BRIGHT, Jeremy, Richard (Look West, High Street Harwell, Oxon OX11 0EU, GB)
THURSZ, Mark (Toads on the Green, The Green Potten End, Hertfordshire HP4 2RY, GB)
BRIGHT, Jeremy, Richard (Look West, High Street Harwell, Oxon OX11 0EU, GB)
1. Recombinant IFNa 8 polypeptide having an antiviral activity of EC50 value 10 "4 ng/ml or less and having an apparent molecular weight of 17IcD or less.
2. The recombinant IFNαs polypeptide of claim 1 wherein the polypeptide is obtainable by the method of Claims 13 to 21.
3. The recombinant IFNαg polypeptide of claim 1 or 2 wherein the interferon is human interferon.
4. The recombinant IFNαs polypeptide of any one of claims 1 to 3 wherein the IFNαs is PEGylated and/or conjugated to albumin.
5. A pharmaceutical preparation comprising recombinant IFNαs polypeptide of any one of claims 1 to 4 and a pharmaceutically acceptable excipient.
6. A recombinant IFNαg polypeptide of any one of claims 1 to 4 or a pharmaceutical preparation of claim 5 for use in medicine.
7. Use of recombinant IFNa 8 polypeptide of any one of claims 1 to 3 or a pharmaceutical preparation of claim 5 in the manufacture of a medicament for . treating a viral infection.
8. The use of claim 7 wherein the viral infection is caused by HBV, HCV 5 HDV (delta virus), human papilloma virus, FfJV, yellow fever, West Nile virus or Dengue fever.
9. The use of claim 7 wherein the viral infection exacerbates chronic obstructive pulmonary disease.
10. The pharmaceutical composition of claim 6 or the use of claim 7 to 9 wherein the composition or medicament further comprises an anti-viral therapeutic agent.
11. Use of a recombinant interferon polypeptide of any one of claims 1 to 4 or a pharmaceutical preparation of claim 5 in the manufacture of a medicament for treating an oncological indication.
12. The use of claim 11 wherein the oncological indication is hairy cell leukaemia, chronic myeloid leukaemia, hepatocellular carcinoma, renal carcinoma, melanoma or prostate carcinoma.
13. A method of preparing recombinant interferon polypeptide comprising:
i) adding a refolding buffer, comprising a non-ionic detergent and a redox reagent, to microbially expressed denatured recombinant interferon polypeptide;
ii) incubating the recombinant interferon polypeptide and refolding buffer mixture of step i) at a temperature between 4 0 C to 25 0 C;
iii) removing the components of the refolding buffer from the soluble refolded recombinant interferon polypeptide of ϋ);
iv) optionally, purifying monomeric soluble refolded recombinant interferon polypeptide from the polypeptide of step iii).
14. The method of claim 13 wherein the denatured recombinant interferon polypeptide of step i) is bacterially expressed.
15. The method of claim 14 comprising the prior steps of:
i) suspending bacterially expressed insoluble recombinant interferon polypeptide in a stringent wash buffer comprising an alkaline pH buffer; a cliaotropic agent; a readily dissociable salt; and a reducing agent;
ii) recovering the insoluble recombinant interferon polypeptide from the stringent wash buffer of step i);
iii) optionally, repeating steps i) and ii) up to three times;
iv) incubating the recovered insoluble recombinant interferon pol y peptide of step ii) or iii) in a solublisation solution comprising a denaturant, an alkaline pH buffer, a reducing agent and optionally a detergent;
v) optionally, purifying monomeric soluble denatured recombinant interferon pol3φeptide from the polypeptide of step iv) .
16. The method of claim 14 or 15 comprising the prior steps of:
i) expressing a nucleic acid molecule (optionally optimised for expression in bacteria) encoding recombinant interferon polypeptide in bacteria;
H) harvesting the bacteria and isolating recombinant interferon polypeptide.
17. The method of any of the previous claims wherein the non-ionic detergent is selected from a group consisting of oligo - or poly-alkylene glycol sorbitan acyl esters; sorbitan acyl esters; oligo- and poly-alkylene glycol acyl esters; oligo- and poly-alkylene glycol alkyl/aryl ethers; alkyl p)τanosides; N-alkanoyl-N- allcylaldamines; BigCHAPS.
18. The method of claim 17 wherein oligo - or poly-alkylene glycol sorbitan acyl esters include TWEEN detergents 20, 21, 40, 60, 61, 65 and 80; sorbitan acyl esters include Span detergents 20, 40, 60, 65, 80 and 85; oligo- and poly-alkylene
glycol acjd esters include Myrj detergents; oligo- and poly-alkylene glycol alkyl/aiyl ethers include Brij detergents 35 . , 56, 5S 5 72, 76, 93 and 97, Igepal CA- 630, Tergitol detergents Tj'pe 15 and Type NP 5 Triton-X detergents 15, 45, 100, 114, 151, 165, 200, 207, 305, 405; alkyl pyranosides include octyl-β,D- glucopyranoside; N-aUcanoyl-N-allqdaldamines include N-nonano}4-N- metli3dglucamine and N-decanoyl-N-meth34glucamine
19. The method of claim 18 wherein the non-ionic detergent is Tween 80.
20. The method of any of the previous claims wherein the recombinant interferon polypeptide is IFN-αg.
21. The method of claim 20 wherein the interferon is human IFN-αg.
METHOD FOR PRODUCING RECOMBINANT INTERFERON ALPHA-8
The present invention relates to recombinant human interferon alpha-8 (IFN-αs) and a method of producing soluble and refolded interferon polypeptide from a sample of recombinant protein recovered from a microbe. The invention also relates to the production of medicaments for the treatment or prophylaxis of viral infections and also for the treatment of certain oncological indications.
Type I interferons are a family of closely related glycoproteins comprised of thirteen IFN-α subtypes as well as IFN-β, IFN-K, IFN-λ, IFN-τ and IFN-ω. The different human IFN-α subtypes have been identified by analysis of human cDNA libraries and by protein analysis of the IFNs produced by stimulated lymphoblastoid cells; the reasons for their heterogeneity remain unclear. Early studies indicated that all subtypes bind the same receptor from which it was inferred that they must elicit identical responses. Subsequently, comparative studies of both purified and recombinant subtypes revealed a spectrum of antiviral, antiproliferative and immunomodulatory responses. The early assumption was also confounded by the isolation of a mutant cell line that responds only to IFN-αs and EFN-β 5 implying that either another receptor or a modified binding mechanism must be involved in the mediation of responses generated by these species. Structural analyses have since suggested that the binding mechanism may be subtype-dependent.
A number of studies have compared the ability of different IFN-α subtypes to elicit anti-viral responses in human cell lines. Zoon et al., J. Biol. Chem.
267:15210 - 15216 (1992) studied subtypes that were obtained from HPLC purification of natural IFNs and found no gross differences in their antiviral activities. In contrast, Sperber et al., J Interferon Res, 12: 363 - 368, (1992) examined the effects of different recombinant IFN-α subtypes on cells infected with the human immunodeficiency virus (HTV) and found marked differences in their anti-viral properties. WO95/24212 disclosed that different IFN-α subtypes elicit different anti-viral responses in different cell types. Thus it is possible to
target viral infections of, eg. the liver, by the use of a particular subtype, eg. IFN-
O 8 .
The relative anti-viral potencies of different IFN-α subtypes disclosed in WO95/24212 were determined using preparations of each subtype purified from Wellferon using methods described by Zoon et al (1992) supra.
The mechanism underlying the augmented anti-viral potency of IFN-αg is not currently understood. It is known that the pleiotropic effects of the Type I interferons are mediated by a number of intra-cellular signaling pathways (reviewed by Brierley & Fish, 2002, J Interferon and Cytokine Res. 22:835 - 845) and it is not implausible to suggest that the IFN-αg subtype might be capable of activating some or all of these pathways more readily than other subtypes.
We have deterrnined that the most active form of the recombinant interferon polypeptide may be the form having the lowest apparent molecular weight of monomeric interferon species. Where the recombinant interferon polypeptide is IFN-αg, the apparent molecular weight of the most active form of the polypeptide is 17IcD or less as measured by SDS-PAGE.
The recombinant IFN-αg of the present invention displays an anti-viral potency in vitro that is several orders of magnitude greater than the preparations of IFN-α (most commonly α 2 ) that are currently commercially available and used clinicalfy in the treatment of chronic viral infection, eg. infection with hepatitis B or hepatitis C virus.
The present invention also describes a scaleable process for the microbial production of recombinant interferon polypeptide.
Accordingly, the present invention provides a more biologically active recombinant IFN-αg polypeptide and an improved method of preparing interferon polypeptide.
A first aspect of Hie invention provides recombinant IFN-αg pol y peptide having an antiviral activity of EC50 value 10 "4 ng/nil or less and having an apparent molecular weight of 17IdD or less.
As mentioned above, the inventors have identified a recombinant IFN-α 8 polypeptide having an anti-viral potency in vitro (expressed using EC50 values) that is several times greater than currently available preparations of ocg. For example, the maximum value for the IFN-αg polypeptide produced according to the method disclosed by Di Marco et al (1996) J Biotechnology 50, 63-73 is 4.1 x 10 s U/ml representing an EC50 value of 6 x 10 " ° iig/ml (Figure 6).
EC50 is an internationally accepted standard for assessing the activity of a therapeutic agent. The methods used to determine the EC50 value of a polypeptide are usual in the art and could be performed without inventive contribution from a skilled person.
The EC50 value of the TFN-CX 8 polypeptide of the invention is 10 "4 ng/ml or less, for example 10 '5 , 10 "6 , 10 "7 , 10 "s , 10 "9 , 10 '10 or 10 "11 ng/ml or less as measured using standard assays well known in the art. An example of such a standard assay is provided in example 2. Typically the EC50 value of the TFN-α 8 polypeptide of the invention is within the range of 10 4 ng/ml to 10 "π ng/ml: for example, 10 "4 ng/ml to 10 '9 ng/ml.
The apparent size of the recombinant TFN-αg polypeptide may of the invention vary depending on the. method used to determine the size of the polypeptide. The
SDS-PAGE gel/buffer system is a commonly used method to determine polypeptide size, as would be appreciated by the skilled person. The apparent size of the interferon polypeptide may also vary depending on the SDS-PAGE gel/buffer system used, as would be appreciated by the skilled person. The preferred sizes set out above are apparent when the SDS-PAGE conditions include a Bis-Tris buffered gradient gel and MES running buffer as set out in example 4.
Preferably, the apparent molecular weight of the most active form of IFN-α,g polypeptide is 17IcD or less as measured by SDS-PAGE 5 for example between 15IdD and 17IdD. Preferably the most active form of the IFN-αg polypeptide having an apparent molecular weight of 15.5IdD to 16.5IdD as measured by SDS-PAGE.
The present invention has established that the lowest apparent molecular weight of monomeric interferon species is the most active. This has not been reported before. In particular, it is worth noting that the IFN-αg polypeptide produced according to the method of Di Marco et al supra has an apparent molecular weight under reducing conditions of 27 IdD. Zoon et al supra HPLC purified and characterised a raft of IFN-α subtypes from Sendai virus stimulated Namalwa cells and found that all of the subtypes had apparent molecular weights in the range 17.5 - 23.3 IdD on non-reducing SDS-PAGE. Activities of all subtypes were in the range 0.3 - 4.6xlO s IU/rαg. Platis & Foster Protein Expr. & Purif. 31(2): 222 - 230, (2003) report the expression of IFN-αg in E.coli and describe refolding it to yield a protein with activity 3xlO "3 ng/ml; under reducing conditions its apparent molecular weight on SDS-PAGE was approximately 28kD. Acosta-Rivera et al, Biochem. Biophys. Res. Commun. 296:1301 - 1309, 2002, report expression of IFN-αs at very low levels in E.coli with an apparent molecular weight on reducing SDS-PAGE of 27IdD.
The IFN-αg polypeptide of the invention may be obtained using the protein expression and purification procedure provided herein.
An embodiment of this aspect of the invention is wherein the recombinant IFN-αs polypeptide is obtainable, preferably obtained, by the method of the invention set out below.
The recombinant IFN-αg polypeptide may be derivatised. For example, the recombinant IFN-αg polypeptide of the invention may be PEGylated or conjugated to albumin. Such derivatisation may improve the pharmacokinetic or immunogenic properties of the recombinant IFN-αs polypeptide.
PEGj'lation is a method well known to those skilled in the art wherein a polypeptide or peptidomimetic compound is modified such that one or more polyethylene glycol (PEG) molecules are covalently attached to the side chain of one or more amino acids or derivatives thereof. It is one of the most important molecule altering structural chemistry techniques (MASC). Other MASC techniques may be used; such techniques may improve the pharmacodynamic properties of the molecule, for example extending its half life in vivo. A PBG- protein conjugate is formed by first activating the PEG moiety so that it will react with, and couple to, the polypeptide of the invention. PEG moieties vary considerably in molecular weight and conformation, with the early moieties (monofunctional PEGs; mPEGs) being linear with molecular weights of 12kDa or less, and later moieties being of increased molecular weights. PEG2, a recent innovation in PEG technology, involves the coupling of a 3OkDa (or less) mPEG to a lysine animo acid (although PEGylation can be extended to the addition of PEG to other amino acids) that is further reacted to form a branched structure that behaves like a linear mPEG of much greater molecular weight (Kozlowski et al., (2001), Biodrugs 15, 419 - 429). Methods that may be used to covalently attach the PEG molecules to the polypeptide of the invention are further described in Roberts et al, (2002) Adv Drug Deliv itev54, 459 - 476, Bhadra et al, (2002) Pharmazie 57, 5 - 29, Kozlowski et al, (2001) J Confrol Release 72, 217 - 224, and Veronese (2001) Biomaterials 22, 405 - 417 and references referred to therein.
The advantages of PEG}4ation to the polypeptide of the invention include reduced renal clearance which, for some products, results in a more sustained adsorption after subcutaneous administration as well as restricted distribution, possibly leading to a more constant and sustained plasma concentration and hence an increase in clinical effectiveness (Harris et al, (2001) Clin Pharmacolάnet 40, 539
- 551). Further advantages include reduced immunogenicity of the therapeutic compound (Reddy, (2001) Ann Pharmacother 34, 915 - 923), and lower toxicity
(Kozlowski et al, (2001), Biodrugs 15, 419 - 429). Examples of PEGylated proteins with clinical applications include adenosine deaminase .(Hershfϊeld,
(1995) CHn Immunol Immunopathol 16, S228 - S232), L-asparaginase (Holle,
(1997) Ann Pharmacother 31, 616 - 624), interleuldn-2 (Chen et a!., (2000) J
Pharmacol Exp Ther 293, 248 - 259), granuloc3^te-macophage colony-stimulating factor (Knusli et aL, (1992) Br J Haematol 82, 654 - 663), tumor necrosis factor alpha (Tsunoda et aL, (1992) J Pharmacol Exp Ther 290, 368 - 372), human growth, hormone (Clark et aL, (1996) J Biol Chem 27I 5 21969 - 21977) and Interferon α (Kozlowski et al, (2001), Biodrugs 15, 419 - 429).
Therapeutic proteins such as interferons and growth hormones, in their native state or when recombinant^ produced, can be labile molecules exhibiting short shelf- lives, particularly when formulated in aqueous solutions. The instability in these molecules when formulated for administration dictates that the molecules may have to be lyophilized and refrigerated at all times during storage, thereby rendering the molecules difficult to transport and/or store. Storage problems are particularly acute when pharmaceutical formulations must be stored and dispensed outside of the hospital environment. Many protein and peptide drugs also require the addition of high concentrations of other protein such as albumin to reduce or prevent loss of protein due to binding to the container. This is a major concern with respect to proteins, such as interferons.
The role of albumin as a carrier molecule and its inert nature are desirable properties for use as a carrier and transporter of polypeptides in vivo. The use of albumin as a component of an albumin fusion protein as a carrier for various proteins has been suggested in WO 93/15199, WO 93/15200, and EP 413 622. The use of N-terminal fragments of HA for fusions to polypeptides has also been proposed (EP 399 666). Fusion of albumin to the therapeutic protein may be achieved by genetic manipulation, such that the DNA coding for albumin, or a fragment thereof, is joined to the DNA coding for the therapeutic protein. A suitable host is then transformed or transfected with the fused nucleotide sequences, so arranged on a suitable plasmid as to express a fusion polypeptide. The expression may be effected in vitro from, for example, prokaryotic or eukaryotic cells, or in vivo e.g. from a transgenic organism.
A further aspect of the invention provides a pharmaceutical preparation comprising a recombinant IFN-αg polypeptide of the previous aspect of the invention and a pharmaceutically acceptable excipient,
Whilst it is possible for a polypeptide or compound to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be "acceptable" in the sense of being compatible with the compound and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free.
The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.
A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert
liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile,
Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such, as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which ma}' contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient. Also preferred are unit dosage formulations suitable for tri-weekly, weekly or monthly administration.
It should be understood that in addition to the ingredients particularly mentioned- above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
In an embodiment of this aspect of the invention the pharmaceutical composition is formulated for slow-release of the interferon polypeptide.
The recombinant interferon may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various types of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
A further aspect of the invention provides a recombinant IFN-αg polypeptide of the invention or a pharmaceutical composition of the invention for use in medicine.
A further aspect of the invention provides the use of a recombinant IFN-αg polypeptide of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for treating viral infections, including HBV or HCV infection, HDV (delta virus), human papilloma virus, HTV 5 yellow fever, West Nile virus, Dengue fever and exacerbations of chronic obstructive pulmonary disease.
In an embodiment of the invention the pharmaceutical composition or medicament may further comprise anti-viral therapeutic agents, e.g. targeted at the viral helicase, protease or pofymerase. For example, the pharmaceutical composition or medicament may further comprise a therapeuticalfy appropriate quantity of ribavirin.
A further aspect of the invention provides the use of a recombinant interferon polypeptide of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for treating oncological indications, including hairy cell leukaemia, chronic myeloid leukaemia, hepatocellular carcinoma, renal carcinoma, melanoma and prostate carcinoma.
The present invention also describes a scaleable process for the microbial production of recombinant interferon polypeptide., in particular IFN-αg polypeptide.
As set out below, the method of the present invention provides a number of surprising advantages over the method of preparing IFN-αg from yeast reported in Di Marco et al supra.
The expression yield in yeast for IFN-αs is cited in Di Marco supra as 77mg IFN per litre, while the present method can routinely achieve 300mg IFN per litre when the microbial expression is conducted in E.coli, even using unoptimised growth conditions.
The IFN-αg refolding protocol disclosed in Di Marco supra lasts three days, whereas the interferon polypeptide refolding conditions used in the method of the invention can take only up to five hours.
The process yield for the method disclosed in DiMarco supra is 20% (15mg ex 77mg per litre), while the yield achieved by the method of the invention can be as high as 50%.
The method of the present invention favours production of purified interferon polypeptide of lower apparent molecular weight than that produced by DiMarco supra (as judged by SDS-PAGE) and is considered to be significantly more biologically active than the interferon produced using the protocol presented in DiMarco supra.
Accordingly, the present invention provides an improved method of preparing interferon polypeptide, and a preparation of more biologically active interferon polypeptide.
A further aspect of the invention provides a method of preparing recombinant interferon polypeptide comprising:
i) adding a refolding buffer, comprising a non-ionic detergent and a redox reagent, to microbially expressed denatured recombinant interferon pofypeptide;
ii) incubating the recombinant interferon polypeptide and refolding buffer mixture of step i) at a temperature between 4 0 C to 25 0 C;
iii) removing the components of the refolding buffer from the soluble refolded recombinant interferon polypeptide of ii);
iv) optionally, purifying monomeric soluble refolded recombinant interferon polypeptide from the polypeptide of step iii).
In many cases, the heterologous expression of protein in microbial cells can lead to the expressed protein forming "inclusion bodies" of insoluble and incorrectly folded recombinant protein. While it is possible to isolate inclusion bodies relatively cleanly from other proteins in the microbial cell, a big disadvantage is that the recombinant protein is not in a native state, and some solubilisation/refolding step is needed. In general inclusion bodies occur more frequently when expressing secreted eukaryotic proteins in E. coli than when expressing cytosolic proteins.
The method of the invention provides a method of preparing recombinant interferon polypeptide.
The most active form of the recombinant interferon polypeptide may be the form having the lowest apparent molecular weight of monomeric interferon species generated. Where the recombinant interferon polypeptide is σFN-αs, the apparent molecular weight of the most active form of the polypeptide is ITkD or less as measured by SDS-PAGE, for example between 15kD and 17IcD. Preferably the method of the invention produces the most active form of the IFN-(X 8 polypeptide having an apparent molecular weight of 15.5kD to 16.5IcD as measured by SDS- PAGE.
The apparent size of the recombinant interferon polypeptide may vary depending on the SDS-PAGE gel/buffer system used, as would be appreciated by the skilled person. The preferred sizes set out above are apparent when the SDS-PAGE conditions include a Bis-Tris buffered gradient gel and MES running buffer as set out in example 4.
The inventors have determined that recombinant interferon polypeptide obtained from the method of the present invention displays an anti-viral potency in vitro (expressed using EC50 values) that is several times greater than currently available preparations of IFN-α (most commonly α 2 ).
As set out in example 2 below, EC50 is an internationally accepted standard for assessing the anti-viral activity of a therapeutic agent. The methods used to determine the EC50 value of a polypeptide are usual in the art and could be performed without inventive contribution from a skilled person.
The EC50 value of the interferon polypeptide produced using the method of the invention is in the range 10 "π ng/ml to 10 "4 ng/ml, for example 10 "10 , 10 '9 , or 10 "8 , 10 "7 5 10 '6 , 10° or 10 "4 ng/ml as measured using standard assa3's well known in the art. An example of such a standard assay is provided in example 2. Preferably the method of the invention produces a recombinant IFNαg polypeptide having an antiviral activity of EC50 value 10 ng/ml or less and having an apparent molecular weight of 17kD or less.
The "recombinant interferon polypeptide" required for step i) can be provided using standard recombinant molecular biology techniques.
In general, DNA encoding the desired interferon polypeptide is expressed in a suitable microbial host cell. Thus, DNA encoding interferon polypeptide may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of interferon
polypeptide. Such techniques include those disclosed in US Patent Nos. 4,440,859 issued 3 April 1984 to Rutter et al, 4,530,901 issued 23 July 1985 to Weissman, 4,582,800 issued 15 April 1986 to Crawl, 4,677,063 issued 30 June 1987 to Mark et al, 4,678,751 issued 7 July 1987 to Goeddel, 4,704,362 issued 3 November 1987 to Itakura et al, 4,710,463 issued 1 December 1987 to Murray, 4,757,006 issued 12 JuI)' 1988 to Toole, Jr. et al, 4,766,075 issued 23 August 1988 to Goeddel et al and 4,810,648 issued 7 March 1989 to Stalker, all of which are incorporated herein by reference.
The DNA encoding interferon polypeptide may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.
Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. Ji " necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. Thus, the DNA insert may be operatively linked to an appropriate promoter. Bacterial promoters include the E.coli laclmά lacZ promoters, the T3 and T7 promoters, the gpt promoter, the phage λ PR and PL promoters, the phoA promoter and the tηp promoter. Eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters and the promoters of retroviral LTRs. Other suitable promoters will be known to the skilled artisan. The expression constructs will desirably also contain sites for transcription initiation and termination, and in the transcribed region, a ribosome binding site for translation. (Hastings et al, International Patent No. WO 98/16643, published 23 April 1998).
The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector and it will therefore be necessary to select for transformed host cells. One selection technique involves incorporating into an expression vector containing any necessary control elements a
DNA sequence marker that codes for a selectable trait in the transformed cell. These markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture, and tetracyclic kanamycin or ampicillin resistance genes for culturing in E.coli and other bacteria. The selectable markers could also be those which complement auxotrophisms in the host. Alternatively, the gene for such a selectable trait can be on another vector, which is used to co-transform the desired host cell.
Host cells that have been transformed by DNA encoding interferon polypeptide are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of interferon polypeptide.
Many microbial expression systems are known, including S3 r stems employing: bacteria (eg. E.coli and B. subtϊlis) transformed with, for example, recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeasts (eg. Saccaromyces cerevisiae) transformed with, for example, yeast expression vectors; insect cell systems transformed with, for example, viral expression vectors (eg. baculovirus).
The vectors can include a prokaryotic replicon, such as the Col El ori, for propagation in a prokaryote. The vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription) of the genes in a bacterial host cell, such as E.coli, transformed therewith, and a translation initiation sequence, such as the Shine-Dalgarno consensus ribosome- binding sequence, usuaUy adjacent to the promoter sequence, that forms part of the resulting transcript and from which translation of the cloned gene transcript can commence.
A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
Typical prokaiyotic vector plasmids are: pUCIS, pUC19, pBR322 and pBR329 available from Biorad Laboratories (Richmond, CA, USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 available from Pharmacia (Piscataway, NJ 5 USA); pBS vectors, Phagescript vectors, Bluescript vectors, pNHSA, pNHl 6A 5 pNHl 8A 5 ρNH46A available from Stratagene Cloning Systems (La JoIIa 5 CA 92037, USA). Preferred prokaiyotic vector plasmids include pET26b (Novagen, Nottingham, UK).
Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems (La JoUa, CA 92037, USA). Plasmids pRS403, pRS404, pRS405 and ρRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRJPl, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (YCps).
Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence and, for example appropriate transcriptional or translational controls. One such method involves ligation via homopolymer tails. Homopolymer polydA (or polydC) tails are added to exposed 3 ' OH groups on the DNA fragment to be cloned by terminal deoxynucleotidyl transferases. The fragment is then capable of annealing to the polydT (or polydG) tails added to the ends of a linearised plasmid vector. Gaps left following annealing can be filled by DNA polymerase and the free ends joined by DNA ligase.
Another method involves ligation via cohesive ends. Compatible cohesive ends can be generated on the DNA fragment and vector by the action of suitable restriction enzymes. These ends will rapidly anneal through complementary base pairing and remaining nicks can be closed by the action of DNA ligase.
A further method uses synthetic molecules called linkers and adaptors. DNA fragments with blunt ends are generated by bacteriophage T4 DNA polymerase or E.coli DNA polymerase I which remove protruding 3 ' termini and fill in recessed 3 ' ends. Synthetic linkers, pieces of blunt-ended double-stranded DNA which contain recognition sequences for defined restriction enzymes, can be ligated to blunt-ended
DNA fragments by T4 DNA ligase. They are subsequently digested with appropriate restriction enzymes to create cohesive ends and ligated to an expression vector with compatible termini. Adaptors are also chemically synthesised DNA fragments which contain one blunt end used for ligation but which also possess one preformed cohesive end.
Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc., New Haven, CN 5 USA.
A desirable way to modify DNA encoding the interferon polypeptide is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491. In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
DNA sequences encoding interferon polypeptides are well known in the art. For example, DNA sequence encoding IFN-αs is given in association with GenBank Accession numbers XM_005505, K01900, X03125 and NM_002170; a further such DNA sequence is provided in Figure 1.
Accordingly, the procedures outlined above can be used to prepare a microbial expression system for the preparation of recombinant interferon polypeptide. An example of such a microbial expression system is provided in the accompanying examples.
The recombinant, interferon polypeptide can be recovered from microbial expression systems using a number of different well known methods, including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite
chromatography, lectin chromatography, dye-ligand chromatography and reverse phase high performance liquid chromatography ("HPLC").
Such methods may include the step of lysing the microbial host cells (unless the expression system directed the recombinant polypeptide to be secreted from the cell).
Soluble denatured recombinant interferon can be prepared from host cells of the microbial expression S3'stems described above using an appropriate quantity and concentration of a chaotropic agent, such as urea or guanidine hydrochloride.
Therefore the procedures outlined above can be used to prepare soluble denatured recombinant interferon for use in step i) of the method of the invention.
Step i) of this aspect of the invention requires that microbially expressed denatured recombinant interferon polypeptide is added to a refolding buffer comprising a non-ionic detergent and a redox reagent. Both a solution of denatured recombinant interferon polypeptide or lyophilised denatured recombinant interferon polypeptide can be used in this step of the method. Typically, soluble denatured recombinant interferon polypeptide is used.
The denatured interferon polypeptide is added to refolding buffer and, optionally, stirred, at a temperature between 4° to 25 0 C to allow the denatured soluble interferon to refold.
hi this aspect of the invention, extensive screening of refolding conditions was undertaken to identify the key components necessary to maximise the yield, homogeneity and activity of the refolded interferon polypeptide.
The present invention has determined that the presence of an appropriate quantity of a non-ionic detergent and a redox reagent in the refolding buffer is necessary to provide suitable refolding conditions for the soluble denatured interferon polypeptide and to maximise the yield of the refolded interferon polypeptide.
By "non-ionic detergent" we include oligo- and poly-alkylene glycol sorbitan ac3'l esters eg. the TWEEN detergents 20, 21, 40 . , 6O 5 61, 65 and 80; sorbitan acyl esters eg. the Span detergents 2O 5 40, 6O 5 65, 80 and 85; oligo- and pofy-alkylene glycol acyl esters eg. the Myrj detergents; oligo- and poly-alkylene glycol alkyl/aryl ethers eg. the Brij detergents 35, 56, 58, 72, 76, 93 and 97, Igepal CA- 630, the Tergitol detergents Type 15 and Type NP, the Triton-X detergents 15, 45, 100, 114, 151, 165, 200, 207, 305, 405; alkyl pyranosides eg. octyl-β,D- glucopyranoslde, N-alk-anoyl-N-alkylaldamines eg. N-nonanoyl-N- methylglucam ine and N-decanoyl-N-methylglucamine and other non-ionic detergents eg. BigCHAPS. The amount of non-ionic detergent used may be 0.001% to 2°/o (w/v), preferably 0.02%. Such detergents are commonfy available as standard laboratory reagents.
By "redox agent" we include cysteine, cystine, dithiothreitol, dithioerythritol, oxidised and reduced forms of glutathione, 2-mercaptoethanol and hydroxyethyl disulphide. The amount of redox agent used may be typically in the range 0.01 — 1OmM. Where the redox agent is DTT 5 preferably the amount of DTT in the refolding buffer is ImM. Such redox agents are commonly available as standard laboratory reagents.
Further components of the refolding buffer may include a polar excipient (e.g. arginine); an alkaline pH buffer (e.g. Tris pH >7); divalent cations (e.g. Ca + * or Mg +"1" ). An example of a buffer including such components is presented in the accompanying examples 1 and 3.
Step ii) of the method of the invention requires that the recombinant interferon polypeptide is incubated with the refolding buffer at between 4 0 C to 25 0 C. It is preferred that the incubation time for this step is sufficient to allow at least 10% of the soluble polypeptide to be refolded.
The percentage of refolded polypeptide may be assessed using SDS-PAGE analysis or biological activity of the polypeptide. It is preferred that the assay to
deterniine the percentage of refolded polypeptide is conducted after step iii) of the method of the invention. It has been determined that 5 hours is a sufficient period of time in which to allow step ii) of the method to proceed.
Methods for determining whether the recombinant interferon polypeptide is soluble and refolded include SDS-PAGE analysis of the recombinant interferon polypeptide/refolding buffer mixture under non-denaturing conditions, and size- exclusion chromatography under non-denaturing conditions. Capillary electrophoresis may also be used to distinguish between different monomeric forms of the interferon polypeptide. Alternatively, the methods for determining whether the recombinant interferon polypeptide is soluble and refolded include where the biological activity of the polypeptide is assessed. An example of an assay for biological activity of interferon polypeptide is provided in the accompanying example 2.
As mentioned above, the method of the invention may produce interferon polypeptide having the lowest apparent molecular weight of any of the monomeric interferon species generated.
Where the method of the invention is used to prepare IFN-αs, bioassay data (further discussed in the accompanying examples) has revealed that the most active form of the refolded IFN-αs polypeptide migrated with an apparent molecular weight of 17IcD or less as measured by SDS-PAGE 5 for example between 15kD and 17kD; preferably the recombinant interferon polypeptide has an apparent molecular weight of 15.5IcD to 16.5kD measured by SDS-PAGE. Further it has been determined that a form of approximately 18kD as measured by SDS-PAGE retains a high proportion of this activity. Accordingly, it is preferred that the conditions used in the method of the invention produces IFN-αs polypeptide having an apparent molecular weight of approximately 17IcD or less as measured by SDS-PAGE. Preferably, the conditions used in the method of the invention produces IFN-αs polypeptide having an apparent molecular weight of approximately 15kD to 17kD, for example 15.5kD to 16.5kD measured b3 r SDS- PAGE.
As discussed above, it has been determined that the most active form of IFN-αg polypeptide prepared using the method has the lowest apparent molecular weight as measured by SDS-PAGE of any of the monomeric interferon species generated. Where the interferon polypeptide is IFN-α 8 , the apparent molecular weight of the most active form of interferon is 15.5IcD to 16.5kD The apparent size of the interferon polypeptide may vary depending on the SDS-PAGE gel/buffer system used, as would be appreciated by the skilled person. The 15.5IcD to 16.5IcD size is apparent where the SDS-PAGE conditions include a Bis-Tris buffered gradient gel and MES running buffer and is further discussed in the accompanying examples.
Step iii) of the method of the invention provides removing the refolding buffer from the soluble refolded recombinant interferon polypeptide of ii).
Refolding buffer components can be removed from the soluble refolded recombinant interferon polypeptide using, for example, buffer-exchange chromatography in group-separation mode (e.g. using Sephadex G25 chromatography, Amersham Biosciences/GE Healthcare). The soluble refolded recombinant interferon polypeptide is at the same time reformulated into a biologically acceptable buffer; for example a PBS solution containing an appropriate quantity of a non-ionic detergent. An example of a buffer including such components is presented in the accompan y ing examples.
Step iv) of the method of the invention provides, optionally, purifying monomeric soluble refolded recombinant interferon polypeptide from the polypeptide of step iii).
After refolding and buffer exchange, the interferon polypeptide is soluble and substantially pure but some heterogeneity exists in the preparation, caused by a proportion of dimeric and aggregated protein species formed during the refolding process. Accordingly, where the formulation of the soluble refolded recombinant interferon polypeptide after buffer-exchange chromatography permits, the soluble heterogeneous interferon can optionally be applied to anion exchange media (eg.
Q-Sepharose, Amersham Biosciences/GE Healthcare). After application of the soluble interferon protein, two column washes using buffers with different ionic strength selectively elute the monomeric interferon protein and the higher molecular weight aggregated protein. A non-ionic detergent, for example Tween 8O 5 can be included in the buffers to guard against an)' further aggregation of the interferon pofypeptide arising, for example, from localised extremes of protein concentration when binding and eluting from the chromatography matrix. Fractions collected during the chromatography were analysed by SDS-PAGE to determine the best fractions to pool and subsequent to pooling, were concentrated and, if required, simultaneously reformulated by standard diafiltration methods known in the art.
Step iv) can be mandatory where it is necessary to ensure that the method of the invention provides monomeric recombinant interferon polypeptide.
All of the techniques discussed above are routine, as would be appreciated by a person skilled in the art.
As discussed above, many microbial expression systems are known. However, it is preferred that the denatured recombinant interferon polypeptide of step i) -is bacterially expressed.
Suitable bacterial expression systems that may be used to generate bacterially expressed denatured recombinant interferon are provided above and in the accompanying examples.
In an embodiment of the invention the method comprises the prior steps of:
i) suspending bacterially expressed insoluble recombinant interferon polypeptide in a stringent wash buffer comprising an alkaline pH buffer; a chaotropic agent; a readily dissociable salt; and a reducing agent;
ii) recovering the insoluble recombinant interferon polypeptide from the stringent wash buffer of step i);
iii) optionally, repeating steps i) and ii) up to three times;
iv) incubating the recovered insoluble recombinant interferon polypeptide of step ii) or iii) in a solublisation solution comprising a denarurant, an alkaline pH buffer, a reducing agent and optionally a detergent;
v) optionally, purifying monomeric soluble denatured recombinant interferon polypeptide from the polypeptide of step iv).
As discussed above, the heterologous expression of protein in microbial cells can lead to the expressed protein forming "inclusion bodies" of insoluble and incorrectly folded recombinant protein. Although inclusion bodies are comprised of relatively pure recombinant protein, nonetheless specific contaminating bacterial proteins are often present. This embodiment of the invention provides a process for removing some of the contaminating bacterial proteins from the recombinant interferon polypeptide.
The insoluble inclusion bodies comprising the bacterially expressed interferon polypeptide are washed in a stringent wash buffer to remove some of the contaminating insoluble cellular proteins and debris.
Washing can be accomplished by thorough resuspension of the insoluble pellet followed by harvesting of the remaining insoluble material containing the interferon polypeptide. Typically this process can be repeated up to three times and, at each stage, the suspension is passed through a homogeniser to ensure the complete dispersal of the insoluble material. The remaining insoluble material, substantially pure IFN-ccs, can be finally recovered by centrifugation. Such techniques are well known to those skilled in the art.
We have found that the following components are required in the stringent wash buffer: an alkaline pH buffer (pH > S. O), a chaotropic agent, a readily dissociable salt and reducing agent. An example of such a stringent wash buffer is provided in • the accompanying examples.
The stringent wash buffer comprises a chaotropic agent. Preferably this is > 4M urea, preferably between 4.0M to 8.9M urea. Alternatively, the stringent wash buffer may comprise guanidine hydrochloride at a concentration of between 1.5 and 4.5M.
The amount of salt that can be used in the stringent wash buffer can be 0.1 — 2.0M.
Examples of suitable reducing agents that can be used in the wash buffer include 2-mercaptoethanol, tris(2-carboxyethyl)phosphine hydrochloride, ditniothreitol or ditbioerythritol. The amount of reducing agent used may be between I uM to IM. Where the reducing agent is 2-mercaptoethanol, preferabfy the amount of 2- mercaptoethanol is 0.1 IM.
Once the insoluble fraction washing is complete, the remaining material is substantially pure insoluble interferon polypeptide. The insoluble interferon polypeptide may be analysed by SDS-PAGE and quantified using a commercially available colorimetric protein assay modified for use in the presence of SDS.
The procedure in step iv) of this embodiment of the invention solubilises the insoluble recombinant interferon polypeptide.
In this step the washed insoluble interferon polypeptide is dissolved in a solublisation solution containing a denaturant, an alkaline pH buffer, a reducing agent and optionally a detergent. It is preferred that the incubation time for this step is sufficient to allow at least 10% of the soluble polypeptide to become monomeric.
Examples of suitable denaturaiits that can be used in the solublisation buffer include guanidine hydrochloride or urea. Preferably the solublisation solution comprises 7M guanidine hydrochloride.
Examples of suitable reducing agents that can be used in the solublisation buffer include 2-mercaptoethanol, tris(2-carboxyethyl)phosphine hydrochloride, dithiothreitol or dithioerythritol. Preferably the solublisation buffer comprises 0.1% (v/v) 2-mercaptoethanol.
Denaturing size-exclusion chromatography studies examined the effect of time and temperature on the yield of monomeric denatured interferon polypeptide obtained by this step. Vi 7 MIe the formulation given in the accompanying example was found to maximise production of the soluble interferon polypeptide monomer essentially irrespective of the duration or temperature of the incubation, this was not true of other formulations (e.g. where there was no pH buffer or acidic pH buffer). Therefore, the composition of the solublisation solution can vary from that in the accompanying example and still be able to solubilise interferon polypeptide.
In optional step v), the monomeric component of the solublised denatured interferon polypeptide sample was separated from other forms (eg. higher molecular weight aggregates) by denaturing size-exclusion chromatograph}^ (eg. using Superdex 200 matrix, Amersham Biosciences/GE Healthcare) with the solublisation solution in the mobile phase.
The presence of monomeric interferon polypeptide in the fractions generated during size-exclusion chromatography was confirmed by SDS-PAGE analysis under non-reducing conditions and those containing monomeric interferon polypeptide were pooled. The approximate concentration of monomeric interferon polypeptide in the pool could be estimated by integration of the monomeric peak area on the UV-absorbance chromatogram generated during chromatography or by direct UV absorbance measurement of the pool. Ultrafiltration using filter units (eg. centrifugal, tangential-flow or pressurised stirred cell) comprising a low protein-binding membrane (eg. polyethersulphone) with a molecular weight cut-
off of 5000 daltons was then used to concentrate the pool to approximately lmg/ml for subsequent refolding.
Step v) can be mandatory where it is necessary to ensure that the method of the invention provides monomeric recombinant interferon polypeptide.
AU of the techniques discussed above are routine, as would be appreciated b)' a person skilled in the art.
In a further embodiment of this aspect of the invention the method also comprises the prior steps of:
i) expressing a nucleic acid molecule (optionally optimised for expression in bacteria) encoding recombinant interferon polypeptide in bacteria;
ii) harvesting the bacteria and isolating recombinant interferon polypeptide.
The steps provided in this embodiment of the aspect of the invention provide a source of the recombinant interferon polypeptide used in subsequent steps in tiie method of the invention.
Suitable methods for providing bacterial expression systems are described above, as are examples of nucleic acid molecules encoding interferon polypeptide.
By "nucleic acid" we include DNA, e.g., cDNA and genomic DNA 5 RNA 5 e.g. mRNA, and peptide nucleic acids (i.e. PNAs). Preferably the nucleic acid is DNA.
A segment of double-stranded DNA encoding the amino acid sequence of interferon can be synthesised (optionally to incorporate a codon-bias compatible with the microbial cell within which the interferon is to be expressed). An example of a DNA sequence encoding the amino acid sequence of wild-type secreted human IFN-αg and incorporating a codon-bias compatible with microbial
cells is given in Figure 1. Methods by which to optimise the codon-bias of a nucleic acid molecule are well known to those skilled in the art, and are provided in, for example, Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2001. 3rd edition.
The DNA can be ligated into plasmids designed for expression of proteins in cultured cells, using standard molecular biological methods known in the art. Plasmids were propagated in suitable laboratory strains (eg. strains of E. colϊ) and maintained during culture by a plasmid-bome selectable marker (eg. antibiotic resistance) compatible with production of clinical grade biopharmaceuticals.
The plasmids used preferably contain promoters, preferably strong tightry- regulated inducible promoters (eg. tip, lac, tac, λ) controlling transcription of the DNA encoding interferon polypeptide and a ribosome-binding site and translation initiation codon to permit production of the polypeptide. The design of the plasmid determines whether production of the polypeptide is directed to the extracellular environment, towards an internal compartment (eg. the periplasmic space) or towards the cytosol. These factors, as well as culture conditions, the intracellular environment and the nature of the polypeptide being produced determine whether the polypeptide accumulates in a soluble or insoluble form or partitions between the two.
Plasmid-bearing cells can be cultured to high density in a bioreactor (fermenter), optionally using animal-product free media. For high-density culture of microbial cells, media commonly comprise a peptone, yeast extract, a carbon source (eg. glucose), a pH buffer (eg. potassium phosphate), vitamin and trace element supplements and the target of the plasmid-borne selectable marker (eg. antibiotic); inert antifoam agents are also commonly added. As well as medium composition, fermentation conditions are determined empirically for the particular micro- organism being cultured but considerations will include the volume and cell density of the inoculum, temperature, pH, the rate of aeration (expressed as volumes of air per unit volume of culture per minute - wm), the level of dissolved ox3'gen (DO - expressed as a percentage) and the impeller speed (rpm). Samples
of the culture are taken at intervals both to assess the culture density, by measurement of optical absorbance, and for analysis using sodium dodec)'! sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, to monitor production of interferon polypeptide.
Cells are then typically recovered from fermentation cultures by centrifugation or filtration methods or a combination of the two. The resulting cell paste can be used immediately or stored frozen; cycles of freezing and thawing can be used to aid lysis of the cells. The cell paste is typically resuspended in cold, pH-neutral buffer containing deoxyribonuclease and the suspended cells lysed mechanically (eg. by several passes through a French-press or a pressurised shear-disrupter). The density at which the cells are resuspended prior to performing the lysis procedure is determined empirically and is dependent upon the method of lysis chosen. The soluble and insoluble components of the resulting suspension are separated (eg. by centrifugation) and the insoluble fraction containing the recombinant interferon polypeptide reserved.
By "interferon polypeptides" we include all human or non-human interferon polypeptides known in the art. For example, the thirteen TFN-α subtypes as well as IFN-β, IFN-K 5 IFN-λ, IFN-τ and IFN-ω subtypes discussed above. Preferably the interferon polypeptide is IFN-αg, preferably human IFN-αg.
As will be appreciated, the method of this aspect of the invention can be readily scaled up to produce large quantities of biologically active interferon polypeptide.
All documents referred to herein are hereby incorporated by reference.
The invention will now be described by reference to the following, non-limiting Examples and Figures.
Figure 1: A synthetic DNA sequence encoding IFN-cts codon-optimised for expression in E.coli and the amino acid sequence of recombinant IFN-αg. The
initial encoded methionine translation initiation residue is omitted from both sequences.
Figure 2: Growth curves and inducible IFN-αs expression profile. (A) Growth curve from a small-scale culture using an animal-product free medium of the E.coli strain expressing IFN-αs. The temperature shift from 3O 0 C to 42°C was performed at zero time; (B) SDS-PAGE analysis of the expression profile of IFN- αs. Taken from the small-scale culture shown in Figure 2A, samples were taken at different times after induction of the culture (shown in hours above the gel). The gel illustrates the emergence of a 23kD species after 4h and the presence of the same species in the crude inclusion body (IB) fraction.
Figure 3: SDS-PAGE analysis of size-exclusion chromatography of the denatured solublised IFN-αg. The denatured monomelic protein (23kD) is the predominant species in Lanes 7-9.
Figure 4: Purification of IFN-αg; SDS-PAGE analysis of anion-exchange chromatography of the refolded IFN-αg. Lane 1 shows the refolded IFN-αg before purification and Lanes 4 and 5 show the purified monomeric IFN-αs and the higher molecular weight aggregated forms, respectively. Lane 4 also illustrates the presence of a small quantity of the 18kD isoform, migrating above the main 16IcD isoform.
Figure 5: Production of the different IFN-αs isoforms; SDS-PAGE analysis of various samples from a refolding screen showing production variously of the 16kD, 18kD and 23kD species.
Figure 6: Anti-viral activity data; EMCV/A549 viral cytopathic assay. Viable cells are measured by their ability to take-up a staining compound. A high optical absorbance therefore indicates a large number of viable cells. The concentration of interferon tested is plotted as a logarithmic scale. The graph shows the EC50 curves for the 16kD, 18IcD and 23kD species generated in the refolding screen as well as the EC50 curve for IFN-α 2 . IFN-αg and IFN-α2 have approximately the
same molecular weight so the concentrations of each, measured in mass/unit volume, are directly comparable.
The assay presented above is based on a different virus/host combination to that in DiMarco supra. Commercially sourced IFN-α/? has an activity of approximately 2.7x10 s U/mg and, in the present assay, has an EC50 value of approximately 6xlO "3 ng/ml. The maximum value for the IFN-αs produced by
DiMarco supra is 4.IxIO 8 U/mg representing an increase in potency over IFN-σ. 2 of 1.5 fold, theoretically reducing the EC50 value to 4xlO '3 ng/ml. The EC50 for IFN-αg produced by the present method is approximately 1.7x10 "9 ng/ml in the same assa}', which theoretically equates to an activity of 9.5x10 14 U/mg.
Example 1: Large scale production of IFN-αs
A synthetic DNA fragment encoding IFN-αs was constructed with a codon-bias favouring expression of the encoded protein in E.coli cells. The DNA fragment was ligated into a proprietary plasmid at a unique Nde I site such that its transcription was under the control of the temperature-inducible λ promoter and such that the plasmid' s ribosome-bindϊng sequence and translation initiation codon were adjacent to the S3'nthetic fragment. The plasmid was used to transform E.coli B834 cells, upon which it conferred resistance to growth in the presence of kanamycrn.
The plasmid-bearing cells were grown at 30 °C in a 10 litre bioreactor and the cell density of the culture monitored by measurement of its optical density (OD) at a wavelength of 600nm (Figure 2A). On reaching a density of 3.7 OD unit, the temperature of the culture was shifted to 42°C to induce transcription of the synthetic DNA encoding IFN-α 8 from the promoter. The culture was maintained at
42°C for up to 18h to maximise the culture density. Expression of IFN-αs was directed to the cytosol, where it accumulated in the insoluble inclusion body (IB) fraction.
AB animal-product free medium was used for the fermentation, the principal components of which were a peptone made from pea-flour digested with fungal enzymes, yeast extract, glucose (as the carbon source), potassium phosphate (as a pH buffer) and kanamycin. The medium was aerated at 0.5wm and the impeller speed adjusted automatically up to a maximum of 800rpm in order to maintain DO at 40%. The wet weight yield of cells equated to 1.7g per litre per unit of OD. SDS-PAGE analysis of samples revealed that the product first appeared 4 hours after induction, migrating as a single band increasing hi intensity over time relative to the host cell proteins until about 7 hours after induction and migrating with an apparent molecular weight of approximately 23IcD 5 by comparison with protein standards of known size (Figure 2B).
Cells were harvested from the fermentation medium by centrifugation at 9,00Og for 20 min at 4°C and resuspended in ice-cold buffer containing 5OmM Tris/HCl pH8.0, 15OmM NaCl and 40U/ml benzonase at a suspension density of approximately 0.25g cells (wet weight) per ml. After three passes through a pressurised Emulsiflex C5 shear-disrupter, the insoluble fraction containing cell debris and IB was harvested by centrifugation at 9 3 000g for 15min at 4°C. The pellet of insoluble material was then resuspended in stringent wash buffer at a suspension density of 0.25g pellet (wet weight) per ml and the suspension passed- through the shear-disrupter to ensure complete dispersal of the insoluble material. The remaining insoluble material was recovered by centrifugation at 9 5 000g for 15 min at 4 0 C and wash procedure repeated using the stringent wash buffer. The formulation of the stringent wash buffer was 55mM Tris/HCl pH 8.0, 8.9M urea, 1. IM NaCl and 0.1 IM 2-mercaptoethanol. The insoluble material was sampled for analysis by SDS-PAGE (Figure 3) and for quantification using an SDS-Bradford assay. Typically, 20mg of washed protein was recovered per litre of culture per OD unit (βOOnm) at harvest.
The washed insoluble IFN-αg was dissolved at a concentration of lOmg/ml in a solublisation solution containing 5OmM Tris/HCl pH S. O 5 7M guanidine hydrochloride and 0.1% (v/v) 2-mercaptoethanol and incubated at 22°C for 1 hour before being subjected to size-exclusion chromatography, collecting fractions.
Those fractions identified by SDS-PAGE analysis as containing the monomelic
IFN-αs (Figure 3) were pooled. The mononieric protein represented at least 40% of the total protein applied to the chromatography column and underwent a dilution of approximately 10-fold during size-exclusion chromatography. The pool of denatured monomelic IFN-αg was concentrated by ultrafiltration to 1 - 2 mg/ml then diluted in 20 volumes of a refolding solution, pre-equilibrated to 4 0 C 5 containing 52 mM Tris/HCl pH 8.0, 1OmM NaCl, 0.4mM KCl 5 2mM CaCl 2 , 2mM MgCl 25 522mM L-arginineHCl, ImM DTT and 0.02% Tween 80. The refolding solution was incubated stirring for 5h at 4°C then subjected to Sephadex G-25 desalting chromatography to remove- the refolding buffer components and reformulate the soluble TFN-αg in 2OmM Tris/HCl pH8.0, 15OmM NaCl and 0.02% (v/v) Tween 80. In this invention, the refolded reformulated IFN-αs was applied to Q-Sepharose Fast Flow (Amersham Biosciences) pre-equilibrated in 2OmM Tris/HCl pH8.0 containing 15OmM NaCl and 0.02% (v/v) Tween 80 and the monomeric IFN-αs selectively eluted in 2OmM Tris/HCl pH 8.0 containing 25OmM NaCl and 0.02% (v/v) Tween 80. Any aggregated higher molecular weight IFN-αs was then washed from the column using 2OmM Tris pH 8.0 containing 55OmMNaCl and 0.02% (v/v) Tween 80 (Figure 4).
Determination of the EC50 value to assess the relative anti-viral potency Of different IFNs requires the preparation of a dilution series for each sample. The degree of dilution necessary to obtain the EC50 value renders the initial formulation of the IFN almost irrelevant as the highest concentration of protein in a dilution series is typically 1000 ng/ml (10 "6 g/ml) and EC50 values are typically less than 1 ng/ml (10 "9 g/ml). In a typical assa3', the EC50 value for IFN-α 2 is in the range 10 "2 - 10 "3 ng/ml whether assaying the International Standard or a sample of clinical grade material. In the same assay, the EC50 value for IFN-αs prepared according to this invention is typically in the range 10 '11 - 10 "4 ng/ml (Figure 6).
Example 2: Assay of anti-viral activity of IFN-αs
The anti-viral potency of type I interferons is determined by measuring the concentration of interferon required to afford 50% protection against the
cytopatliic effect of a given virus (eg. encephalomyocarditis virus, EMCV) on interferon-responsive, virus-sensitive, cultured cells (eg. human lung carcinoma- derived A549 cells). Typically, growing cells are seeded at a uniform cell density in a multi-well culture plate containing a range of dilutions of the test interferon and of a standard interferon. Certain wells are used as controls for the experiment and contain no interferon. After a 24 hour incubation the culture supernatant is replaced to remove the interferon, and the cells are challenged with virus, prepared at a standard concentration (measured in plaque-forming units/ml, pfu/ml) demonstrated to kill any untreated or unprotected cells. Again virus is omitted from certain wells to ensure that untreated, uninfected cells remain viable during the assay. The potency can be expressed as an EC50 value (the concentration that is 50% inhibitory) or in terms of the International standard interferon unit (IU).
International Standard samples of interferons which have received regulatory approval (eg. IFN-CX 2 ) are available for comparative purposes. The monomeric IFN-αs produced from the methods of the invention was found to have an EC50 value lower than the international standard sample of IFN-α 2 .
Example 3: Refolding of IFN-α$ by a two-step method
The pool of denatured monomeric IFN-αs was diluted in a cold refolding buffer and left stirring at 4°C to allow the denatured soluble IFN-αg to refold. In this invention, extensive screening of refolding conditions was undertaken to identify the key components necessary to maximise the jάeld, homogeneity and activity of the refolded IFN-αg. Refolding buffers that yielded monomeric refolded protein, assessed by SDS-PAGE under non-reducing conditions and by size-exclusion chromatography under non-denaturing conditions, were found variously to generate species migrating on SDS-PAGE with apparent molecular weights of 16kD, 18kD and 23kD (Figure 5). Bioassay data revealed that the most potent form of the refolded IFN-αs migrated with an apparent molecular weight of approximately 16kD, and that the 18kD species retained a high proportion of this bioactivity. The 23kD species was found to be only moderatefy active in comparison (Figure 6). It was found that refolding buffer that contained a polar
J-3 excipient (eg. arginine) and a detergent (eg. Tween 80) favoured the production of soluble refolded protein and that buffers containing an alkaline pH buffer (eg-.Tris) divalent cations (eg. Ca +"1" or Mg +4 ) and a redox reagent (eg. cysteine, cystine or DTT) favoured the production of the 16IcD species; lower pH buffers appeared to favour production of the 18IcD species. The target concentration range for the protein after dilution was 50 - 100 ug/ml, Dilution of the denatured monomeric IFN-αg into the refolding buffer formed the first phase of the refolding process. After incubation at 4 0 C 5 refolding buffer components were removed using buffer- exchange chromatograplry in group separation mode (eg. using Sephadex G25, Amersham Biosciences/GE Healthcare) and simultaneously trie soluble IFN-αg was re-formulated in preparation for the subsequent stage of the process.
Example 4: SDS-PAGE analysis of IFN-α s
The different isoforms of IFN-αg referred to as 16IcD 5 18IcD and 23kD and illustrated in Figure 5 5 were distinguished by SDS-PAGE analysis using the commercially available system NuPAGE (Invitrogen). Recipes for gels, running buffers and sample buffers are also provided by Invitrogen. An alternative BisTris SDS-PAGE gel system that could be used to determine the size of IFN-αs isoforms is Criterion XT 5 supplied by Bio-Rad Laboratories (www.bio- rad.com).
Protein samples were first prepared for analysis under non-reducing conditions by mixing with distilled water and gel sample buffer (NuPAGE 4xLDS, supplied at 4x working concentration and diluted four-fold during sample preparation), incubated at 95 0 C for 5 min then at 4 0 C or on ice for 5 min. Samples were collected by brief centrifugation, eg.l min at 13,00Og at room temperature, then vortex-mixed before gel anafysis. The final concentration of protein in the samples was typically in the range 20ng/ul - lug/ul and typically 5ul or lOul of sample were applied per lane on a gel.
lmm thick, pre-cast NuPAGE BisTris-buffered gels containing a gradient of polyacrylamide concentration from 4% - 12% and typically formed with 12
sample wells, were used for sample electrophoresis in combination with NuPAGE
MES running buffer, supplied at 2Ox working concentration and diluted with distilled water immediately prior to use (5OmM MES; 5OmM Tris base; 0.1% SDS; ImM EDTA; pH 7.3). Each sample well was flushed with running buffer immediately prior to sample application and proteins typically separated by electrophoresis at 200V (constant voltage) for up to 45 min. After electrophoresis, gels were stained by immersion and gentle agitation for at least 1 hr in a solution of Coomassie Brilliant Blue R-250 dissolved at a final concentration of 0.2% (w/v) in an aqueous solution of 40% (v/v) methanol and 15% (v/v) acetic acid. The gels were then background-destained, typically over a period of 24h ; by immersion and gentle agitation in a solution of 20% (v/v) methanol and 7.5% (v/v) acetic acid, which was changed several times.
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