9f15g or ser157ser.59 form thereof. The LCSF or an NV2 of NV3 truncated mutein thereof may also have a truncated carboxy terminus that is selected from the group consisting of CV150, CV190, CV221 and CV223.

Alternatively one or both of the monomeric human CSF-1 subunits may comprise a human SCSF or an NV2 or an NV3 truncated mutein thereof, and wherein the residue at position 59 is optionally Asp. The SCSF or said NV2 or NV3 truncated mutein thereof may have a carboxy truncated terminus that is selected group consisting of CV15O CV158. from the and One or both of said monomeric human CSF-1 subunits in the dimeric CSF-1 of the invention may be selected from the group consisting of LCSF/NV3CV221 , as,,59SCSF/NV3CV150, as,,59SCSF/NV3CV158, se,157LCSF/NV3CV221, 5er159LCSF/NV3CV221 and ser157ser159LCSF/NV3CV221.

The dimeric CSF~1 of the invention may comprise refolded CSF-1.

The invention also provides a clinically pure, biologically active refolded CSF-1 dimer comprising a dimeric CSF-1 as hereinbefore defined having an endotoxin content of less than 1.0 ng/mg of CSF-1 and substantially free of pyrogens, the dimeric CSF-1 from CSF-1 recombinantly in bacteria. being prepared produced The invention also includes a biologically active refolded human CSF-1 dimer comprising two monomeric units selected from the group consisting of LCSF monomers and muteins and C- or N-terminal truncations thereof, and SCSF monomers and muteins and C- or N-terminal truncations thereof, and wherein the monomeric units of said dimer are not identical.

The invention also provides a composition comprising the dimeric CSF-1 as hereinbefore defined, optionally in admixture with a pharmaceutically acceptable excipient.

The invention includes a dimeric CSF~1 as hereinbefore defined tor use as a pharmaceutical.

Brief Description of the Drawings Figure 1 shows the partial purification of one type of monomeric CSF-1 using molecular sieve chromatography.

Figure 2 shows the extent of dimerization as assayed using molecular sieve chromatography.

Figure 3 represents RP—HPLC analysis. of one refolded recombinant: E type of CSF-1. denatured and . coli Figure 4 shows a spectral analysis to determine the solubility of one type of denatured and refolded recombinant E. coli CSF-1.

Figure 5 shows the cDNA and deduced amino acid sequence for a cDNA clone encoding a "short" form of human CSF-1 designated pcCSF-17.

Figure 6 shows the CDNA and deduced amino acid sequence ‘for a» CDNA clone encoding a human CSF-1 designated pcCSF-4. i Figure 7 shows the results of a reducing and nonereducing SDS-PAGE analysis of dimeric asp59SCSF/CVl50 csr—1. "long" form of Modes of Carrying out the Invention A. Definitions As used herein, 'chaotro ic environment" P refers to an environment which contains appropriate chaotropic agents, such as urea in concentration to sufficient disrupt the tertiary structure of is maintainad at a temperature or other condition which causes such disruption. proteins, or which Chaotropic agents or conditions such as temperature and pH may disrupt structure in a variety of ways, including the disruption of hydrogen bonds. Suitable chaotropic include 2-8 M urea,. 4-7 M guanidinium, detergents such as SDS at concentrations around 0.1% by environments weight, and acids such as acetic acid at concentrations of about 1 M, basic conditions of, e.g., pH 11 and above, and elevated temperatures. When placed in ‘a chaotropic enyironment, the normal physiological conformation of proteins may be reversibly as well as irreversibly altered, "unfolded" to and the primary structure may be varying degrees, depending on the concentration of the chaotropic agent and the degree of It should be understood that agents and/or conditions which create severity of other chaotropic conditions. chaotropic environments can be used in combination or in sequence. For example, mixtures of chaotropic agents can be used, or the CSF-l may first be placed in a chaotropic environment created by one chaotropic agent, and then subjected to a second chaotropic environment created by another agent or by temperature.

As used herein, "reducingwagent" specifically refers to a reducing agent which is capable of reducing disulfide linkages to sulfhydryl groups. mildly conversion A variety of reducing materials capable of effecting this is available, but the most common comprises thiol-containing moieties such as B-mercaptoethanol or dithiothreitol. Additional functional reducing agents include reduced glutathione and free cysteine itself.

While emphasis is placed on thiol-containing compounds, any material which is capable of the disulfide to thiol conversion reactions is without undesirable side included in this definition. conditions the CSF-1 If the CSF-l is produced in an environment which places it initially in "Reducing conditions” refers to which maintain or place, as the case may be, protein in the monomeric reduced form. reduced form (i.e., the cysteines are in said form, not cystine) milder conditions may suffice than would be required if the protein were initially in oxidized form.

"Refolding conditions" refers to conditions wherehi a denatured protein is permitted to assume a conformation associated This with physiological activity. specifically includes formation of disulfides and/or association into dimeric or multimeric structures which are functionally identical to those of the native protein. Such conditions include slow removal of or step-wise dilution of chaotropic agents in the presence or absence of agents which permit the formation of disulfide bonds present in the conformation. If high concentrations of chaotropic agents are used for solubilization, or if the protein is otherwise denatured by virtue of these normally active agents, the chaotropic substances included in the chaotrope may be removed by simple dilution, by dialysis, by hollow fiber diafiltrafibn, or by a number of other means known in the art by which the concentration of small molecules may effectively be lowered, with or in the without a corresponding decrease concentration of the protein.

It is desirable to promote disulfide formation during this process. bond This can be accomplished by air oxidation or by including reagents suitable for this purpose in the refolding conditions. include Such reagents 'redox systems‘ which permit the continuous oxidation and reduction of the thiol/disulfide pairs.

One of the most commonly used of glutathione, these systems is in both oxidized and reduced forms. It is known that oxidized glutathione and reduced glutathione are naturally occurring constituents of mammalian cells and may, in fact, in addition to or in conjunction with isomerases catalyzing this reaction, promote thiol/disulfide bond exchange in vivo (Tietze, F Biochem (1969) g1:so2). other pairs of (disulfide) and reduced (thiol) reagents may also be indeed, the disulfide and thiol need not be from the same molecule. In addition, new disulfide bonds may be formed by sulfitolysis, followed by oxidation of the sulfonated thiol groups. This ., Anal oxidized used; derived process is described in U.S. Patent 4,620,948 to Builder et al, sup a.

The purification methods referred to herein include a variety of procedures. which Among several types useful are size may be fractionation using molecular sieve chromatography; ion-exchange chromatography under suitable conditions; affinity chromatography using, for example, monoclonal antibodies directed to the biologically active form of the protein; adsorption chromatography using nonspecific supports, such as hydroxyapatite, silica, alumina, and so forth; and also gel-supported electrophoresis.

CSF-l, such as using phenyl-Sepharose or phenyl-TSK, has been shown to be particularly useful. In addition, purification of CSF—l ion-exchange chromatography (such as DEAE-Sepharose chromatography) has been shown to be a particularly effective procedure to increase the purity of the dimeric CSF-l protein.

In the case of hydrophobic interaction chromatography, initial monomeric using These purification techniques are, in a general sense, well known in the art, and a detailed description of the of their specific application to CSF-l proteins is described in the examples below. pecularities As used herein, "biologically active‘ means a preparation of human CSF-l produced recombinantly in bacteria which has essentially the same specific activity in human and mouse bone marrow colony—forming assays as native human CSF-l produced by mammalian cells.

"Clinically pure" CSF—1 means a preparation of biologically active human CSF-l produced recombinantly in bacteria which is 95% csr-1 either by RP—HPLC or by either reducing or non-reducing SDS-PAGE at least _l5- than 1.0 ng/mg CSF-1 as assayed by standard LAL assay. and has an endotoxin content of less about B. CSF-1 Proteins As set forth in the background section, CSF-l is biologically active in its dimeric form. It has been possible to obtain encoding CSF-l monomers consisting of a variety of amino acid sequences and lengths. recombinant DNA Figures 5 and 6, respectively, show the DNA and amino acid sequences for the short and long forms, both of which are preceded by a 32-amino acid signal sequence. The sequences of monomeric CSF-l protein are considered herein for convenience to be the 224-amino-acid short form (SCSF) and the 522-amino-acid long form (LCSF) shown in these figures.

Plasmids encoding a variety of CS?-l forms are currently available, and can be expressed in bacterial As described the gene encoding the long form of CSF-l can be expressed in its systems. immediately above, entirety, or the gene can be truncated to express C-terminally deleted forms. In addition, the first two or three N-terminal codons can be deleted so that the resulting protein is more homogeneous. Specifically, the N-terminal methionine encoded upstream of the mature (which unless native sequence N-terminus is retained in the protein as "N-terminal met" removed by post- translational processing), has been found to be more readily removed from these N-terminal deletion constructs. Furthermore, significant heterogeneity (resolvable by RP-HPLC analysis of the reduced monomer) is found when the gene encoding the “native” N-terminal (for SCSF/CV150) is eliminated when the corresponding CSF-l gene lacking the short This sequence example, of the form, mutein expressed. heterogeneity is two glutamic acid N-terminal codons is expressed.

Correspondingly, N-terminal truncated forms of other short and long CSF-l gene constructs can also be primary monomeric various notation, as follows: tyrosine residue at position 59, defined by the genomic clone has been found to encode Asp59SCSF denotes a mutein of the disclosed short form having this (The disclosed LCSF clone encodes Asp at aspartic acid at that position. Therefore, modification. position 59.) substitutions within the "native" sequences depicted are Muteins corresponding to amino acid correspondingly designated by the substitution subscripted with the position. Mutein forms of CSF-l are disclosed in European Patent Application No. 87309409.8, (EP 0 272 779 A2) filed 23 October 1987. when constructs putatively encoding these proteins are expressed as mature proteins in bacteria, they may also retain an N-terminal methionine. Since the presence or absence of .the N-terminal methionine cannot be predicted, this possibility is not included in the notation. -17..

C-terminal and N-terminal truncations of these basic SCSF and LCSF sequences will be designated as CV or NV, The C-terminal deletions will be followed by the number representing the number of amino acids of the respectively. native structure remaining; for the N-terminal deletions, NV will be followed by the number of amino acids deleted from the N terminus.

LCSF/CVl5O denotes a construct encoding a protein which contains the first 150 amino acids of the long CSF SCSF/CVl5B construct encoding a protein which contains the first 158 amino SCSF/NV2 denotes a construct encoding the short form with two N-terminal (As set forth above, the LCSF and SCSF diverge beginning at position 150 and reconverge near the C-termini.) LCSF/NVZCVISO denotes a form which is the LCSF/CVlS0, that the two N-terminal glutamic acid residues are deleted.

Thus,'for example, sequence; denotes a acid residues of the short form; amino acids deleted. same as except Particularly preferred constructions which result in CSF-1 proteins subjected to the process of the invention, include genes encoding LCSF/CVl50, LCSF/CVl90, LCSF/CV22l, LCSF/CV223, LCSF, and their corresponding NV2, NV3, tyr5g, ser157, serlgg, and ser157ser15g forms. Also preferred are SCSF/CVl5B, SCSF/CVISO, SCSF, and their corresponding NV2 and NV3 and asp59 forms.

Particularly preferred starting materials include the products of the genes encoding SCSF/NV3CVl50, LCSF/NV3CV22l, ser157LCSF/NV3CV22l, ser157LCSF/NV3CV22l, and ser157ser159LCSF/CV22l.

The resulting proteins may or may not retain the length prescribed by the gene, due to processing by various host systems used for expression. Therefore, although the starting material proteins for refolding are referred to by the same designation, it should be understood that these designations, in reality, refer to the gene the length of the starting material for the process disclosed herein may it has N-terminal Met) than construct, and actual be shorter or longer (if that specified by the C-terminal amino acid number.

C. General Procedure The starting material for the procedure of the invention is CSF-l CSF-l-encoding DNA transformed The CSF-l gene can be expressed as a mature protein by utilizing the appropriate CSF-l-encoding DNA which is immediately preceded by an ATG Met-encoding codon or as a fusion protein wherein the CSF-1 sequence is placed in reading frame with a protein-encoding sequence, or in a secreted form by utilizing a signal sequence which is functional in the selected host. If the construct encodes the "mature" form of the protein, the N—terminal not at all, or protein produced - from the into a bacterial host. methionine may be processed entirely, partially; Methionine is, of course, not present at the N-terminus of secreted forms expressed from genes having operably linked signal sequences. Signal sequences are generally those derived from bacterial systems such as penicillinase or phosphatase A. If the secreted form is employed, whether or not secretion is successful, generally the protein is produced in a form more soluble than that obtained when produced as a mature or not without fusion protein. This generalization is exceptions.

If the secreted protein is already soluble, the chaotropic environment may be needed, nonetheless, to affect the refolding procedure. If the protein is formed in insoluble form, initial solubilization is required.

In general, therefore, the process begins with the solubilized monomer in a chaotropic environment, which Such maintenance may involve the use of a suitable reducing is maintained under reducing conditions. agent such as B-mercaptoethanol or dithiothreitol (DTT) but the CSP—l may already be reduced, and exclusion of The solubilized for example, 8 M urea or 7 M guanidinium hydrochloride, at a pH of about oxidizing agents may be sufficient. protein is typically maintained in, 7-8.6, in the presence of about 2-100 mM thiol compound.

Starting with this solubilized form, the monomer may either be refolded directly or partially purified from remaining proteins by a suitable purification procedure such as chromatography on an adsorbent gel, chromatography using an ion exchange column, or gel- permeation chromatography prior to refolding. Use of a purification step prior to refolding has the advantage of removing contaminating host proteins and inaterials that CSF-l. chromatography is useful, as it permits an easy size may degrade or alter Gel-permeation separation of the desired monomer length, which is generally known in advance, from impurities of differing molecular weights. As the volume of materials increase, the capacity of gel-permeation columns becomes limiting.

For larger volumes, ion exchange chromatography, for is preferable. It is purification be conducted example, DEAE chromatography, that the reducing conditions in order to prevent the formation of Thus, used, a required under disulfide-linked aggregates. regardless of the chromatographic procedure suitable reducing in the solutions used to in the agent is preferably included load the chromatographic columns or batches and eluting solutions. In some instances, low pH, such as pH 6, may be substituted for the reducing agent, as low pH will essentially prevent disulfide bond formation in some chromatographic systems, even hi the absence of reducing agent.

The partially purified monomer is then subjected to refolding conditions for the formation of the dimer. The protein concentration during this step is of considerable importance. Final percent yields of dimer per volume of refolding reaction are increased if the protein concentration is less than about 2 mg/ml of the CSF-1 protein; mg/ml is preferred. The use of protein concentrations high higher-order a concentration range of 0.03-0.5 which are too may result in formation of The refolding conditions may include gradual removal of the chaotropic undesirable oligomers. environment over an appropriate time period (usually several hours) or dilution of the sample to the desired concentration of protein and chaotropic agent. Also possible are methods which provide a constant protein concentration, such as dialysis or hollow fiber diafiltration while the chaotrope is slowly removed. At the end of the process, when the chaotropic environment is depleted, a nondenaturing level is reached. For example, if guanidine hydrochloride is chaotropic agent, about 2 M, urea is used as a final concentration of less than and preferably 0.1-l M used as the is attained and if chaotropic agent, a final concentration at less than about 1. M, .1-0.5 M, and preferably is attained.

The environment refolding during removal of chaotropic is conducted in a manner so as to permit oxidation of the sulfhydryl groups to disulfides in order to establish the resultant biologically active dimeric configuration which, in the case of CSF-1 is stabilized by the formation of disulfides, one or more of which may link the two chains. Intrachain disulfides are also formed. Suitable redox conditions which encourage this formation of dimer include the sulfhydryl/disulfide reagent combinations, such as oxidized and reduced glutathione. The ratio of reduced to oxidized glutathione or other sulfhydryl/disulfide typically from about 2. mH/0.1 mM to Alternative methods for providing this combination is 0.5 mM/l.0 mM. oxidation are also acceptable. For example, simple removal or dilution of the reducing agent without precautions to exclude air and metal ions affect formation of desirable disulfide linkages. In any event, the pH of the solution during the refolding process should be maintained at about pH 7.5-9.0. It is clear that in the process of refolding, the highly reducing conditions under which the initial purification was conducted are no longer employed. Minimizing the concentration of salts, such as sodium chloride, during the refolding process, permits the use of ion exchange and/or subsequent concentration chromatography as a purification step.

During the refolding process, several dimeric and higher oligomeric species of CSF-l may be formed including those which have lowered solubility in high salt and higher order oligomers which can be resolved by size exclusion chromatography. This aggregation process is minimized through temperature control, temperatures of about 0-4°C are preferable to higher wherein low temperatures of 2S~37°C.

Less stable dimeric forms of CSF-l which can be resolved as an early eluting peak on reverse-phase HPLC under certain conditions may also form during the refolding process. These less stable forms may result disulfide bonds.

Cysteine residues at positions 157 and 159, present in CSF—l, are not DNA constructs from the formation of undesirable long form required for biological activity. encoding CS?-l containing serine substitutions for one or both of these cysteines produce higher yields in the present purification process and may also change solubility characteristics in a desirable fashion.

Residual redox reagents if present in refolded’ CSF-l may generate problems during subsequent purification steps. There are many ways to block or prevent the disulfide exchanges which might occur in the presence of such residual redox reagents (e.g., example, diafiltration or dialysis; dilution; and lowering the pH glutathione) including removal by, for of the solution appropriately. Of the above procedures, two of the more preferred procedures are lowering the pH to below pH 7.0 and diafiltration.

After and/or the initial purification steps are completed, the dimer is refolding, concentration further purified from residual redox material and from other proteins using procedures similar to those set forth above for the monomer. It is, of course, not necessary to choose the same purification procedure; indeed it may be preferred to use a different approach than that employed for solubilized monomer purification.

Suitable means, in particular, include gel filtration, hydrophobic interaction chromatography, ion exchange chromatography, and reverse—phase HPLC.

For example, prior to further purification of the refolded, CSF-l, material, if present, and concentration of the refolded dimeric removal of the redox proteins may be performed by direct loading of the refolded material onto an ion-exchange chromatography column using, for example, DBAE Sepharose. at pH's however, lowering the pH into the range of 5.5 to 7.0 Frequently, such procedures are carried out around 8, was found to reduce oligomer formation and increase yield of dimeric CSF-l.

The purification of the dimer is required to remove impurities, in particular, pyrogens or other endotoxins xhich result from the bacterial production of the protein. A particularly successful protocol for removal of these undesirable impurities uses phenyl-TSK chromatography chromatography on a or The conditions and with reagents which are endotoxin-free.

The desired dimeric CSF—l is soluble and‘ stable approximately 1.5 M ammonium sulfate at neutral pH, and phenyl-Sepharose column. is carried out under is loaded onto the columns under these conditions at low temperatures, of about 2°C-10°C, 4°C. In addition, refolded CSF-l dimeric and preferably about aggregates and unstable forms of stable of a are apparently removed from refolded csr-1 by precipitate that forms upon the addition of ammonium forms of removal sulfate. The desired dimeric protein may be eluted using a gradient of decreasing ammonium. sulfate with The CSF-l dimer elutes at approximately 0.6 M ammonium sulfate, % the phenyl-TSK Alternative can also be used, increasing ethylene glycol in neutral buffer. ethylene glycol from column. supports and phenyl- Sepharose, may be preferred for larger scale production of the purified CSF-l dimeric protein. specific resulting dimer is of clinical purity.

The approximately equivalent to that of native human CSF—l activity of such preparations is produced by mammalian cells; In situations where the -24.. starting CSF~l is of lower final purity are purity, or where higher an additional purification step (such as DEAR chromatography following degrees of required, refolding) may be employed. which include the solubilizing the In those embodiments additional monomeric form of the protein, preliminary step of the starting materials which can be separated from soluble bacterial proteins by the.~cells are obtained as insoluble intracellular protein, conditions and recovery of the insoluble protein by centrifugation. The lysis of under suitable recovered insoluble protein is then placed directly into a chaotropic environment to disassemble aggregates and effect solubilization/denaturation. shown to ‘be biologically active using any of several recovered, purified dimeric forms are proliferation assays. A standard assay which meets the formation of predominantly macrophage colonies. results in the Another assay is increase in cell proliferation, as measured by presence of system H thymidine incorporation in a CSF-l-dependent cell line such as the mouse macrophage line BAC. In another form of this assay, a colorimetric detection system based on the reduction of the tetrazolium salt, MTT, can be used. The CSF-1 dimers resulting from the process of the invention are active in such assays and are essentially free of other proteins produced by the bacteria.

Importantly, the CSF—l preparations are clinically pure. They are substantially free of endotoxin, having less than about 1.0 ng endotoxin/mg of CSF-l as assayed by the standard limulus amebocyte lysate (LAL) assay, Associates of Cape Cod, Inc., woods Hole, MA. preparations of approximately 95% or Further purification may be desired, but i more purity in as determined by SDS-PAGE, are obtained Further, the specific activity is approximately equivalent to or higher than that of the native protein.

CSF-l protein, by the method of the invention.

D. Pharmaceutical Compositions The refolded and CSF—l preparations can then be formulated for administration clinically pure by bconventional protocols and regimens, preferably systemic, including intravenous administration. The compositions may include conventional excipients, such as water for injection, buffers, solubilizing agents, and stabilizers, as is known in the art. A summary of formulation techniques for pharmaceutical compositions, including protein, is found, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., PA, latest edition.

Baston, E. Heterogimer Formation It should be noted that the process of the invention permits the formation of heterodimers from among various monomeric units of CSF-1. For example, the large number of CSF-1 proteins formed by variations in C-terminal processing provides a variety of starting materials which can be utilized in dimer formation.

Thus, novel heterodimeric materials can readily be formed. For example, the monomeric form of SCSF/CVl5D, along with the monomeric form of LCSF/CVl90, according to the can be mixed and treated method of the invention; the heterodimer can then be separated from the homodimer side products by various chromatographic -26.. methods. Similar mixtures subjected to the method of the invention lead to heterodimers of components having amino acid substitutions -- e.g., glu52 LCSF and LCSF/CV190.

The differing monomers may be mixed in vitro If produced in the same expression of each monomer is or produced in the same cell. cell, a construct for introduced into the same host; in such embodiments, it is preferred that each construct bear a different marker (such as TcR and AmpR) so that cotransformed hosts are selected. The cotransformed cells are then grown and induced to obtain mixtures of the two forms.

Examples The following examples are intended to illustrate, but not to limit, the invention.

Example 1 This example describes the recovery of purified, biologically active protein expressed from a construct encoding asp59SCSF/CV15O in E. coli. under control of the PL promoter in a vector contructed as described in European Patent Application No. 87309409.8, (EP 0 272 779 A2) filed October 1987, assigned to the same assignee and incorporated herein by reference. The protein is produced in a monomeric, insoluble form intracellularly.

An E. coli X lysogen, DGll6, transformed with (O/E) pPLSCSFaspS9/CVl50, 2948, in a 10 l fermenter in basal medium containing 72 mM.(NH4)2SO4, 20 mM KHZPO4, 2.0 ml/l TK9, g/l glucose, 3.0 mM MgSO4°7H2O, thiamine°HCl, and 50 the plasmid over-expresser CMCC cell line deposit no. were grown with sterile additions of 10 72 uM FeSO4, 20 mg/l mg/l ampicillin.

The cells were grown at 30°C to OD53onm of 12; _added to 2%; and CSF-l induced by shifting to 42°C. The cells were then grown for 3 more hours to a final OD530nm of 16.5. casamino acids were then expression was The cells were harvested by centrifugation and 4°C. The homogenate was then centrifuged and the cell debris retained. The debris contained the insoluble protein, which was resuspended in 30% sucrose and centrifuged at homogenized using 30 min sonication at ,000 x g for 10 min at 4°C to enrich for the insoluble protein.

The pellet from the solubilized in centrifugation was 7 M guanidine HCl in 0.1 M sodium phosphate, pH 7, containing 50 mM DTT and 5 mM EDTA for min. The suspension was then heated to 40°C for 5 min and the supernatant recovered after centrifugation.

The recovered supernatant was loaded onto a 90 x 2.6 cm (s-200) buffer, but containing 2 mM DTT rather than 50 mM. The column was_run using the same buffer, Sephacryl column equilibrated in the same and the protein concentration was monitored by 280 nm adsorption with the results shown in Figure 1. The majority of the bacterial proteins were separated from CSF-l, which was recovered as a 17 kd peak representing approximately 80% pure CSF-1 monomer.

The CSF-l pool was then diluted to 0.25 mg/ml protein in a corresponding buffer containing 7 M guanidine hydrochloride, 50 mM Tris, pH 8.5, and 5 mM EDTA which contained a redox system, consisting of 2 mM reduced glutathione (GSH) and 1 mM oxidized glutathione (cssc). pool from the 8-200 column was dialyzed against buffer To refold the partially purified CSF-1, the this (containing 7 M guanidine hydrochloride and GSH/GSSG), and then allowed to fold by slowly adding a solution of 50 mM Tris, pH 8.5, 5 mM EDTA, and the GSH/GSSG in 0.1 )4 NaCl to the dialysis ‘vessel. The addition was carried out at 4°C over 48 hr until the final guanidine concentration was approximately 0.2 M.

The dialyzate at this’ point contained dimeric CSF-1, which was loaded directly onto a Sepharose 12 molecular sizing column equilibrated in phosphate-buffered saline for further purification. Elution was again followed by 280 nm absorption. The elution pattern Figure 2. Before exposure to refolding conditions, the CSF-1 eluted as would be expected for the (Figure 2a); when the protein was exposed to refolding conditions at 0.3 mg/ml, as described above (or, at 0.1 mg/ml), results show the formation of the dimer-sized material, as Figures 2b and 2c, respectively.

The dimeric is= shown‘ in monomer however, alternatively, indicated in product chromatographed as a single peak on reverse-phase HPLC, as shown in Figure 3b. The dimeric product is over 90% a single species on RP-HPLC (see Figure 3b) and shows satisfactory stability and full biological activity. with respect to other proteins the CSF-l is shown to be over 95% pure by reducing and non—reducing SDS-PAGE analysis (Figure 7).

Results for the S-200 pool starting material before refolding, shown in Figure 3a, indicate a predominance of monomer (which elutes as two major peaks of CSF-l).

However, the single dimer peak illustrated in Figure 3b was shown to consist of two major components following re-reduction to the monomer (Figure 3c) as separated by RP-HPLC. ‘ characterized for The protein product was solubility by UV—visib1e spectroscopy. Spectra were recorded at 30-min intervals following dilution of the purified dimeric pool in phosphate-buffered saline, as shown in Figure 4. the constant, As shown in panel A, over a 2—hr period final spectrum of the that the was stable and soluble under physiological conditions. In CO1’1tI’3St, product remained indicating refolded protein a similar spectral analysis on the monomeric starting material, shown as panel B in Figure 4, at 90- sec intervals showed that the protein was unstable and rapidly formed insoluble, light-scattering aggregates.

The purified dimeric material prepared above was assayed in the mouse bone-marrow colony assay in duplicate, along with a "control" consisting of purified recombinant CSF obtained from a gene of similar sequence (SCSF) active secreted molecule of approximately 158 amino acids in the mammalian cell line ~CV—l. The refolded E. coli CSF-1 has a mouse bone marrow assay specific activity (in U/mg) of 2-4 x lo7, as compared to about 3 x 107 U/mg for CSF-1 obtained from CV-1 The purified unrefolded starting material had a specific activity approximately l0O0-fold expressed as an cells. lower. (The mouse bone marrow assay was described by Moore, R., et al, J Immunol (1983) l;l:2397 and by Prystowsky, M., et al, Am J Pathol (1984) l;g:l49. Human CSF-l shows about 10-fold greater activity in a murine bone marrow assay as compared to activity in a human bone marrow assay.) Native CSF-l, purified from MIAPaCa cells had a mouse bone marrow assay specific activity of 4-8 x 107 U/mg.

The circular dichroism refolded E. coli (CD) essentially spectrum of the identical "naturally folded" protein was within experimental error to that of CSF-l from CV-l cells.

Example 2 Twenty grams of frozen E. coli from cells expressing a construct encoding asp59SCSF/CVl50 under control of the PL promoter were resuspended in 200 ml of 50 mM Tris, 10 mM EDTA (pH 8.5) and sonicated for 30 min ice bath, 60% pulse, intensity of 9.

DGll6 paste in an The cell debnis was retained following 10 min x 15,000 x cell debris was resuspended in 200 ml of 30% sucrose (in 10 mM EDTA, pH 8.0) and sonicated 3 min to break up clumps and free The suspension was then centrifuged for 15 min x 15,000 x g, and the pellet was retained.

The sucrose-purified insoluble ‘protein was then solubilized in 15 'ml of 0.45 1.1 filtered 7 M guanidine HCl (GuHCl), 0.1 M sodium phosphate, 5 mM EDTA, 50 mM DTT (pH 7.5-8.0) for approximately 15 min and then heated to approximately 37-40°C for 10 min to The solubilized material was then centrifuged for 10 min x 15,000 x g. g centrifugation. The insoluble protein. insure reduction of disulfide bonds.

Six to ten ml of the clarified, solubilized CSF-l was loaded onto a 2.6 x 95 cm S-200 in filter-sterilized S-200 buffer mM EDTA, at room column (7 M 2 mM DTT, pH temperature at equilibrated GuHCl, 0.1 M sodium phosphate, 6.8) and 1 ml/min. sized overnight The protein eluted as a well-resolved peak, and when pooled, contained 40-70 mg of protein at about 1.2-1.5 mg/ml (40-60 ml).

The protein content was determined by that 1 A230 equals The solution was then diluted to 0.l—0.l5 mg/ml protein, 0.5-0.7 M GuHCl, mM Tris (pH 8.5), 100 mM Nacl, absorbance at 280 nm, assuming 1 mg/ml. in buffer-containing 50 mM EDTA, 2 mM reduced ._3l.. glutathione (GSH), l mM oxidized glutathione (GSSG), by the appropriate buffer to the solution and letting it sit 24 hr at 4°C.

Solid ammonium sulfate was added to 1.2 M final concentration and the pH was then adjusted to 7.0.

At this point addition of protein a precipitate formed which contained This can be at least The CSF-l removal of incorrectly folded forms of CSF-l. partially recovered and.recycled (see below). preparation was then prepared for further pyrogens/endotoxins and residual contaminants on a phenyl-TSK column. All buffers and reagents are prepared pyrogen-free. The CSF-l preparation was centrifuged 10 min x 15,000 x g and filtered through a 0.45 p filter (500 ml) disposable unit before being pumped onto a phenyl-TSK HPLC column equilibrated in 1.5 M ammonium sulfate, 0.1 M sodium phosphate (pH 7.0) run at 4°C.

After loading the CSF—l, the column was washed for 30 min. The protein was then eluted with a 45-min gradient of decreasing ammonium sulfate, increasing ethylene glycol B buffer (B buffer = 60% ethylene glycol, 0.01 M sodium phosphate (pH 7.0)). The CSF-l protein eluted at approximately 0.6 M ammonium sulfate, % ethylene glycol.

The first major peak that eluted was biologically active, dimeric CSF-l. The CSF-l peak was pooled and then extensively dialyzed against 5% mannitol,' 25 mM sodium phosphate (pH 7.4), filter sterilized, and stored at 4°C. Endotoxin content varied from 0.1-1 ng/mg.

In a similar manner, E. coli protein produced under control of the from DNA encoding asp5gSCSF/NV2CV150, asp5gSCSF/NV3CVlS0, NV3CVl58, LCSF/CVl90, and LCSF/CV22l was refolded and purified.

PL promoter -32..

The final preparations contained 6-15 mg of purified CSF-1 with an approximate overall yield of 15-30%, and a specific activity of 5410 X 107 U/mg in the mouse bone marrow assay (using A230 and assuming a value of 1.0 corresponds to 1; mg CSF-l per ml). The preparations also have approximately the same specific activity in human bone marrow assay CSF-1.’ as purified native MIAPaCa Example 3 Direct Refolginq sf Solubilized Refractile Bodies solubilized asp59SCSF/CVl50 refractile bodies were prepared as in Example 2, and had Sucrose-purified, a protein concentration of 29 mg/ml. For refolding, the protein concentration was decreased by diluting to l.5 mg/ml asp59SCSF/CVl50 (total CSF-1 was 38 mg) in 7 M GuHC1, 0.1 M sodium phosphate (pH 7.0), 5 mM EDTA, 1 mM DTT. Refolding was initiated by diluting tenfold to 0.15 mg/ml in 50 mM Tris (pH 8.5), 100 mM NaCl; 5 mM EDTA, 2 mM GSH, and 1 mM GSSG (same refolding buffer as above) at 4°C and allowed to proceed 24 hr.

CSF-l refolded into dimeric form (based on the known retention time of dimeric CSF-l) as detected by RP-HPLC. The purity of the refolded dimers was estimated to be about 63% by RP-HPLC.

Approximately 35% of the monomer Example 4 Recycling Aqqreqates The precipitate described in Example 2 incorrectly folded forms of CSF—l. when formed from refolding of about 38 mg of protein, it presumably contains constituted about 10 mg of pelletable precipitate. This precipitate was dissolved in the S-200 buffer containing 7 M GuHCl and 2 mM DTT (described in Example 2). The suspension was heated at 37°C for 15 min to reduce any disulfide bonds, and the resulting clear solution was cooled to 4°C. The solution was then diluted to 0.7 M GuHCl in refolding buffer and allowed to refold, as described above. Ammonium sulfate was then added and the CSF-1 refolded dimer was purified from the resulting solution to remove pyrogens/endotoxins by phenyl-TSK HPLC as described above. soluble, dimeric CSF-1.

This recycling process, This yielded over 3 mg of when carried out at larger scale, is expected to significantly improve the overall yield of the process for producing refolded CSF-1.

Example 5 strain DG1l6 was with plasmid vector pLCSF22lA, a plasmid containing the gene encoding asp5gLCSF/NV3CV22l. coli DGll6 was with the Type Culture Collection under accession no. ATCC 67390, on l4 April 1987. in a 100 1 standard air-sparged Rushton turbine fermenter in basal medium containing 96 mM (NH4)2SO4, 28 mM KH2PO4, 4 mM Na3 citrate°2 H20, l.7 ml/l TK9 (30 mM znSO4, 30 mM MgSO4, 1 mM CuSO4), with sterile additions of 6.5 g/l glucose, 2.2 mM MgSO4°7 H20, 95 um FeSO4°7 H20 and 26 mg/l thiamine° HCl at 30°C until an 0D5ggnm of 10 was then E. coli transformed The transformed E. strain deposited American The transformed host was grown The culture was to 37°C reached. shift induced by temperature with concurrent sterile additions of casamino acids to 2.3% (w/v) MgSO4°7 H20 to 1.7 mm final concentration. the cells were and diafiltered against 10 volumes of 5 IM4 EDTA, pH 8.5, using Dorr- final concentration and Four hours post-induction, harvested by five-fold concentration Oliver tangential cross-flow microporous filtration. The cells were disrupted by three passes at 7,500 psi in a Manton-Gaulin high pressure mechanical cell homogenizer. l—Octanol was added to 0.1% (v/v) held overnight at 4°C.

The addition of and the homogenate % a 63% w/v sucrose solution. homogenate was made sucrose by The insoluble protein fraction (refractile bodies) was separated from cell debris by continuous flow disk stack centrifugation ‘(Westphalia SB7) at 9000 x 9. l liter/minute and 4-6°C.

The wet pellet was mixed 50:50 (w/v) in deionized water and stored at -20°C in 45 g aliquots.

Ninety’ grams refractile body suspension was thawed at room temperature and homogenized in 200 ml 0.1 M Tris, pH 8.5, containimg 25 mM EDTA and 10 mM DTT using a Tekmar tissumizer for 1 minute at 50% speed. The suspension was adjusted to 1 liter 8 M urea,'2 mM DTT, 5 mM EDTA and 20 mM Tris, pH 8.5 and stirred for approximately 30 minutes at room temperature. Insoluble debris was removed using a 1 sq. ft. 0.8—0.2 um Sartorius disposable membrane filter cartridge.

Following filtration, the suspension containing reduced CSF-1 monomer was partially purified Sample at an A230 of 10 (500 ml) was applied to each of two 5 x 45 cm DEAE Sepharose by DEAB chromatography. fast flow columns equilibrated in 0.1 M Tris, pH 8.5.

Each column was developed using a 3600 ml, 0-0.4 M NaCl gradient in 4 M urea, 0.1 M Tris, pH 8.5, 5 mM EDTA, and mM DTT. Based on the assumption that l A390 equals 1 mg/ml, 4.5 g of protein were recovered.

DEAE purified CSF-l monomer was cooled to 4°C and diluted 1:10 in pre-chilled 50 mM ‘Tris, pH 8.5, containing 5 mM EDTA, 2 mM reduced glutathione, and 1 mM oxidized glutathione to a final estimated protein A230 absorbance of 0.2. Although initial dimer formation was essentially complete within 24 hours as judged by SD5- PAGB, the refolding mixture (22.5 liters) was held for five days at 4°C to maximize yield of CSF-1 dimer with the correct conformation. The conformation of dimeric CSF-l in the refolding mixture was assessed by reverse- phase HPLC. Using a C4 column and a 35-55% acetonitrile gradient, dimeric CSF-1 eluted as two discrete species; stable active CSF-l was the more hydrophobic. This stable, represented 65% of the protein after five days incubation.

Reduced and oxidized glutathione were removed by diafiltration against 20 mM sodium phosphate, pH 7, and the protein concentrated to an A230 absorbance of PMIO hollow fiber ‘added to the concentration of 1.2 M.

Precipitated unstable conformer (the less hydrophobic species detected by reverse-phase HPLC) was removed by filtration. The filtrate (2 g stable dimeric CSF—1) was applied to a 5 x 20 cm bed of fast flow phenyl Sepharose equilibrated in l.2 M active CSF-l species .2 using an Amicon 10 sq. ft.

Ammonium sulfate was diafiltered material to a cartridge. ammonium sulfate containing 0.0025 M sodium phosphate, pH 7.0, and eluted in 6 hours in a simultaneously decreasing (0.72 M to O M ammonium sulfate) and increasing (24% to 60% v/v ethylene glycol) gradient of 1500 ml in 0.01 M sodium phosphate buffer, pH 7.0. Dimeric CSF—l eluted at approximately 30-35% ethylene glycol and was well separated from tetrameric CSF-l and endotoxin, both of which eluted later. Dimeric CSP—1 was diafiltered against 20 mM sodium phosphate, pH 7.5, and concentrated to an A230 of 10 using a 1 sq. ft.

Amicon spiral cartridge (YMl0). The recovery was 1.3 g stable dimeric CSF-1 based on A230. CSF-1 produced had a biological activity of about 6 x 107 U/mg busing an CSF-lrdependent cell proliferation assay to determine The final product was 98.6% dimer and 93% reducible dimer, determined by nonreducing and reducing SDS—PAGE analysis. of CSP-l as determined by LAL assay and A230 nm. activity.

Example 6 DEAE Chromatography Following Refoldinq An E. coli strain HW22, transformed with the plasmid pJN653 containing the asp59SCSF/NV3CVl58 gene was grown in a 10-liter fermenter in the same medium described in Example 5. The cells were grown at 30°C to an absorbance at 680 nm of 10, then added to 2%. induced by shifting the temperature of the culture to 37°C. After hr the absorbance at 680 nm reached 79; the cells were and casamino acids were CSF-l expression was harvested, homogenized and refractile bodies were prepared as described in Example 5.

Twenty-five grams of refractile body suspension (approximately 390 g of protein) were solubilized in 250 ml of 8 M urea containing 25 mM Tris, l0 mM sodium phosphate buffer (pH 8.4), 1 mM EDTA and 4 mM DTT. After 2 hr at room temperature, the solution was clarified by centrifugation at 15,000 x g for 15 min. A lS0 ml aliquot of the solubilized CSF-1 was then loaded Onto a 5 x 8 cm DBAE-Sepharose (Pharmacia) column equilibrated in 6 M urea containing 25 mM Tris and 10 mM The endotoxin content was 0.01 ng/mg. sodium phosphate buffer (pH 7.0). The column was washed with 1 bed volume of the above solution which had been modified to contain 1 mM DTT and 1 mM EDTA, and the CSF—l was then eluted with a 1.4 1 salt gradient of 0-0.6 M sodium chloride in the wash buffer. The CSF-1 peak eluted at approximately 0.06 M sodium chloride.

The remaining 90 ml of solubilized -refractile bodies were then purified over the DEAE-Sepharose column in identical fashion. The combined CsF—l pools (165 ml) contained approximately 250 mg of protein at a purity of approximately 50%.

The CSF-l was then refolded by diluting the DEAE pool 10-fold into refolding buffer containing 50 mM Tris (pH 8.5), 5 mM BDTA, 2 mM reduced glutathione, 1 mM oxidized glutathione, precooled to 4°C. The CSF-1 was allowed to refold for 30 hrs at 4°C. The pH of the refolded CSF-1 was adjusted to 6.8 using 8.5% phosphoric acid solution. The solution was clarified by centrifugation for 10 min at 15,000 x g and loaded onto a S )( 4 cm DBAE-Sepharose column pre-equilibrated in mM sodium phosphate, 25 mM Tris (pH 6.8). The column was washed with 300 ml of this buffer and eluted with a 700 ml 0-0.6 M sodium chloride gradient buffer system. in the same The CSF-1 eluted at approximately 120 mM Ammonium sulfate (4 M stock, pH 7.0) was added to the 95 ml DEAE pool to a final concentra tion of 1 M. The CSF-1 was filtered through a Nalgene 0.45 micron filter and loaded (at 4°C) 150 mm Bio-Rad TSK PhenylPW column equilibrated in depyrogenated 1.5 M ammonium sulfate and 0.1 M sodium phosphate (pH 7.0). of this sodimn phosphate sodium chloride. onto a 21.5 x The column was washed with two bed and eluted in 0.1 M using a 45-min gradient in volumes loading buffer (pH 7.0) which the ammonium sulfate concentration decreased from .5 M to 0 M and the ethylene glycol concentration increased from 0—60%. All operations were carried out at 4°C under essentially pyrogen-free conditions. The CSF—l eluted at approximately 0.6 M ammonium sulfate in % ethylene glycol. The CSF-1 was extensively dialyzed into 10 mM HEPES buffer (pH 7.5) containing 150 mM sodium chloride and filter sterilized through a Millex 0.45 micron filter.

Approximately 50 mg of purified asp59SCSF/NV3 CVl58 CSF—l was obtained. The final CSF-1 product was greater than 90% single species by SDS—PAGE analysis and approximately 96% pure by RP—HPLC in acetonitrile/TFA.

The specific activity was 1.7 x 108 u/mg (units determined as colony forming units equivalents using a CSF-l-dependent cell determined using line, and protein concentration and an coefficient of 1.0). extinction This specific activity is at least if not greater than, assumed equivalent to, Paca CSF-1. The endotoxin content, assay was 0.5-1 ng/mg of CSF—l. that of native Mia determined by LAL Example 7 An alternative purification method was used to process a refolding reaction of LCSF/NV3 CV22l prepared according to method of Example 5 up to and including the refolding step. In this modified method, the refolded CSF—l was directly loaded onto an anion exchange column.

At pH 6.8, the redox system reagents flowed directly through the anion while the CSF-l remained bound and concentrated on the column. In this manner, the CSF—l was separated from the redox system at a pH thio-disulfide thus exchange column, where exchange reactions were minimized, preventing the significant oligomer -39? formation that was found to occur performed at higher pH (8.5). , Five ml of refolded CSF-1 (1 mg total protein from the refolding reaction described in Example 5) was directly loaded onto a’ 7.5 x 75 mm Bio-Rad TSK DEAEPW column after lowering the pH of the refolded CSF-1 to 6.8 using a l 14 phosphoric acid solution. The DEAE column had been equilibrated in 10 mM sodium phosphate, mM Tris (pH 6.8). After loading the CSF-1, the column was washed with two bed volumes of this buffer and then eluted with a 45 min 0-0.6 M sodium chloride gradient if this step was in the same buffer. The column separated dimeric CSF-1 front monomeric and oligomeric forms of CSF-1 (as determined by nonreducing SDS-PAGE and Western analysis of the DEAE fractions).

CSF-1 was approximately 70%.

The yield of dimeric This is a 5-fold greater yield than that obtained when the same purification was performed at pH 8.5. Subsequent to this DEAE— purification step, the CSF-1 would be purified away from contaminating endotoxins and. the unstable fornl of the CSF-1 dimer as described beginning with the ammonium sulfate addition which precedes the phenyl- Sepharose step. in Example 6, Example 8 An alternative method for the refolding of CSF-1 has been utilized. Plasmid pLCSF22lA was induced and the expressed protein processed in substantial accordance with the teaching of Example 5 in E. coli with some modifications. For example, the harvested cells were diafiltered against 5 mM EDTA with no pH After the through the homogenizer, the pH was adjusted to 6 with acetic acid. adjustment. second pass In addition, air oxidation was relied upon for formation of disulfide« bonds refolding of the CSF-1 molecule. during DEAE—purified CSF-1 monomer was diluted to a final concentration of 0.2 mg/ml in 50 mM Tris pH 8.5, 5 mM EDTA, and refolded for 4 days at 4°C in the presence or in the absence of the glutathione redox system. The refolded proteins were further purified in substantial accordance with the procedures described in Example 5, again with some modification. The refolded dimeric mixture was diafiltered and concentrated to an OD of 1.

After the ammonium sulfate precipitation, the sample was applied to a phenyl-Sepharose fast flow column and then eluted in a decreasing (0.78 to 0.18 M ammonium sulfate) gradient of 1800 ml in 0.0114 sodium phosphate buffer (pH 7). The dimer "0.6 M ammonium sulfate.

Lastly, the dimeric CSF—l was diafiltered against 0.588% sodium citrate and 0.645% NaCl at pH 7. elutes at In the absence of the glutathione redox system, the diafiltration step required for glutathione removal may be omitted.

Final products from the refoldings done in the presence or in the absence of a redox system were compared by SDS-PAGE, RP/HPLC, isoelectric focusing and bioassay. Similar molecular weights and purities (95% both reducing and non-reducing conditions ¢of 12% SDS-PAGE visualized’ by Coomassie both refolded Reverse—phase HPLC analysis was also used to compare the refolding kinetics after 5 or 12 days of CSF-1 glutathione by densitometry scanning) under staining were observed for samples. refolding in the presence or These samples were immediately run on a C4 Vydac column with a 35-55% acetonitrile, 0.1% TPA gradient elution developed over minutes. Both systems resulted in two major dimeric absence of the redox system. -41.. species having similar retention times and which appeared to be in a relatively stable equilibrium over the time period analyzed. Phast (Pharmacia) isoelectic focusing (IEF) gels of 1.0 pg each of the refolded CSF—l preparations showed similar ionic patterns, containing a major ionic species with a pl of approximately 4.7 and a slightly more acidic minor species. Both spontaneously refolded CSF-1 and CSF-1 refolded using the redox system had specific activities of 1.2 x 105 U/mg in the NSF—6O cell proliferation assay. Thus the CSF-1 produced by these two refolding systems appeared to be essentially identical in product purity and biological activity, as assayed by the criteria described. Overall yields were also comparable for the two processes.

In addition to deleting the diafiltration step for glutathione removal, the concentration step may be replaced by an alternative purification step the large volume of refolded dimer CSF—l applied to a in which is directly second anion exchange column for concentration prior to ammonium sulfate precipitation and subsequent purification by hydrophobic chromatography. interaction Example 9 CSF-1 constructs in which certain cysteines have been changed to serines have also been successfully refolded. These refolded proteins are fully active in yitgg, but have slightly different RP-HPLC retention times. For example, the double-serine ser157ser15gLCSF/NV3CV22l, was refolded using the procedure described in Example 5, and this resulted in a construct, CSF-1 preparation RP-HPLC. which displays a single peak on When either of the single-serine constructs, ,eluted SeF157LCSF/NV3CV22l or ser159LCSF/NV3CV22l, were refolded, a modified refolding protocol was required in order to obtain a product which was homogeneous when analyzed. on RP-HPLC. These two products both eluted with a later retention time than the ser157ser15gLCSF/NV3CV221 refolded product, yet again fully active in vitro.

DG1l6 was transformed with pLCSF22lB or pLCSF22lC, plasmids containing the gen encoding ser157LCSF/NV3CV22l or ser15gLCSF/NV3CV22l, respectively. These two E. coli strains were grown in shake flasks at 30°C in 500 ml of the same medium desribed in Example 5 (final Aggonm of 0.2). CSF-1 expression was induced by shifting the temperature of the culture to 42°C. After 4 hr, the culture was harvested by centrifugation and the cells resuspended in 30 ml of 50 mM Tris buffer (pH 8.5), 5 mM were strain plasmid E. coli either the vector EDTA. The cells were lysed by sonication and the cell debris retained following centrifugation. Refractile bodies were then isolated by resuspending the cell debris in bodies %, sucrose by centrifugation. and pelleting the refractile The refractile bodies were solubilized in 10 D4 urea, 10 mM Tris (pH 8.5), 1 mM EDTA, and 5 mM DTT. Insoluble material was removed by centrifugation, followed by filtration through a 0.2 micron Millex filter. The CSF-l monomers were then filtrate, using ion exchange chromatography on a Bio-Rad TSK DEAE-5—PW column (7.5 x 75 mm) equilibrated in 6 M urea, 10 mM Tris (pH 8.5) containing 1 mM EDTA and 1 mM DTT. The CSF-1 was eluted with a 45 min, 0-0.4 M Sodium chloride gradient. CSF—l in the gradient as the single, major The protein was pooled and the absorbance at 280nm determined. The CSF-1 was refolded by diluting purified from the early protein peak. -43f into a solution containing 50 mM Tris (pH 8.5), 5 mM EDTA, 2 mM and 1 mM oxidized glutathione to a final Azgonm value of 0.2 as calculated from the undiluted DBAE pool Aggonm absorbance. The CSF—l was allowed to refold for 48 hr at 4°C.

At this point an additional oxidation step was added to the refolding protocol in order to obtain a product which was essentially homogeneous by RP-HPLC analysis. The refolded CSF—l protein was dialyzed at 4°C for 24 hr in 0.4 M urea, 50 mM Tris (pH 8.5), 5 mM EDTA containing only reduced glutathione (2 mM). This step may remove glutathione bound to the protein through a mixed disulfide. l M phosphoric acid was then used to adjust the pH to 6.5, thereby decreasing the rate of thio—disulfide exchange. The CSF-1 was purified by ion Bio-Rad TSK DEAEPW column equilibrated in 10 mM sodiunl phosphate, 25 mM reduced glutathione, exchange chromatography on a Tris vbuffer (pH 6.5). This step removes residual glutathione and further purifies the protein. The protein was eluted with a 45 min, 0-0.6 M sodium chloride gradient. The refolded, CSF-l dimer pool was then subjected to cupric chloride oxidation using a modification of the Inethod taught hi U.S. Patent No. 4,572,798. The CSF—1 was diluted to 0.2 absorbance (Azgonm) in 10 mM sodium phosphate, 25 mM Tris buffer (pH 6.5) and treated with 50 micromolar cupric chloride for 2 hr at room temperature.

The oxidized CSF-1 dimer was soluble in 1.2 M ammonium sulfate. units found to be Further purification by hydrophobic interaction phenyl-Sepharose column as described in Example 5 may be chromatography on a performed.

Claims (14)

1. An isolated and purified, recombinant, unglycosylated and dimeric CSF-1, said dimeric CSF—1 being biologically active and essentially endotoxin and pyrogen— free, said dimeric CSF-1 consisting CSF—l subunits being the same or different, with the proviso of two monomeric human subunits, said two monomeric that when said two monomeric subunits are the same, said monomeric subunits are an NV2 or an NV3 deletion mutein of human mature CSF-1.

2. A dimeric CSF-1 as claimed in claim 1, wherein said two monomeric human CSF~l subunits are different.

3. A dimeric CSF—1 as claimed in claim 1, wherein said two monomeric human CSF~l subunits are the same and are an NV2 or an NV3 deletion mutein of human mature CSF—l.

4. A dimeric CSF—1 as claimed in any one of claims 1 to 3, wherein one or both of said monomeric human CSF-1 subunits comprises a‘ human LCSF or an NV2 or an NV3 truncated mutein thereof, and optionally a tyrgm seruy, S€r15g OI ser157ser159 form thereof.

5. A dimeric CSF-1 as claimed in claim 4, wherein said LCSF or an NV2 of NV3 truncated mutein thereof also has a truncated carboxy terminus that is selected from the group consisting of CVl50, CVl90, CV22l and CV223.

6. A dimeric CSF—1 of any one of claims 1 to 3, wherein one or both of said monomeric human CSF—1 subunits comprises a human SCSE’ or an IQVZ or an NV3 truncated mutein thereof, and wherein the residue at position 59 is optionally Asp.

7. A dimeric CSF—l as claimed in claim 6, wherein said SCSF or said NV2 or NV3 truncated mutein thereof has a carboxy truncated terminus that is selected from the group consisting of CV15O and CV158.

8. A dimeric CSF-1 as claimed in any one of claims 1 to said monomeric human CSF—l the 3, wherein one or both of subunits is selected from group w,59LCSF/NV3CV22l consisting of LCSF/NV3CV221, aqfi9SCSF/NV3CVl50, sex-157LCSF/NV3CV22l, and .

9. A dimeric CSF-1 as claimed in any preceding claim, wherein said CSF-1 comprises refolded CSF—l.

10. A clinically pure, biologically active refolded CSF- l dimer comprising a dimeric CSF—l of any one of claims 1 to 9 having an endotoxin content of less than 1.0 ng/mg of CSF—l and substantially free of pyrogens, said dimeric CSF—l being prepared from CSF—l produced recombinantly in bacteria.

11. A. refolded CSF—l dimer as further comprising the features of any of claims 2 to 9. claimed in claim 10,

12. A biologically active refolded human CSF—l dimer comprising’ two monomeric units selected from the group consisting of LCSF monomers and muteins and C— or N- terminal truncations thereof, and SCSF monomers and muteins and C- or N-terminal truncations thereof, and 10 the wherein monomeric units of said dimer are not identical.

13. A composition comprising the dimeric CSF-1 of any one of claims 1 to 12, optionally in admixture with a pharmaceutically acceptable excipient.

14. A dimeric CSF—1 of any of claims 1 to 12 for use as a pharmaceutical. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS