WO2025210641A1 - Interleukin 1-receptor antagonist with increased solubility and process of preparation thereof - Google Patents

Interleukin 1-receptor antagonist with increased solubility and process of preparation thereof

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Publication number
WO2025210641A1
WO2025210641A1 PCT/IN2024/050358 IN2024050358W WO2025210641A1 WO 2025210641 A1 WO2025210641 A1 WO 2025210641A1 IN 2024050358 W IN2024050358 W IN 2024050358W WO 2025210641 A1 WO2025210641 A1 WO 2025210641A1
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ira
interleukin
seq
receptor antagonist
expression
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French (fr)
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Rahul Sharad BHAMBURE
Tejas Prakash Chirmade
Sunil Rana
Neeraj Nainwal
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Council of Scientific and Industrial Research CSIR
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Council of Scientific and Industrial Research CSIR
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Priority to PCT/IN2024/050358 priority Critical patent/WO2025210641A1/en
Publication of WO2025210641A1 publication Critical patent/WO2025210641A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/545—IL-1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the present disclosure relates to the field of recombinant protein expression and purification using bacterial hosts.
  • the present invention relates to a production process for obtaining high levels of soluble recombinant interleukin 1 -receptor antagonist (IL- IRa) protein from E. coli.
  • the invention also relates to purification and characterization methods for interleukin 1-receptor antagonist (IL-IRa) as well as uses of the interleukin 1-receptor antagonist (IL-IRa) produced by the method.
  • IL-1 and IL-IRa The balance between IL-1 and IL-IRa in local tissue influences the possible development of inflammatory disease and resultant structural damage.
  • inflammatory and autoimmune diseases may develop in many organs, such as the joints, lungs, gastrointestinal tract, central nervous system (CNS), or blood vessels.
  • CNS central nervous system
  • Treatment of human disease with IL-IRa has been carried out by injection of recombinant protein or using gene therapy approaches. This chapter will review background information on the members of the IL-1 family, the role of IL-1 and IL-IRa in normal physiology and disease, and the results of clinical trials with the administration of IL-IRa in human disease.
  • the primary object of the present invention is to provide soluble form of interleukin 1 -receptor antagonist.
  • Another object of the present invention is to provide a gene sequence for synthesis of soluble form of interleukin 1 -receptor antagonist.
  • Another object of the present invention relates to a process for the synthesis of soluble form of interleukin 1 -receptor antagonist.
  • Another object of the present invention relates to a pharmaceutical composition comprising soluble form of interleukin 1 -receptor antagonist.
  • the present invention overcomes the problems and disadvantages associated with current strategies and designs and provide new compositions and methods for producing interleukin 1 -receptor antagonist (IL-IRa).
  • IL-IRa interleukin 1 -receptor antagonist
  • the present invention relates to a high-level soluble expression of interleukin 1 -receptor antagonist (IL-IRa) and method of producing a recombinant interleukin 1 -receptor antagonist (IL-IRa) protein (SEQ ID NO. 1) in a E. coli, said method comprising: ligating into a vector a-nucleotide sequence encoding a interleukin 1 -receptor antagonist (IL-IRa); transforming the E. coli. host cell; followed by culturing the transformed E.
  • IL-IRa interleukin 1 -receptor antagonist
  • coli host cell in a culture media suitable for the expression of the recombinant interleukin 1-receptor antagonist (IL-IRa) protein through which a recombinant interleukin 1-receptor antagonist (IL-IRa) protein is obtained.
  • IL-IRa interleukin 1-receptor antagonist
  • a first aspect provides a codon-optimized polynucleotide sequence encoding interleukin 1-receptor antagonist comprising SEQ ID NO. 2.
  • a second aspect provides an expression vector comprising a codon -optimized polynucleotide according to the first aspect.
  • a third aspect provides a process for producing IL-IRa, comprising the steps of: a) inserting or transforming into a host cell a codon-optimized polynucleotide according to the first aspect ligated into a suitable vector or an expression vector according to the second aspect; b) culturing the transformed host cell in a culture medium for high-level expression of IL-IRa; c) maintaining an induction temperature between 10 to 40°C to produce IL-IRa; d) extracting IL-IRa from the transformed host cell; and e) purifying IL-IRa to obtain IL-IRa polypeptide with high yield using multimodal chromatography.
  • a fourth aspect provides IL-IRa when produced by a process according to the third aspect.
  • IL-IRa Disclosed herein is an optimized polynucleotide for high-level expression of IL-IRa.
  • Also disclosed is a process for the production of a polypeptide comprising steps of: a) selecting a codon optimized polynucleotide sequence essentially consisting of SEQ ID NO. 2 or its variants which are at least 70% homologous to SEQ ID NO.2, b) optionally ligating the polynucleotide sequences of step (a) into a suitable vector, c) inserting or transforming the polynucleotide sequence into Escherichia coli host cell, d) culturing the transformed host cell in a culture media for high-level expression of the polypeptide, e) maintaining the induction temperature between 10 to 40 °C to produce polypeptide, f) extracting the bacterial polypeptide from the host cell, followed by purification to obtain pure polypeptide with high yields.
  • the purification of the folded IL-IRa comprises: a) subjecting the supernatant of step (g) to multimodal anion exchange chromatography phase I in a flow-through mode, by feeding the supernatant having pH in the range of 3.0 to 5.0 to multimodal anion exchange chromatography resin, with a buffer comprising 10 - 50 mM sodium acetate and 0.1 - 1.0 M sodium chloride and eluting in the presence of an elution buffer using a step salt gradient having a salt concentration gradient of 5-10 CV; and b) subjecting the folded and soluble IL-IRa obtained from step a having pH of 3.0 to 5.0to multimodal anion exchange chromatography phase II in a flow-through mode, and eluting in the presence of an elution buffer using a step salt gradient having a salt concentration gradient of 5-20 CV to obtain folded and solubilized IL-IRa polypeptide.
  • Fig. 3a and 3b illustrates the SDS-PAGE analysis of soluble fraction and insoluble pellet of E. coli expressing IL-IRa.
  • Fig. 13 illustrates intact mass analysis based on the MALDI-TOF profiles for both the present IL-IRa and the innovator molecule.
  • the nucleotide sequence has been optimized for expression in the E. coli host cell. In related embodiments, the nucleotide sequence has been optimized for expression in the E. coli host cell. In other related embodiments, the nucleotide sequence has been optimized for expression in the E. coli host cell.
  • the invention provides an optimized polynucleotide sequence (SEQ ID NO. 2) and its structural variants selected from but not limited to SEQ ID NO. 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 and 16 having equal to or more than 70% similarity to SEQ ID NO. 2 useful for high level expression of polypeptide for IL-lRa.
  • SEQ ID NO. 2 an optimized polynucleotide sequence
  • structural variants selected from but not limited to SEQ ID NO. 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 and 16 having equal to or more than 70% similarity to SEQ ID NO. 2 useful for high level expression of polypeptide for IL-lRa.
  • Induction of expression refers to the step performed to induce the expression from the polynucleotide so that the product is obtained at an accelerated rate, this may involve addition of suitable inducing agent such as IPTG, arabinose, maltose, lactose, galactose and the like.
  • polynucleotide of the invention may also be associated with polynucleotides of tag polypeptides.
  • a tag is also known to enhance the stability and solubility of the protein in the cytoplasm and for its subsequent purification.
  • a hydrophobic tag can also be introduced at the N-terminal to induce the expression in the form of inclusion bodies.
  • These tags can be associated at 5’ terminus or 3’ terminus, singly or in combination pertinent to multi-tagging, with an oligonucleotide sequence that encodes a tag polypeptide to facilitate its cytoplasmic stability and/or subsequent purification using matrices and resins with a high affinity for the various tag peptides.
  • the host cell is a gram negative host cell. Host cell expression systems like Escherichia coli, Bacillus sp., Pseudomonas sp., have been extensively discussed in the production of proteins.
  • the polynucleotide of the invention is preferably used for the intracellular soluble expression of present IL-lRa in Escherichia coli wherein the host strain is selected from BL21 (DE3), BL21 Al, HMS174 (DE3), DH5a, W3110, B834, origami, Rosetta, NovaBlue (DE3), Lemo21 (DE3), T7, ER2566 and C43 (DE3).
  • the present invention provides a polynucleotide sequence (SEQ ID NO. 2) encoding interleukin 1 -receptor antagonist which is optionally ligated into a vector, followed by its insertion in a host cell.
  • the insertion into host cell may be performed by any of the methods known in the art. Such an insertion or transformation may be performed by a physical or a chemical method of transformation. Subsequently, the converted colonies are selected on petri dishes with the added antibiotic.
  • Suitable vectors used in the present invention include but not limited to pET9a, pET3a, pET3b, pET3c, pET5a, pET5b, pET5c, pET9b, pET9c, pET12a, pET28a, pET28b, pET28c, pET30a, pET30b, pET30c, pTWIN 1, pTWIN2, pET12b, pET12c, pET17b and in general, all the vectors that have a strong phage T7 promoter (e.g.
  • the protein can also be expressed in the vectors derived from the vectors mentioned with a change in the regulatory region and/or the promoter and may include but not limited to pL, rhaBAD, and rhaT.
  • the polynucleotide encoding the full length IL-lRa protein is cloned adjacent to T7 lac promoter that drives the expression of protein in T7 polymerase positive strains of Escherichia coli.
  • the expression of polynucleotide is stringently controlled by T7 promoter which is induced in the presence of IPTG, natural sugars (other than glucose) or in auto-induction mediums.
  • the selected IL-lRa product is produced using a batch, fed-batch, or continuous fermentation process.
  • the complex fermentation media used for the expression of IL-IRa is HiVeg Tartoff Broth.
  • the harvested material is an E. coli (BL-21 DE3) harvest with an expressed IL-IRa in soluble form.
  • the ingredients of media includes, but is not limited to, a carbon source such as, e.g., glucose, sucrose, or glycerol, organic nitrogen source, such as peptone, tryptone, amino acids, or a yeast extract, inorganic nitrogen source is used and this may be selected from among, e.g., ammonium salts, aqueous ammonia, and gaseous ammonia, supplements as supplemented with, e.g., low levels of amino acids, vitamins, peptones, or other ingredients,
  • a carbon source such as, e.g., glucose, sucrose, or glycerol
  • organic nitrogen source such as peptone, tryptone, amino acids, or a yeast extract
  • inorganic nitrogen source is used and this may be selected from among, e.g., ammonium salts, aqueous ammonia, and gaseous ammonia, supplements as supplemented with, e.g., low levels of amino acids, vitamins, peptones, or
  • the expression of Interleukin- 1 -receptor is induced using IPTG.
  • Interleukin- 1 -receptor is induced using natural sugars like galactose and lactose.
  • the transformed host cells may be tested for expression on small volumes such as 5- 100 ml in LB, terrific broth or chemically defined medium.
  • the expression may be subjected to different concentrations of inducers ranging from about 0.01 mM, about 0.05 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM and about 1 mM.
  • the expression may be subjected to different concentrations of inducers ranging from about 1 mM, about 2.5 mM, about 5 mM, about 7.5 mM, about 10 mM, about 12.5 mM, about 15 mM, about 17.5 mM, and about 20 mM.
  • the polypeptide expression is determined in an electrophoretic set up, preferably in SDS PAGE electrophoresis and viewed as over-expressed bands stained with coomassie brilliant blue (-).
  • the host cells are inoculated in 500 mL flasks cultures or small scale bioreactors with media volume of about 500 mL, about 1000 mL, about 1500 mL and about 2000 mL; and allowed to grow under optimal conditions, during which IL-IRa expression continued from 16 hours to 32 hours under batch or fed -batch condition.
  • the cells After culturing in constant agitation and preferably under aerobic conditions, the cells are harvested, and lysed. Any of known methods may be applied to lyse cells, preferred method includes a lysis buffer containing a detergent at an appropriate concentration. After lysis, the protein component is pooled in one or more centrifugation steps.
  • Lysis is carried out in a buffer containing Tris-HCl 20-50 mM pH 7.0-9.0, NaCl 100-150 mM, detergent 0.5-1.5% and protease inhibitor 0.5-1.5%, with agitation.
  • the lysis is carried out in a buffer containing sodium acetate 10-50 mM, pH 3.0- 5.5, NaCl 50-300 mM, glycerol 1-5%, EDTA l-5mM using a cell disrupter.
  • induction temperatures for expression is carried out between 10 to 40 °C.
  • the expression of IL-IRa is in soluble form over the broad range of induction temperatures unless accompanied with a hydrophobic tag, where the protein expression is in insoluble inclusion bodies.
  • the invention thus involves more than one subsequent purification steps, and also exploits pl value of interleukin 1 receptor antagonist in mixed-mode or multimodal ion exchange chromatographic step, whereby it is separated from other contaminating proteins. Finally, the quantity of IL-IRa is quantified by RP-HPLC and visualized inl2% acrylamide gel (SDS-PAGE).
  • the HCP log reduction achieved using the step low pH cell lysis is not less than 0.5.
  • the lysate is continuously centrifuged at 6000-9000 rpm for 40 min for single or multiple times.
  • the Interleukin- 1 receptor antagonist (IL-IRa) centrifuged supernatant is always purest as compared to homogenized cell harvest.
  • there suspended cell pellet is homogenized using a high-pressure homogenizer and the outlet can be directly connected to the inlet of the continuous centrifuge to separate soluble fractions and the centrifuge outlet can be connected to multimodal anion exchange chromatography.
  • residence time for multimodal anion exchange chromatography and multimodal cation exchange chromatography is more than 15 seconds.
  • multimodal anion exchange chromatography or multimodal cation exchange is performed using the axial or radial flow chromatography column.
  • the multimodal anion exchange chromatography includes a resin/membrane selected from a group consisting of HEA HypercelTM, PPA HypercelTM, and BAKERBONDOXWP 500 Poly PEI-35, CaptoTM adhere or CaptoTM adhere ImpRes resin.
  • the total amount of interleukin 1 receptor antagonist (Il-IRa) product passed through the single multimodal anion exchange chromatography column is not less than 10 g/L resin.
  • the IL-IRa polypeptide is produced in a continuous fermentation process.

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Abstract

The present invention is directed to the cells, compositions, and methods for the production of and purification of recombinant protein. In particular, the invention is directed to have enhanced soluble expression of interleukin 1-receptor antagonist by means of an optimized novel polynucleotide sequence and host transformed with the said polynucleotide. The invention also provides a method for increased production and purification of interleukin 1-receptor antagonist (IL-1Ra) wherein, the polynucleotide of the present invention is used to transform a suitable host resulting in over-expression of corresponding proteins and a method for isolating the expressed polypeptide. The invention also relates to the purification method for of interleukin 1-receptor antagonist (IL-1Ra) using multimodal chromatography as well as characterization of properly folded of interleukin 1-receptor antagonist (IL-1Ra) protein.

Description

INTERLEUKIN 1-RECEPTOR ANTAGONIST WITH INCREASED SOLUBILITY AND PROCESS OF PREPARATION THEREOF
FIELD OF THE INVENTION
The present disclosure relates to the field of recombinant protein expression and purification using bacterial hosts. In particular, the present invention relates to a production process for obtaining high levels of soluble recombinant interleukin 1 -receptor antagonist (IL- IRa) protein from E. coli. The invention also relates to purification and characterization methods for interleukin 1-receptor antagonist (IL-IRa) as well as uses of the interleukin 1-receptor antagonist (IL-IRa) produced by the method.
BACKGROUND OF THE INVENTION
The IL-1 family of molecules consists of two agonists, IL- la and IL-ip, a specific receptor antagonist called IL-IRa, and two different receptors, IL-1R type I (IL-1RI) and IL-1R type II (IL-1RII). IL-1 plays an important role in host resistance against microorganisms that divide inside cells, such as mycobacteria or listeria. The IL-1 family is an important part of the innate immune system, which regulates the functions of the adaptive immune system.
The balance between IL-1 and IL-IRa in local tissue influences the possible development of inflammatory disease and resultant structural damage. In the presence of an excess amount of IL-1, inflammatory and autoimmune diseases may develop in many organs, such as the joints, lungs, gastrointestinal tract, central nervous system (CNS), or blood vessels. Treatment of human disease with IL-IRa has been carried out by injection of recombinant protein or using gene therapy approaches. This chapter will review background information on the members of the IL-1 family, the role of IL-1 and IL-IRa in normal physiology and disease, and the results of clinical trials with the administration of IL-IRa in human disease.
An important proinflammatory role for IL- 1 in many human diseases has been described over the past 10 years. The balance between IL-1 and IL-IRa has been extensively studied in a variety of experimental animal models of disease, including arthritis, inflammatory bowel disease (IBD), granulomatous and fibrotic lung disorders, kidney diseases, diseases of the liver and pancreas, graft-versus-host disease (GVHD), leukemia and cancer, osteoporosis and diabetes, central nervous system diseases, infectious diseases, and arterial diseases. In each of these diseases, either local overproduction of IL-1 and/or underproduction of IL-IRa predisposes to the development of disease and the therapeutic administration of IL-IRa is efficacious in preventing tissue damage.
Extensive studies indicate the presence of IL-IRa in the joints of rheumatoid patients, but the amounts produced are insufficient to inhibit the injurious effects of IL-1. IL-IRa is present in the rheumatoid synovium, particularly in the lining layer cells and in the sublining area in a perivascular distribution within macrophages. High levels of IL-IRa were present in rheumatoid synovial fluid, produced by both neutrophils and macrophages. The balance between IL-1 and IL-IRa in synovial fluids is clinically important as acute knee arthritis in patients with Lyme disease resolved more rapidly in the presence of an excess amount of IL-IRa over IL-1. IL-IRa blocked the in vitro effects of IL-1 on PGE2 and collagenase production by human synovial cells and reversed the inhibition of proteoglycan synthesis in articular cartilage. Although IL-IRa is abundant in the rheumatoid synovial fluid and tissue, insufficient IL-IRa is produced by rheumatoid synovium to effectively block the biological effects of the locally produced IL- 1.
IL-IRa can be produced at a commercially relevant scale using recombinant DNA technology. The gene encoding IL-IRa is either isolated or synthesized and incorporated into an expression vector. This construct is introduced into a suitable expression host by one of the many methods available for DNA transformation. The protein expression in the host is enabled by an action of an inducer. The expressed recombinant protein is later on extracted from the cell lysate and further purified to yield the drug substance.
One of published PCT application WO1996009323A1 discloses DNA molecules coding for improved IL-1 antagonists inserted into expression vectors and host cells transformed with the said vectors containing the DNA coding for improved IL-1 antagonists and a method for the production of improved IL-1 antagonists in essentially pure form. Another patent CA2843197A1 covers methods and compositions for the preparation and delivery of chimeric cytokine proteins, including cell cultures, methods of purification, and purified compositions. However, the soluble expression of the gene sequence of of IL-IRa in said PCT application and CA patent is different than the IL-IRa in the present application, and that to reported with lesser yields and purity.
Thus, there is need for solving said requirement and problem to produce IL-IRa in higher yield, soluble form and with better purity, which the inventors of present case accomplished by providing highly soluble and purified form of interleukin 1 -receptor antagonist with high-level soluble expression of interleukin 1 -receptor antagonist (IL-IRa). OBJECTS OF THE INVENTION
The primary object of the present invention is to provide soluble form of interleukin 1 -receptor antagonist.
Another object of the present invention is to provide a gene sequence for synthesis of soluble form of interleukin 1 -receptor antagonist.
Another object of the present invention relates to a process for the synthesis of soluble form of interleukin 1 -receptor antagonist.
Another object of the present invention relates to a pharmaceutical composition comprising soluble form of interleukin 1 -receptor antagonist.
SUMMARY OF THE INVENTION
The present invention overcomes the problems and disadvantages associated with current strategies and designs and provide new compositions and methods for producing interleukin 1 -receptor antagonist (IL-IRa).
The present invention relates to a high-level soluble expression of interleukin 1 -receptor antagonist (IL-IRa) and method of producing a recombinant interleukin 1 -receptor antagonist (IL-IRa) protein (SEQ ID NO. 1) in a E. coli, said method comprising: ligating into a vector a-nucleotide sequence encoding a interleukin 1 -receptor antagonist (IL-IRa); transforming the E. coli. host cell; followed by culturing the transformed E. coli host cell in a culture media suitable for the expression of the recombinant interleukin 1-receptor antagonist (IL-IRa) protein through which a recombinant interleukin 1-receptor antagonist (IL-IRa) protein is obtained.
A first aspect provides a codon-optimized polynucleotide sequence encoding interleukin 1-receptor antagonist comprising SEQ ID NO. 2.
A second aspect provides an expression vector comprising a codon -optimized polynucleotide according to the first aspect.
A third aspect provides a process for producing IL-IRa, comprising the steps of: a) inserting or transforming into a host cell a codon-optimized polynucleotide according to the first aspect ligated into a suitable vector or an expression vector according to the second aspect; b) culturing the transformed host cell in a culture medium for high-level expression of IL-IRa; c) maintaining an induction temperature between 10 to 40°C to produce IL-IRa; d) extracting IL-IRa from the transformed host cell; and e) purifying IL-IRa to obtain IL-IRa polypeptide with high yield using multimodal chromatography.
A fourth aspect provides IL-IRa when produced by a process according to the third aspect.
Disclosed herein is an optimized polynucleotide for high-level expression of IL-IRa.
Also disclosed is a tunable process for extraction of the polypeptide to obtain IL-IRa in soluble form, excluding major E. coli host cell proteins.
Also disclosed is a tunable process for controlling the expression of the polypeptide as to obtain an IL-IRa.
Also disclosed is a high-level expression process for commercial production of IL-IRa in pure form with high yield.
Also disclosed is a process for the production of a polypeptide, comprising steps of: a) selecting a codon optimized polynucleotide sequence essentially consisting of SEQ ID NO. 2 or its variants which are at least 70% homologous to SEQ ID NO.2, b) optionally ligating the polynucleotide sequences of step (a) into a suitable vector, c) inserting or transforming the polynucleotide sequence into Escherichia coli host cell, d) culturing the transformed host cell in a culture media for high-level expression of the polypeptide, e) maintaining the induction temperature between 10 to 40 °C to produce polypeptide, f) extracting the bacterial polypeptide from the host cell, followed by purification to obtain pure polypeptide with high yields.
Accordingly, the present invention provides a codon-optimized polynucleotide sequence encoding IL-IRa, said sequence comprising SEQ ID NO. 2 or structural variants thereof having at least 70% homology to SEQ ID NO.2. structural variants are selected from a group consisting of SEQ ID NO. 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 and 16. The present invention provides an expression vector construct comprising (a) codon-optimized polynucleotide sequence encoding IL-IRa, said sequence comprising SEQ ID NO. 2 or structural variants thereof having at least 70% homology to SEQ ID NO.2; and (ii) one or more regulatory sequences selected from the group consisting of a suitable promoter, origin of replication, ribosomal binding site, transcription termination sequence, selectable markers and multiple cloning sites.
In a preferred embodiment of the present invention, the expression vector construct is a plasmid selected from the group consisting of pUC57, pET9a, pET3a, pET3b, pET3c, pET5a, pET5b, pET5c, pET9b, pET9c, pET12a, pET28a, pET28b, pET28c, pET30a, pET30b, pET30c, pTWIN 1, pTWIN2, pET12b, pET12c, pET17b, pRSET A, pRSET B, pRSET C, pTYBl, pTYB2, pTYB3, pTYB4, pL, rhaBAD, and rhaT.
The present invention provides a process for producing soluble form of interleukin 1 -receptor antagonist (IL-IRa), said process comprising the steps of: a) providing an expression vector construct comprising a codon-optimized polynucleotide sequence encoding IL-IRa comprising SEQ ID NO. 2 or structural variants thereof having at least 70% homology to SEQ ID NO.2; b) transforming a host cell with the expression vector construct of step a); c) selecting the transformed host cells; d) culturing the transformed host cell in a culture medium followed by inducing the expression of IL-IRa; e) maintaining an induction temperature between 10 to 40°C in the culture medium to produce IL-IRa; f) harvesting the the transformed host cells from the culture medium of step e), followed by resuspension in lysis buffer followed by cell lysis and centrifugation at 6000-9000 rpm to obtain soluble fraction of IL-IRa in homogenate cell supernatant solution; g) filtering the homogenate cell supernatant solution containing the soluble fraction of IL-IRa; and h) purifying the folded and soluble IL-IRa from the homogenate cell supernatant solution to obtain the folded and soluble IL-IRa polypeptide with >70% yield using multimodal chromatography.
In an embodiment of the present invention, the host cell is selected from the group consisting of Escherichia coli strains BL21 (DE3), BL21 Al, HMS174 (DE3), DH5a, W3110, B834, Lemo21 (DE3), T7, ER2566 and C43 (DE3).
In an embodiment of the present invention, the codon-optimized polynucleotide sequence encoding IL-IRa is cloned adjacent to T7 lac promoter; wherein the T7 lac promoter is induced by the presence of IPTG, galactose, lactose or auto-induction medium.
In an embodiment of the present invention, the purification of the folded IL-IRa comprises: a) subjecting the supernatant of step (g) to multimodal anion exchange chromatography phase I in a flow-through mode, by feeding the supernatant having pH in the range of 3.0 to 5.0 to multimodal anion exchange chromatography resin, with a buffer comprising 10 - 50 mM sodium acetate and 0.1 - 1.0 M sodium chloride and eluting in the presence of an elution buffer using a step salt gradient having a salt concentration gradient of 5-10 CV; and b) subjecting the folded and soluble IL-IRa obtained from step a having pH of 3.0 to 5.0to multimodal anion exchange chromatography phase II in a flow-through mode, and eluting in the presence of an elution buffer using a step salt gradient having a salt concentration gradient of 5-20 CV to obtain folded and solubilized IL-IRa polypeptide.
The purification of the folded IL-IRa comprises the steps of: a) subjecting a supernatant containing folded and soluble IL-IRa from step (g) to multimodal chromatography phase I in a flow-through mode, said chromatography phase I process comprising: (i) feeding and eluting the supernatant containing folded and soluble IL-IRa having pH in the range of 3.0 to 5.0 to multimodal anion exchange chromatography resin, with a buffer comprising 10 - 50 mM sodium acetate and 0.1 - 1.0 M sodium chloride; and (ii) eluting soluble fraction of IL-IRa in the presence of an elution buffer comprising 10 - 50 mM sodium acetate along with 0.1 - 1.0 M sodium chloride with pH ranging from 3.0 to 5.0 into an elution pool, wherein, the elution is effected using a step salt gradient having a salt concentration gradient of 5-10 CV; and b) Subjecting the folded and soluble IL-IRa obtained from step a(ii) to multimodal chromatography phase II in bind-elute mode, said chromatography phase II process comprising: (i) feeding and eluting the properly folded and solubilized IL-IRa from step a(ii) having pH of 3.0 to 5.0 to multimodal anion exchange chromatography resin in a flow-through mode, with a buffer comprising 20 mM sodium acetate with or without 1.0 M sodium chloride; and (ii) eluting soluble fraction of IL-IRa in the presence of an elution buffer comprising 20 mM sodium acetate with or without 1.0 M sodium chloride with pH ranging from 3.0 to 5.0 into an elution pool, wherein, the elution is effected using a step salt gradient having a salt concentration gradient of 5-20 CV. In an embodiment of the present invention, the multimodal chromatography is in batch, integrated continuous or pseudo -continuous mode. In a preferred embodiment of the present invention, the anion exchange resin is selected from a group consisting of sulfonate group, sulfopropyl group, and sulphonic acid linked to bead-based, membrane-based, hydrogel-based, and fiber-based chromatography matrix.
In an embodiment of the present invention, the folded and solubilized IL-IRa polypeptide contains no more than 0.3 % soluble aggregate and no more than 10 ng/mL of DNA.
Thus, the present disclosure discloses a novel soluble form of IL-IRa such as anakinra. The present disclosure also provides a novel gene sequence for synthesis of soluble form of IL-IRa. Further, the present disclosure discloses a process for the synthesis of novel soluble form of IL-IRa. Furthermore, the present disclosure relates to a pharmaceutical composition comprising soluble form of IL-IRa.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1: illustrates the vector map for the expression of IL-IRa in Escherichia coli along with agarose gel electrophoresis of Nde\ treated expression vector.
Fig. 2: illustrates the method for purifying IL-IRa in batch, integrated continuous or pseudo-continuous mode.
Fig. 3a and 3b: illustrates the SDS-PAGE analysis of soluble fraction and insoluble pellet of E. coli expressing IL-IRa.
Fig. 4: illustrates the biosimilarity between the present IL-IRa and the innovator KINERET based on the CD spectra. The secondary structure prediction has been performed for both based on these spectra.
Fig. 5: illustrates the biosimilarity between the present IL-IRa and the innovator KINERET based on the UV fluorescence spectra.
Fig. 6: illustrates the biosimilarity between the present IL-IRa and the innovator KINERET based on the intact mass analysis.
Fig. 7: illustrates the biosimilarity between the present IL-IRa and the innovator KINERET based on the peptide mapping analysis.
Fig. 8: illustrates the confirmation of the disulfide bond between SCVK=ESAACPGWFL peptides of present IL-IRa.
Fig. 9: illustrates the purification of the present IL-IRa using multimodal anion exchange chromatography in flow-through mode.
Fig. 10: illustrates the purification of present IL-IRa using multimodal cation exchange chromatography in a bind and elute mode.
Fig. 11: illustrates the RP-HPLC profiles for both the present IL-IRa and the innovator molecule.
Fig. 12: illustrates the SEC-HPLC profiles for both the present IL-IRa and the innovator molecule.
Fig. 13: illustrates intact mass analysis based on the MALDI-TOF profiles for both the present IL-IRa and the innovator molecule.
DESCRIPTION OF THE INVENTION
The invention will now be described in detail in connection with certain preferred and optional embodiments so that various aspects thereof may be more fully understood and appreciated.
The polypeptide expressions for use as pharmaceutical product or vaccines require achieving high biomass and/or productivity of the host cell line. The efficiency of polypeptide production can be significantly diminished in absence of multiple factors, which includes use of an optimal polynucleotide sequence encoding that polypeptide. The genetic code is known to exhibit degeneracy, which amounts to the variance in the polynucleotide sequence encoding the same amino acid sequence. The rate of synthesis of amino acid chain is a determinant factor in the overall expression levels from an individual gene, which effect the design of the expression construct, is of high significance. Thus, the construction of an optimal polynucleotide sequence is important in determining the overall expression levels of a polypeptide and has to be well regulated. It includes but is not limited to the frequency with which the codons are preferred in an organism or in case of artificial vehicles or vectors, the nearest frequency desired. This in turn reflects tRNA abundance or the cognate cellular tRNA frequencies from which the synonymous codon choice patterns has to be carefully selected. Additional factors also include the potential for formation of secondary structures, mRNA levels and RNA stability, subsequent intended manipulations to be carried out, synthesis routes and so on. The occurrence of these structures has to be carefully regulated as the choice of these patterns differs with the optimizations for individual protein of interest and expression hosts.
While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.
Throughout the specification and claims, the following terms take the meanings explicitly associated herein unless the context clearly dictates otherwise. The meaning of "a", "an", and "the" include plural references. The meaning of "in" includes "in" and "on." Referring to the drawings, like numbers indicate like parts throughout the views. Additionally, a reference to the singular includes a reference to the plural unless otherwise stated or inconsistent with the disclosure herein.
The tables, figures, and protocols have been represented where appropriate by conventional representations in the drawings, showing only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having the benefit of the description herein.
Accordingly, the main embodiment of the present invention provides an optimized polynucleotide sequence (SEQ ID NO. 2) and its structural variants.
In an embodiment of the present invention, the codon-optimized polynucleotide sequences were selected for the expression of the recombinant protein.
In preferred embodiment, the recombinant protein expressed is interleukin- 1 -receptor antagonist (IL-IRa).
In another embodiment, the Interleukin- 1 -receptor antagonist is expressed in E. coli.
In certain embodiments, the recombinant protein is produced in soluble and/or active recombinant protein at a yield of about 0.1 grams per liter to about 12 grams per liter.
In specific embodiments, the yield of soluble and/or active recombinant protein is about 0.05 to 12 g/L, specifically, about 0.05 g/L, about 0.1 g/L, about 0.2 g/L, about 0.3 g/L, about 0.4 g/L, about 0.5 g/L, about 0.6 g/L, about 0.7 g/L, about 0.8 g/L, about 0.9 g/L, about 1 g/L, about 1.5 g/L, about 2 g/L, about 2.5 g/L, about 3 g/L, about 3.5 g/L, about 4 g/L, about 4.5 g/L, about 5 g/L, about 5.5 g/L, about 6 g/L, about 6.5 g/L, about 7 g/L. about 7.5 g/L, about 8 g/L, about 8.5 g/L, about 9 g/L, about 9.5 g/L, about 10 g/L, about 10.5 g/L, about 11 g/L, about 12 g/L.
In yet another embodiment of the invention, the nucleotide sequence has been optimized for expression in the E. coli host cell. In related embodiments, the nucleotide sequence has been optimized for expression in the E. coli host cell. In other related embodiments, the nucleotide sequence has been optimized for expression in the E. coli host cell.
In another embodiment, the invention provides an optimized polynucleotide sequence (SEQ ID NO. 2) and its structural variants selected from but not limited to SEQ ID NO. 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 and 16 having equal to or more than 70% similarity to SEQ ID NO. 2 useful for high level expression of polypeptide for IL-lRa.
Induction of expression refers to the step performed to induce the expression from the polynucleotide so that the product is obtained at an accelerated rate, this may involve addition of suitable inducing agent such as IPTG, arabinose, maltose, lactose, galactose and the like.
Optionally the polynucleotide of the invention may also be associated with polynucleotides of tag polypeptides. The presence of a tag is also known to enhance the stability and solubility of the protein in the cytoplasm and for its subsequent purification. Alternatively, a hydrophobic tag can also be introduced at the N-terminal to induce the expression in the form of inclusion bodies. These tags can be associated at 5’ terminus or 3’ terminus, singly or in combination pertinent to multi-tagging, with an oligonucleotide sequence that encodes a tag polypeptide to facilitate its cytoplasmic stability and/or subsequent purification using matrices and resins with a high affinity for the various tag peptides.
The polynucleotide of invention may also be incorporated in a vector construct comprising regulatory sequence, with molecular techniques well known in art (See Sambrook et aL, Molecular Cloning, 2nd ed., (1989)). This includes but is not limited to, a suitable promoter, origin of replication, ribosomal binding site, transcription termination sequence, selectable markers and multiple cloning sites. In particular, a plasmid with an efficient and specific construct is preferred; such as one including T7 Promoter specific for RNA polymerase enzyme of the phage T7.
In one embodiment, the host cell is a gram negative host cell. Host cell expression systems like Escherichia coli, Bacillus sp., Pseudomonas sp., have been extensively discussed in the production of proteins. In one embodiment the polynucleotide of the invention is preferably used for the intracellular soluble expression of present IL-lRa in Escherichia coli wherein the host strain is selected from BL21 (DE3), BL21 Al, HMS174 (DE3), DH5a, W3110, B834, origami, Rosetta, NovaBlue (DE3), Lemo21 (DE3), T7, ER2566 and C43 (DE3).
In a preferred embodiment, the present invention provides a polynucleotide sequence (SEQ ID NO. 2) encoding interleukin 1 -receptor antagonist which is optionally ligated into a vector, followed by its insertion in a host cell. The insertion into host cell may be performed by any of the methods known in the art. Such an insertion or transformation may be performed by a physical or a chemical method of transformation. Subsequently, the converted colonies are selected on petri dishes with the added antibiotic.
Suitable vectors used in the present invention include but not limited to pET9a, pET3a, pET3b, pET3c, pET5a, pET5b, pET5c, pET9b, pET9c, pET12a, pET28a, pET28b, pET28c, pET30a, pET30b, pET30c, pTWIN 1, pTWIN2, pET12b, pET12c, pET17b and in general, all the vectors that have a strong phage T7 promoter (e.g. pRSET A, B and C [Invitrogen]) and pTYBl, pTYB2, pTYB3 and pTYB4. Alternatively, the protein can also be expressed in the vectors derived from the vectors mentioned with a change in the regulatory region and/or the promoter and may include but not limited to pL, rhaBAD, and rhaT.
In another embodiment, Escherichia coli cells are used to express the polynucleotide encoding IL-lRa. The inserted polynucleotide is verified for proper orientation and position by sequencing. The resultant construct is used to transform host cells by any of the known chemical or physical methods. For example, transformation of E. coli host cells by heat shock at 42 °C for 30-90 sec is a common practice for host cell transformation with the desired vector. These cells are allowed to grow for 30 minutes to 120 minutes at 25 to 40 °C in a suitable medium as LB or SOC medium and then transferred to selection media petriplates for 10 to 36 hours, at 25 to 40 °C where the positive colonies containing the polynucleotides of invention are further screened and selected.
The selection of positive colonies can be done with or without markers. Suitable markers which can be used are selected from, but not limited to, antibiotics such as ampicillin, kanamycin and the like.
The polynucleotide encoding the full length IL-lRa protein is cloned adjacent to T7 lac promoter that drives the expression of protein in T7 polymerase positive strains of Escherichia coli. The expression of polynucleotide is stringently controlled by T7 promoter which is induced in the presence of IPTG, natural sugars (other than glucose) or in auto-induction mediums.
In an embodiment of the present invention, the selected IL-lRa product is produced using a batch, fed-batch, or continuous fermentation process.
In another embodiment, the fermentation media used for expression of IL-lRa is chemically defined medium. In another embodiment, the fermentation media used for the expression of IL-IRa is a complex medium.
In another preferred embodiment, the complex fermentation media used for the expression of IL-IRa is HiVeg Tartoff Broth.
In still another embodiment of the present invention, the harvested material is an E. coli (BL-21 DE3) harvest with an expressed IL-IRa in soluble form.
The parameters for culturing the host are optimized for high level expression of IL-IRa protein. In one embodiment, the culture media components, culture conditions including growth temperature, concentration of inducers and induction time is optimized for higher soluble expression of IL-IRa. The culture media used may be selected from, but not limited to, chemically defined media, LB (Luria-Bertani), TB (Terrific Broth), SOB (Super Optimal Broth), SOC (Super Optimal broth with catabolic repressor), YT broth (Yeast Extract and Tryptone), Super broth, rich media, minimal media, mineral media and the like. The ingredients of media includes, but is not limited to, a carbon source such as, e.g., glucose, sucrose, or glycerol, organic nitrogen source, such as peptone, tryptone, amino acids, or a yeast extract, inorganic nitrogen source is used and this may be selected from among, e.g., ammonium salts, aqueous ammonia, and gaseous ammonia, supplements as supplemented with, e.g., low levels of amino acids, vitamins, peptones, or other ingredients, The culture media may be prepared using the methods known in the art.
In one embodiment, the expression of Interleukin- 1 -receptor is induced using IPTG.
In another embodiment, the expression of Interleukin- 1 -receptor is induced using natural sugars like galactose and lactose.
The transformed host cells may be tested for expression on small volumes such as 5- 100 ml in LB, terrific broth or chemically defined medium. The expression may be subjected to different concentrations of inducers ranging from about 0.01 mM, about 0.05 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM and about 1 mM. In the case of natural sugars like galactose or lactose, the expression may be subjected to different concentrations of inducers ranging from about 1 mM, about 2.5 mM, about 5 mM, about 7.5 mM, about 10 mM, about 12.5 mM, about 15 mM, about 17.5 mM, and about 20 mM. The polypeptide expression is determined in an electrophoretic set up, preferably in SDS PAGE electrophoresis and viewed as over-expressed bands stained with coomassie brilliant blue (-). Subsequently, the host cells are inoculated in 500 mL flasks cultures or small scale bioreactors with media volume of about 500 mL, about 1000 mL, about 1500 mL and about 2000 mL; and allowed to grow under optimal conditions, during which IL-IRa expression continued from 16 hours to 32 hours under batch or fed -batch condition. After culturing in constant agitation and preferably under aerobic conditions, the cells are harvested, and lysed. Any of known methods may be applied to lyse cells, preferred method includes a lysis buffer containing a detergent at an appropriate concentration. After lysis, the protein component is pooled in one or more centrifugation steps. Lysis is carried out in a buffer containing Tris-HCl 20-50 mM pH 7.0-9.0, NaCl 100-150 mM, detergent 0.5-1.5% and protease inhibitor 0.5-1.5%, with agitation. Alternatively, the lysis is carried out in a buffer containing sodium acetate 10-50 mM, pH 3.0- 5.5, NaCl 50-300 mM, glycerol 1-5%, EDTA l-5mM using a cell disrupter.
In one embodiment induction temperatures for expression is carried out between 10 to 40 °C. The expression of IL-IRa is in soluble form over the broad range of induction temperatures unless accompanied with a hydrophobic tag, where the protein expression is in insoluble inclusion bodies.
The invention thus involves more than one subsequent purification steps, and also exploits pl value of interleukin 1 receptor antagonist in mixed-mode or multimodal ion exchange chromatographic step, whereby it is separated from other contaminating proteins. Finally, the quantity of IL-IRa is quantified by RP-HPLC and visualized inl2% acrylamide gel (SDS-PAGE).
Accordingly, to accomplish the objectives of the present invention, the inventors propose a method for purifying a protein of interest in a batch, integrated-continuous or pseudo-continuous mode.
In an embodiment of the present invention, the process for purifying a protein of interest in a batch, integrated continuous or pseudo-continuous mode, which is depicted in Figure 2, comprising the steps of, a) Extraction: The harvested microbial cells were resuspended in lysis buffer (20.0 mM sodium acetate, 0.3 M NaCl, 0.1 mM EDTA and 5% sorbitol, pH 4.0-5.0) at a cell pellet: buffer ratio of 1:10 (w/v). The low pH cell lysis was performed using a high-pressure homogenizer at 15000 bar pressure for 10.0 min, and the cell lysate was centrifuged at 10000 rpm for 30.0 min at 4.0 °C to get the soluble fraction of protein in supernatant. Low pH lysis step was given to precipitate the impurities like host cell proteins and host cell nucleic acid. Aliquots of the Supernatant and sediment are analyzed in SDS-PAGE. b) Subjecting the homogenate cell supernatant solution from process step (b) to multimodal anion exchange chromatography is performed in a flow-through mode for separation wherein the said chromatography process comprises;
Loading filtered homogenate cell supernatant solution of step (b) at pH range from 4.0 to 5.0 with the conductivity of more than 5mS/cm to multimodal anion exchange resin to capture various process-related and product-related impurities. c) Subjecting the multimodal anion exchange chromatography flow-through for multimodal cation exchange chromatography. Multimodal cation exchange chromatography was performed in a bind and eluted mode for separation of various product-related impurities at pH of 4.00 to 5.00 with the conductivity of more than 0.5 mS/cm in batch, pseudo-continuous mode, or continuous mode;
In still another embodiment of the present invention, the harvested material is an E.coli (BL-21 DE3) harvest expressed in soluble form.
In a preferred embodiment of the present invention, the downstream processing of the Interleukin- 1 receptor antagonist (IL-IRa) was performed in using a batch, fed-batch, or continuous fermentation process.
In a preferred embodiment of the present invention, the recombinant protein is the Interleukin- 1 receptor antagonist (IL-IRa).
In still another embodiment of the present invention, the process for purifying recombinant protein is Interleukin- 1 receptor antagonist (IL-IRa) in batch, integrated continuous or pseudo-continuous mode includes extraction of soluble protein step performed at pH range from 4.0 to 5.0 with conductivity more than 5 mS/cm.
In a further embodiment of the present invention, the HCP log reduction achieved using the step low pH cell lysis is not less than 0.5.
In another embodiment of the present invention, the lysate is continuously centrifuged at 6000-9000 rpm for 40 min for single or multiple times.
In still another embodiment of the present invention, the Interleukin- 1 receptor antagonist (IL-IRa) centrifuged supernatant is always purest as compared to homogenized cell harvest. In yet another embodiment of the present invention, there suspended cell pellet is homogenized using a high-pressure homogenizer and the outlet can be directly connected to the inlet of the continuous centrifuge to separate soluble fractions and the centrifuge outlet can be connected to multimodal anion exchange chromatography.
In preferred embodiment of present invention, residence time for multimodal anion exchange chromatography and multimodal cation exchange chromatography is more than 15 seconds.
In yet another embodiment of the present invention, multimodal anion exchange chromatography or multimodal cation exchange is performed using the axial or radial flow chromatography column.
In a preferred embodiment of the present invention, the multimodal anion exchange chromatography includes a resin/membrane selected from a group consisting of HEA Hypercel™, PPA Hypercel™, and BAKERBONDOXWP 500 Poly PEI-35, Capto™ adhere or Capto™ adhere ImpRes resin.
In still another embodiment of the present invention, the total amount of interleukin 1 receptor antagonist (Il-IRa) product passed through the single multimodal anion exchange chromatography column is not less than 10 g/L resin.
In yet another embodiment of the present invention, the multimodal cation exchange chromatography is performed using a cation exchange functional groups selected from a group consisting of sulfonate group, sulfopropyl group, and sulphonic acid linked to bead-based, membrane-based, hydrogel-based, and fiber-based chromatography matrix.
In preferred embodiment of the present invention, the multimodal cation exchange chromatography includes a ion exchange resin/membrane selected from the group consisting of HEA Hypercel (cellulose based resin, e.g. https://www.sartorius.com/download/483974/hypercel-hea-ppa-datasheet-en-b-2578760-sarto rius-data.pdf), PPA Hypercel (cellulose based resin e.g. https://www.sartorius.com/download/483974/hypercel-hea-ppa-datasheet-en-b-2578760-sarto rius-data.pdf), BAKERBOND™ XWP 500 PolyCSX-35 (polystyrene based resin e.g. http://ichimarutrading.co.jp/import/download/ultrabioreagent/BAKERBOND_XWP_500_Pol yCSX-35.pdf) or Capto MMC resin (agarose based resin e.g. https://www. scientificlabs.ie/handlers/library Files. ashx?filename=Manuals_l_17531702_A.p df), Fractogel® EMD S03~(M) (crosslinked methacrylate based resin, e.g. http://wolfson.huji.ac.il/purification/PDF/IonExchange/MERCK_SO3.pdf), Capto™ SP ImpRes (agarose based resin, e.g. https://cdn.cytivalifesciences.com/api/public/content/digi-15744-pdf), POROS® XS (polystyrene based resin, e.g. http://tools.thermofisher.com/content/sfs/manuals/cms_085204.pdf), Nuvia™ S (cation exchange media, e.g. https://www.bio-rad.com/webroot/web/pdf/psd/literature/10018215.pdf), SOURCE™ 15S (polystyrene based resin, e.g. http://webhome.auburn.edu/~duinedu/manuals/Sourcel5S.pdf), SOURCE™ 30S (polystyrene based resin, e.g. https://cdn.cytivalifesciences.com/api/public/content/digi-11460-pdf), SP Sepharose® Fast Flow (crosslinked agarose based resin, e.g. https://at.vwr.com/assetsvc/asset/de_AT/id/17886301/contents), Natrix® HD-Sb chromatography membrane (three-dimensional macroporous hydrogel structured membrane, e.g.https://mdpi-res.com/d_attachment/processes/processes-10-01025/article_deploy/processe s-10-01025-v2.pdf?version=1653462580), Sartobind® S (chromatography membrane membrane, e.g. https://www.sartorius.hr/media/itlhlywj/usermanual-en-manual-sartobind-q-s-4-8mm-caps-ca ssettes-sl-6210-e.pdf), and Mustang® S (cross-linked quatemized amine charge polymer coating on membrane, e.g. https://www.pall.com/content/dam/pall/laboratory/literature-library/non-gated/chromatograph y vaccinepurification/capturingchromatography/Mu stang % C2 % AE% 20Chromatography % 20 Capsules%20and%20Cartridges%20.pdf).
In yet another embodiment of the present invention, the gradient elution in multimodal cation exchange chromatography is achieved using a salt gradient and or pH gradient. Preferrably, a salt gradient.
In an embodiment of the present invention, the multimodal cation exchange chromatography column has a dynamic binding capacity for the recombinant protein of more than 5 g/L resin.
In another embodiment of the present invention, the purified recombinant drug substance contains no more than 0.3 % soluble aggregate.
In a preferred embodiment of the present invention, the purified IL-IRa polypeptide contains no more than 100 ppm HCP in the final drug substance.
In still another embodiment of the present invention, the purified IL-IRa polypeptide contains no more than 10 ng/mL of DNA in the final drug substance In an embodiment of the present invention, the IL-IRa polypeptide is produced using a batch, fed-batch, or continuous fermentation process.
In another embodiment of the present invention, the IL-IRa polypeptide is produced in a continuous fermentation process.
In a preferred embodiment of the present invention, the selected recombinant product is anakinra.
In yet one more embodiment of the invention, the mass balance of the process is > 90% and yield of the purified Interleukin- 1 receptor antagonist (IL-IRa) is > 70 % and is independent of the initial concentration.
SEQUENCE IDS; SEQ ID NO.: "1" (IL-IRa polypeptide)
MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHA LFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFES AACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE
SEQ ID NO.: "2" (Codon optimized IL-IRa nucleotide sequence) catatgcgcccgtctggaagaaaatcctctaaaatgcaagccttccgcatctgggatgttaatcaaaaaactttctatctccggaataatc aacttgtagcaggttatttacaagggcctaatgtaaacctcgaagaaaagattgatgttgtacctatagaaccgcacgcactgtttttagg cattcatggagggaaaatgtgtctgagctgcgtaaagagtggagatgaaaccagacttcagttagaagcagtcaatattaccgacctttc ggaaaatcgcaagcaagataaacgtttcgcttttatccggagcgactctggtcctaccacgagttttgagtcagccgcctgtccgggttg gttcttatgcacagctatggaagccgatcagcctgtcagtttaacgaacatgcctgacgaaggtgtcatggtcactaaattctattttcagg aagatgaataataactcgag
SEQ ID NO.: "3" (Variant 1 of Codon optimized IL-IRa nucleotide sequence) atgagaccctccgggaggaagtctagtaagatgcaagctttcagaatatgggacgtaaatcaaaagacattctacctaagaaataatca actagtagctgggtacctacaagggcccaatgtaaatctagaggagaagatagacgtagtacccatagagccccacgctctattccta gggatacacggggggaagatgtgtctctcctgtgtaaagtctggggacgagacaagactacaactagaggctgtaaatataacagac ctatctgagaatagaaagcaagacaagagattcgctttcataagatctgactccgggcccacaacatctttcgagtctgctgcttgtccc gggtggttcctatgtacagctatggaggctgaccaacccgtatctctaacaaatatgcccgacgagggggtaatggtaacaaagttcta cttccaagaggacgag
SEQ ID NO.: "4" (Variant 2 of Codon optimized IL-IRa nucleotide sequence) atgcgcccgagcggccgcaaaagcagcaaaatgcaggcgtttcgcatttgggatgttaaccagaaaaccttttatctgcgcaacaacc agctggttgcgggctatctgcagggcccgaacgttaacctggaagaaaaaattgatgttgttccgattgaaccgcatgcgctgtttctgg gcattcatggcggcaaaatgtgcctgagctgcgttaaaagcggcgatgaaacccgcctgcagctggaagcggttaacattaccgatct gagcgaaaaccgcaaacaggataaacgctttgcgtttattcgcagcgatagcggcccgaccaccagctttgaaagcgcggcgtgcc cgggctggtttctgtgcaccgcgatggaagcggatcagccggttagcctgaccaacatgccggatgaaggcgttatggttaccaaattt tattttcaggaagatgaa sequencelD Number="5" (Variant 3 of Codon optimized IL-IRa nucleotide sequence) atgcgtccttcgggtcgtaaatcatccaaaatgcaggcctttcgtatctgggatgtcaaccagaaaacgttttatttacgtaacaaccagct tgtagcaggttatttgcagggtcctaacgtcaacttagaagaaaaaattgatgtcgttcctatcgaacctcatgccttatttttaggtatccat ggtggtaaaatgtgcttatcgtgcgtcaaatcgggtgatgaaactcgtttacagttagaagccgtcaacatcacggatttatcggaaaac cgtaaacaggataaacgttttgcctttatccgttcagattcgggtcctacgacttcgtttgaatcagcagcctgccctggttggtttttatgca cggccatggaagcagatcagccagtatcattgacgaacatgcctgatgaaggtgtcatggtaactaaattttattttcaggaagatgaa
SEQ ID NO.: "6" (Variant 4 of Codon optimized IL-IRa nucleotide sequence) atgcgtccttcgggtcgtaagtcatccaagatgcaagccttccgtatctgggacgtcaatcaaaagacgttctacttacgtaataatcaac ttgtagcaggttacttgcaaggtcctaatgtcaatttagaggagaagattgacgtcgttcctatcgagcctcacgccttattcttaggtatcc acggtggtaagatgtgtttatcgtgtgtcaagtcgggtgacgagactcgtttacaattagaggccgtcaatatcacggacttatcggaga atcgtaagcaagacaagcgtttcgccttcatccgttcagactcgggtcctacgacttcgttcgagtcagcagcctgtcctggttggttctta tgtacggccatggaggcagaccaaccagtatcattgacgaatatgcctgacgagggtgtcatggtaactaagttctacttccaagagga cgag
SEQ ID NO.: "7" (Variant 5 of Codon optimized IL-IRa nucleotide sequence) atgcgcccgagcggccgcaaaagcagcaaaatgcaggcgtttcgcatttgggacgttaaccagaaaaccttttatctgcgcaacaacc agctggttgcgggctatctgcagggcccgaacgttaacctggaagaaaaaattgacgttgttccgattgaaccgcatgcgctgtttctg ggcattcatggcggcaaaatgtgcctgagctgcgttaaaagcggcgacgaaacccgcctgcagctggaagcggttaacattaccgac ctgagcgaaaaccgcaaacaggataaacgctttgcgtttattcgcagcgacagcggcccgaccaccagctttgaaagcgcggcgtg cccgggctggtttctgtgcaccgcgatggaagcggaccagccggttagcctgaccaacatgccggacgaaggcgttatggttaccaa attttattttcaggaagacgaa
SEQ ID NO.: "8" (Variant 6 of Codon optimized IL-IRa nucleotide sequence) atgcgcccgagcggccgcaaaagcagcaaaatgcaggcgtttcgcatttgggatgttaaccagaaaaccttttatctgcgcaacaacc agctggttgcgggctatctgcagggcccgaacgttaacctggaggagaaaattgatgttgttccgattgagccgcatgcgctgtttctg ggcattcatggcggcaaaatgtgcctgagctgcgttaaaagcggcgatgagacccgcctgcagctggaggcggttaacattaccgat ctgagcgagaaccgcaaacaggataaacgctttgcgtttattcgcagcgatagcggcccgaccaccagctttgagagcgcggcgtgc ccgggctggtttctgtgcaccgcgatggaggcggatcagccggttagcctgaccaacatgccggatgagggcgttatggttaccaaat tttattttcaggaggatgag SEQ ID NO.: "9" (Variant 7 of Codon optimized IL-IRa nucleotide sequence)
Atgcgcccgagcggccgcaaaagcagcaaaatgcaggcgtttcgcatttgggatgttaatcagaaaaccttttatctgcgcaataatc agctggttgcgggctatctgcagggcccgaatgttaatctggaagaaaaaattgatgttgttccgattgaaccgcatgcgctgtttctgg gcattcatggcggcaaaatgtgcctgagctgcgttaaaagcggcgatgaaacccgcctgcagctggaagcggttaatattaccgatct gagcgaaaatcgcaaacaggataaacgctttgcgtttattcgcagcgatagcggcccgaccaccagctttgaaagcgcggcgtgccc gggctggtttctgtgcaccgcgatggaagcggatcagccggttagcctgaccaatatgccggatgaaggcgttatggttaccaaatttt attttcaggaagatgaa
SEQ ID NO.: "10" (Variant 8 of Codon optimized IL-IRa nucleotide sequence) atgcgcccgagcggccgcaaaagcagcaaaatgcaagcgtttcgcatttgggatgttaaccaaaaaaccttttatctgcgcaacaacc aactggttgcgggctatctgcaaggcccgaacgttaacctggaagaaaaaattgatgttgttccgattgaaccgcatgcgctgtttctgg gcattcatggcggcaaaatgtgcctgagctgcgttaaaagcggcgatgaaacccgcctgcaactggaagcggttaacattaccgatct gagcgaaaaccgcaaacaagataaacgctttgcgtttattcgcagcgatagcggcccgaccaccagctttgaaagcgcggcgtgccc gggctggtttctgtgcaccgcgatggaagcggatcaaccggttagcctgaccaacatgccggatgaaggcgttatggttaccaaatttt attttcaagaagatgaa
SEQ ID NO.: "11" (Variant 9 of Codon optimized IL-IRa nucleotide sequence) atgcgcccgagcggccgcaaaagcagcaaaatgcaggcgtttcgcatttgggatgttaaccagaaaaccttttatctgcgcaacaacc agctggttgcgggctatctgcagggcccgaacgttaacctggaagaaaaaattgatgttgttccgattgaaccgcacgcgctgtttctg ggcattcacggcggcaaaatgtgcctgagctgcgttaaaagcggcgatgaaacccgcctgcagctggaagcggttaacattaccgat ctgagcgaaaaccgcaaacaggataaacgctttgcgtttattcgcagcgatagcggcccgaccaccagctttgaaagcgcggcgtgc ccgggctggtttctgtgcaccgcgatggaagcggatcagccggttagcctgaccaacatgccggatgaaggcgttatggttaccaaat tttattttcaggaagatgaa
SEQ ID NO.: "12" (Variant 10 of Codon optimized IL-IRa nucleotide sequence) atgcgcccgagcggccgcaaaagcagcaaaatgcaggcgttccgcatttgggatgttaaccagaaaaccttctatctgcgcaacaac cagctggttgcgggctatctgcagggcccgaacgttaacctggaagaaaaaattgatgttgttccgattgaaccgcatgcgctgttcctg ggcattcatggcggcaaaatgtgcctgagctgcgttaaaagcggcgatgaaacccgcctgcagctggaagcggttaacattaccgat ctgagcgaaaaccgcaaacaggataaacgcttcgcgttcattcgcagcgatagcggcccgaccaccagcttcgaaagcgcggcgtg cccgggctggttcctgtgcaccgcgatggaagcggatcagccggttagcctgaccaacatgccggatgaaggcgttatggttaccaa attctatttccaggaagatgaa
SEQ ID NO.: "13" (Variant 11 of Codon optimized IL-IRa nucleotide sequence) atgcgcccgagcggccgcaaaagcagcaaaatgcaggcgtttcgcatttgggatgttaaccagaaaaccttttacctgcgcaacaacc agctggttgcgggctacctgcagggcccgaacgttaacctggaagaaaaaattgatgttgttccgattgaaccgcatgcgctgtttctg ggcattcatggcggcaaaatgtgcctgagctgcgttaaaagcggcgatgaaacccgcctgcagctggaagcggttaacattaccgat ctgagcgaaaaccgcaaacaggataaacgctttgcgtttattcgcagcgatagcggcccgaccaccagctttgaaagcgcggcgtgc ccgggctggtttctgtgcaccgcgatggaagcggatcagccggttagcctgaccaacatgccggatgaaggcgttatggttaccaaat tttactttcaggaagatgaa
SEQ ID NO.: "14" (Variant 12 of Codon optimized IL-IRa nucleotide sequence) atgcgcccgagcggccgcaagagcagcaagatgcaggcgtttcgcatttgggatgttaaccagaagaccttttatctgcgcaacaacc agctggttgcgggctatctgcagggcccgaacgttaacctggaagaaaagattgatgttgttccgattgaaccgcatgcgctgtttctgg gcattcatggcggcaagatgtgcctgagctgcgttaagagcggcgatgaaacccgcctgcagctggaagcggttaacattaccgatct gagcgaaaaccgcaagcaggataagcgctttgcgtttattcgcagcgatagcggcccgaccaccagctttgaaagcgcggcgtgcc cgggctggtttctgtgcaccgcgatggaagcggatcagccggttagcctgaccaacatgccggatgaaggcgttatggttaccaagtt ttattttcaggaagatgaa
SEQ ID NO.: "15" (Variant 13 of Codon optimized IL-IRa nucleotide sequence) atgcgcccgtctggaagaaaatcctctaaaatgcaagccttccgcatctgggacgttaatcaaaaaactttctatctccggaataatcaac ttgtagcaggttatttacaagggcctaatgtaaatctcgaggagaagattgacgttgtacctatagagccgcacgcactgtttttaggcatt catggagggaaaatgtgtctgagctgcgtaaagagtggagacgagaccagacttcaattagaggcagtcaatattaccgacctttcgg agaatcgcaagcaagacaaacgtttcgcttttatccggagcgactctggtcctaccacgagttttgagtcagccgcctgtccgggttggt tcttatgcacagctatggaggccgaccaacctgtcagtttaacgaatatgcctgacgagggtgtcatggtcactaaattctattttcaaga ggacgag
SEQ ID NO.: "16" (Variant 14 of Codon optimized IL-IRa nucleotide sequence) atgcgcccgtctggaagaaaatcctctaaaatgcaggccttccgcatctgggatgttaaccagaaaactttctatctccggaacaacca gcttgtagcaggttatttacaggggcctaacgtaaacctcgaagaaaagattgatgttgtacctatagaaccgcacgcactgtttttaggc attcatggagggaaaatgtgtctgagctgcgtaaagagtggagatgaaaccagacttcagttagaagcagtcaacattaccgatctttc ggaaaaccgcaagcaggacaaacgtttcgcttttatccggagcgattctggtcctaccacgagttttgaatcagccgcctgtccgggtt ggttcttatgcacagctatggaagccgatcagcctgtcagtttaacgaacatgcctgatgaaggtgtcatggtcactaaattctattttcag gaagatgaa
EXAMPLES
The present disclosure is further explained in the form of the following examples. However, it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention. Procurement details of Materials used in Invention: Galactose, Arabinose, Lactose, Glycine, Tris, Sinapinic acid, and Trifluoroacetic acid: Sigma-Aldrich, Saint Louis, USA; IPTG, Maltose, Ampicillin, LB (Luria Broth) medium, Agarose gel, T4 ligase, Kanamycin, EDTA, and Sorbitol: HIMEDIA, Nashik Maharastra, India; Qiagen QIAQUICK Gel extraction kit: QIAGEN; Coomassie Brilliant Blue dye: SRICHEM.COM SRL; Methanol, Acetic acid, Sodium acetate and Glacial Acetic acid: MERCK LIFE SCIENCE; NaCl and ACN: THOMAS BAKER; Toyopearl® NH2-750F resin: TOSOH BIOSCIENCE; Capto™ adhere resin, Capto™ SP ImpRes resin and Capto™ SP Sepharose Impress resin: CYTIVA; Fractogel®EMD S03“ resin: Merck Millipore. E.Coli BL21 (DE3) & Escherichia coli DH5a: MilliporeSigma, Billerica MA USA
Step (i): Synthesis of novel IL-IRa gene:
Full length IL-IRa gene was optimized according to Escherichia coli codon usage. The following parameters were used for IL-IRa gene optimization: Codon Usage Bias, GC content, mRNA Secondary Structure, Custom Desired Patterns, Custom Undesired Patterns, Repeat Sequences (direct repeat, inverted repeat, and dyad repeat), Restriction Enzyme Recognition Sites (deletion or insertion).
Optimized IL-IRa gene (SEQ ID NO. 2) was cloned at multiple cloning site of pUC57 plasmid vector using BamHl and Sapl restriction sites, generating pUC57_ IL-IRa. The vectors containing IL-IRa gene was transformed in Escherichia coli DH5a host and clones were selected on LB + Ampicillin plate. The presence and correctness of IL-IRa gene in pUC57 was confirmed by restriction digestion of pUC57_ IL-IRa plasmid by Xhol and Ndel present in the flanking regions of the IL-IRa gene. Further the sequence of IL-IRa was confirmed by PCR and DNA sequencing.
Step (ii): Insertion of IL-IRa into expression vector
Escherichia coli DH5a carrying pUC57_ IL-IRa was grown over night in LB + Ampicillin in 50 ml volume. Bacteria were centrifuged and pellet was used for plasmid isolation. Isolation of plasmid was done by using Qiagen plasmid mini-prep kit, using manufacturer instructions. Isolated plasmid was quantified by NanoDrop.
IL-IRa (SEQ ID NO. 2) from pUC57 was excised, 5pg plasmid was digested with restriction endonucleases Ndel and Xhol. The digested plasmid was run on 1 % agarose gel and band corresponding to IL-IRa gene (SEQ ID NO. 2) was purified by using Qiagen Gel extraction kit using manufacturer’s instructions. Subsequently the 5 pg of expression plasmid was also digested with Ndel and Xhol to generate restriction sites in it that is compatible with IL-IRa gene. The digested expression vector was also purified from gel using the Qiagen Gel extraction kit with manufacturer’s instructions.
The digested IL-IRa gene was ligated in expression vector using T4 ligase based DNA ligation kit (Promega) using manufacturer’s instructions. Vector and Insert was mixed in 1 :3, 1:4, 1:5 ratio in the presence of T4 DNA ligase and buffers in a 20pl reaction volume. Ligation mixture was incubated overnight at 16°C. Next morning 5pl of ligation mixture was added/transformed in BL21-DE3 Escherichia coli expression host. BL21 DE3 was transformed by using chemical transformation protocol. The ligation + BL21 DE3 cells were incubated in ice for 30 min. After incubation heat shock was given for 45 seconds at 42°C. Sample was cooled at room temperature and 500pl SOC medium was added into it. The tube with transformants was incubated for 2 hours at 37°C with 200 rpm. From which lOOpl mixture was plated on LB + Kanamycin plate for screening of transformants.
IL-IRa expression BL21-DE3 transformants were selected next morning from LB + Kanamycin plates. Of these 5 clones growing on LB + Kanamycin were selected and grown in 10 ml LB + Kanamycin media for overnight at 37 degrees, 200 rpm. Culture was centrifuged and plasmid was extracted from cell pellet using Qiagen plasmid extraction kit.
To verify the correctness of clone, 2pg plasmid was digested with Ndel and Xhol restriction endonuclease, respectively. The clone was designated as pExp_IL-lRa (BL21-DE3). Furthermore, clones were confirmed by PCR using IL-IRa gene specific primers and DNA sequencing. The glycerol stock of BL21 DE3 expressing IL-IRa was made by growing bacteria in 10ml LB + Kanamycin overnight. Next morning 40% sterilized Glycerol was added into culture and 1ml aliquot was dispensed into cryovial. Vials were stored at -80 degree for further use in expression analysis.
Step (iii): Confirmation of expression of IL-IRa:
BL21 DE3 clone stored at -80 degrees was streaked on Luria Broth+Kanamycin plate. Plate was incubated overnight at 37 degrees. A single colony was picked up and inoculated in 50ml LB + Kanamycin media in 150 ml flask. The flask was incubated at 37 degrees, 200 RPM until OD600 = 1. Once OD reaches to desired point, 5 ml culture was drawn which is used as an uninduced culture. Uninduced culture was kept on ice until use. To induce the expression of IL-IRa gene 0.5mM IPTG was added to remaining 45 ml culture and flask was further incubated for an additional 4 hours at 30 degree and 200 rpm rotation. Induced culture was harvested after 4 hours and expression of IL-IRa was examined by SDS-PAGE (Figure 3a and 3b).
For SDS-PAGE analysis 1ml culture of induced and uninduced culture (both normalized for OD600 = 1) was taken into 1.5ml Eppendorf tube. The tube was centrifuged and the pellet was resuspended into 50pl PBS. In this suspension, 50 pl SDS-PAGE loading buffer with reducing agent (2x) was added. The mixture was boiled at 96 degrees for 5 min.
Sample was cooled at room temperature and 20 pl of un-induced and induced culture was loaded in the 12% Tris Glycine gel. The gel was run for 1.5 hours at 100 volts. Gels were taken out and incubated in Coomassie Brilliant Blue dye for 1 hour. After staining gel was detained in a destaining solution containing 45% methanol, 10 % acetic acid, and 45% water for 3 hours. The IL-IRa expression was visualized as ~17 kDa protein that is only visible in induced culture. This experiment confirms that the clone generated in the present study can express IL-IRa protein. These clone were further used for large-scale production and purification of IL-IRa.
Step (iv): Fermentation of IL-IRa from BL21 DE3 Escherichia coli:
One ml vial of BL21 DE3 Escherichia coli cells was inoculated into 50 ml LB + Kan media and grown overnight at 37 degrees, 200 rpm. Fermentation was done at 1.5-2 L scale. Escherichia coli cells were inoculated to the fermenter and cultivated at 37 degrees centigrade. The culture was induced with 0.5 mM IPTG/ 15 mM Lactose/ 15 mM Galactose at OD600=20. After 12 hours) post-induction fermentation culture was harvested and cell pellet was prepared by centrifugation.
The harvested microbial cells were resuspended in lysis buffer (20.0 mM sodium acetate, 0.3 M NaCl, 0.1 mM EDTA and 5% sorbitol, pH 4.0-5.0) at a cell pellet: buffer ratio of 1:10 (w/v). The low pH cell lysis was performed using a high-pressure homogenizer at 15000 bar pressure for 10.0 min, and the cell lysate was centrifuged at 10000 rpm for 30.0 min at 4.0 °C to get the soluble fraction of protein in supernatant. Low pH lysis step was given to the precipitate impurities like host cell proteins and host cell nucleic acid. Aliquots of the Supernatant and sediment are analyzed in SDS-PAGE. Centrifuged supernatant containing Interleukin- 1 receptor antagonist (IL-IRa) is filtered through 0.22 pm hydrophilic PES filter. Figure 3a and 3b depicts the SDS-PAGE analysis of the Interleukin- 1 receptor antagonist (IL-IRa) after homogenization.
Step (v) Purification of IL-IRa The multimodal chromatography mode is anion exchange chromatography in flow-through mode. The multimodal chromatography resin I is selected from the group consisting of Toyopearl® NH2-750F or Capto™ adhere resin. The pH of the load was adjusted between 3.0 to 5.0 and the conductivity was lowered by dilution to less than 20 mS/cm. The column was regenerated by passing buffer containing 10 - 50 mM sodium acetate along with 0.1 - 1.0 M sodium chloride CIP was performed by passing 0.2 M NaOH. The multimodal chromatography mode employed in phase II is cation exchange chromatography in bind and elute mode. The multimodal chromatography resin I is selected from the group consisting of Capto™ SP Sepharose Impress or Fractogel®EMD SO3“ resin. The flow through of the previous chromatography step was used as the load for multimodal chromatography phase II without any modification.
Multimodal anion exchange chromatography for separation of various process -related impurities
Multimodal anion exchange chromatography was performed using Capto™ Adhere and Toyopearl® NH2-750F as shown in chromatogram figure 9. The output recombinant protein solution of centrifuged supernatant was used as feed material. Multimodal Anion exchange experiment was conducted using 5 CV, 20 mM sodium acetate, pH 4.5 ± 0.50 as an equilibration buffer, 5 CV, 20 mM sodium acetate, pH 4.5 ± 0.5 as a wash buffer, 5 CV, 20 mM sodium acetate containing 1 M NaCl, pH 4.50 ± 0.50 as an elution buffer and 4 CV, 0.5 M NaOH was used for cleaning of resin. Elution was performed using a step salt gradient. All the process steps were performed at six-minute residence time. Also, multimodal anion exchange chromatography was performed using Capto™ adhere resin at pH 4.5 ± 0.50 is shown in figure 9. Recovery % and mass balance % of multimodal anion exchange chromatography are shown in table 1.
Table 1:- Recovery % multimodal chromatography I output.
Multimodal cation exchange chromatography for separation of various product related impurities
Multimodal Cation exchange chromatography was performed using Fractogel® SO3-(M), and Capto™ SP ImpRes resin. The flow-through recombinant protein solution of multimodal anion exchange chromatography was used as feed material. Multimodal cation exchange experiment was conducted using 5 CV, 20 mM Sodium acetate, pH 4.50 ± 0.50 as an equilibration buffer, 3 CV, 20 mM Sodium acetate, pH 4.50 ± 0.50 as a wash buffer, 15 CV, 20 mM Sodium acetate containing 1 M NaCl, pH 4.50 ± 0.50 as an elution buffer. Elution was performed using a linear salt gradient from 10 % to 100% of elution buffer. All the process steps were performed at six-minute residence time. Also, multimodal cation exchange chromatography was performed using Fractogel® SO3-(M) at pH 4.50 ± 0.50. HCP and HCDNA removal by multimodal cation exchange chromatography are as shown in Figure 10 and Table 2.
Table 2: Impurity content in multimodal cation exchange chromatography output
Analytical characterization of interleukin-1 receptor antagonist (IF-lRa) using various analytical techniques i. SDS PAGE analysis of Interleukin-1 receptor antagonist (IF-lRa) samples
SDS PAGE analysis for identification of expression of Interleukin- 1 receptor antagonist (IF-lRa) was carried out using 12 % (Thickness 1.5 mm) of the resolving gel under reducing conditions (Figure 3 a and Figure 3b) at the stacking gel constant voltage 100V and resolving gel constant voltage 100V conditions. Each sample was boiled for 10 min in the starting buffer before being loaded into the gel. 0.05% (w/v) Coomassie brilliant blue G-250 in 4.5: 1:4.5 (Water: Glacial Acetic acid: Methanol) was used to detect proteins after electrophoretic separation on polyacrylamide gels [26]. ii. RP-HPLC analysis for quantification of Interleukin- 1 receptor antagonist (IF-lRa) product.
The concentration of the Interleukin- 1 receptor antagonist (IF-lRa) product in various chromatography outputs was determined using 4.6 mm x 150 mm of particle size 5 pm ZORBAX Eclipse XDB-C18 column on Agilent 1200 HPEC system. The mobile phase consisted of 0.1% TFA Milli Q (Mobile phase A) and 0.1 % TFA ACN (Mobile phase B). The flow rate was maintained at 0.9 mL/min using a gradient of A to B for 25 minutes method at a wavelength of 280 nm. Quantitative estimation of the Interleukin- 1 receptor antagonist (IL-IRa) product in various process outputs was determined using this analytical method, further shown in Figure 11. iii. SEC-HPLC the aggregate analysis of Interleukin- 1 receptor antagonist (IL-IRa) product
Aggregation and fragments in Interleukin- 1 receptor antagonist (IL-IRa) products were determined using a 7.8 mm x 300 mm YARRA™ SEC 2000 column of particle size 3 pm on an Agilent 1200 HPLC system. The mobile phase consisted of sodium phosphate buffer pH 6.5 ± 0.20 (Buffer A). The flow rate was maintained at 0.75 mL/min using an isocratic method at a wavelength of 280 nm. High molecular weight impurities (HMW), the main peak, and low molecular weight impurities (LMW) of purified monoclonal antibody products were determined using this method, further shown in Figure 12. iv. Intact mass analysis, Peptide fingerprinting, and disulfide mapping of purified Interleukin- 1 receptor antagonist (IL-IRa) using mass spectrometry.
Purified Interleukin- 1 receptor antagonist (IL-IRa) and the innovator samples were buffer exchanged into 50 mM ammonium bicarbonate (pH 7.8 and conductivity 5.14 mS/cm) using a Sephadex G-25 desalting column. The final concentration of desalted proteins was adjusted to 1 pg/pL by measuring the OD using Nanodrop™ 2000 (Thermo Scientific, USA) using 50 mM ammonium bicarbonate. Intact mass analysis of purified Interleukin- 1 receptor antagonist (IL-IRa) product was performed using MALD-TOF analysis. 1.0 mg/ml Innovator and purified the Interleukin- 1 receptor antagonist (IL-IRa) were mixed in a 1:1 ratio with sinapinic acid to perform MALDLTOF analysis. Matrix sinapinic acid (20mg/ml) was prepared in ACN: purified water: Trifluoroacetic acid (TFA) (50:50:0.1). 1 pL of a homogenized mixture of sample and matrix was deposited on a clean 384-well MALDI plate. The plate was inserted into AB SCIEX TOF/TOFTM 5800 instruments. The instrument was used in positive ion linear mode. Nitrogen laser at 337 nm radiation was kept as an ionization source. The MALDLTOF range was 5KDa to 35 KDa and laser intensity in between 5000 to 6000 was used for the analysis of samples depicted in Figure 13. BSA was used as a positive control. Result analysis was performed using Data Explorer software. 1.0 pg of desalted Interleukin-1 receptor antagonist (IL-IRa) samples, with a concentration of 1.0 pg/pL, were directly used for intact mass analysis using SYNAPT™ XS (Waters™ Corporation, USA) mass spectrometry. For peptide fingerprinting and disulfide mapping of Interleukin- 1 receptor antagonist (IL-IRa), 50 pg of protein samples (1.0 pg/pL) were initially denatured in 0.1% RapiGest SF (Waters Corporation, USA) and incubated at 37°C for 1 hour. The samples were subsequently reduced by adding 10 mM DTT at 37°C and alkylated by adding 10 mM iodoacetamide, also at 37°C. After incubation, the proteins were digested using 1.0 pg of trypsin enzyme (Roche, USA), followed by further incubation for 16 hours at 37°C. The digestion reaction was terminated by adding 1.0 pL of formic acid (LC-MS grade, Fluka, USA). For disulfide mapping, 50 pL of Fab samples with a concentration of 1.0 pg/pL were processed and digested similarly to peptide mapping but without DTT (i.e., under non-reducing conditions). These samples were analyzed on a SYNAPT™ XS (Waters Corp, USA) mass spectrometer equipped with an ACQUITY™ UPLC I-Class PLUS system connected to an ACQUITY™ UPLC Protein BEH 300 C4 and an ACQUITY™ UPLC Peptide CSH C18 for intact mass and peptide fingerprinting under reduced and non-reduced conditions, respectively. Data acquired from the SYNAPT™ XS (Waters™ Corporation, USA) was analyzed using UNIFI® v 1.9.4.053 (Waters™ Corporation, USA).
The intact mass of the purified Interleukin- 1 receptor antagonist (IL-IRa) and the innovator samples are illustrated in Figure 6. Peptide fingerprinting and disulfide mapping of the SCVK=ESAACPGWFL peptide are presented in Figures 7 and 8, respectively. Regarding the prepared peptide of IL-IRa, the disulfide bond formation within it (refer to Figure 8) is further detailed in the table below: v. Circular Dichroism spectroscopy analysis
JASCO (J-815) was used for CD Spectroscopy analysis of Interleukin- 1 receptor antagonist (IL-IRa) protein samples. All the samples were analyzed at 25°C temperature. Data pitch was set to 0.025 nm and 1.00 nm with continuous scanning mode using 1 mm cuvette for Far UV CD and data pitch was set to 1.00 nm with continuous scanning mode using 1 cm cuvette for Near UV CD. Sample used for analysis was 0.2 mg/mL for Far UV CD. The far UV CD spectra scan was between 190 and 240nm that contains information of the secondary structure of proteins. The Far UV CD spectra of innovator and biosimilar products indicated similar secondary structures. Figure 4 depicts the far CD spectroscopic analysis of the Interleukin- 1 receptor antagonist (IL-IRa). vi. Fluorescence spectroscopy analysis
Intrinsic fluorescence of purified Interleukin- 1 receptor antagonist (IL-IRa) was compared with the standard innovator molecule (Kineret) to confirm the structural integrity of the refolded molecule. Perkin Elmer L 55 Luminescence Spectrophotometer was used for the fluorescence analysis. Absorb wavelength was selected at 280 nm and emitted spectra were collected in the range of 300 to 400 nm. The excitation and emission wavelength slit widths were set to 5 nm each. An average of three scans was plotted against the emission wavelength. Figure 5 depicts the fluorescence spectroscopic analysis of the Interleukin- 1 receptor antagonist (IL-IRa). vii. ELISA analysis for Host Cell Protein (HCP) Quantitation of Interleukin- 1 receptor antagonist (IL-IRa)
Host Cell Protein analysis of process samples was performed using a two-site Immuno-enzymatic assay (Cygnus E. coli HCP ELISA kit F410). Samples containing E. coli HCPs are reacted with a horseradish peroxidase (HRP) enzyme labeled anti-E. coli antibody simultaneously in microtiter strips coated with an affinity purified capture anti-E. coli antibody. The immunological reactions result in the formation of a sandwich complex of solid phase antibody-HCP-enzyme labeled antibody. The microtiter strips are washed to remove any unbound reactants. The substrate, tetramethylbenzidine (TMB) is then reacted. The amount of hydrolyzed substrate is read on a microtiter plate reader and is directly proportional to the concentration of E. coli HCPs present. viii. Host Cell DNA Analysis by using Picogreen Assay
The presence of DNA in various process samples was estimated using Quant- I TTM PicoGreen dsDNA reagent and Kit (Invitrogen, Catalog number: Pl 1496). A standard curve was prepared using double- stranded lambda DNA by diluting 100 pg/mL to 2 pg/mL. The analysis of the process sample was done using 0.1 mL of sample and 0.1 mL of Pico green reagent (used 200 fold diluted reagent). The reaction mixture was incubated for 5 min and fluorescence was measured using fluorescence spectrophotometer, at 480 and 520 nm wavelength (excitation wavelength 480 nm and emission wavelength 520 nm). Advantages of the Invention:
• Provides high-level soluble expression of interleukin 1-receptor antagonist (IL-IRa).
• Using simple, viable and reliable process to produce IL-IRa. • Better control on the expression of the polypeptide to obtain an IL-IRa.
• Provides high-level expression for commercial production of IL-IRa in pure form with high yield.

Claims

We Claim:
1. A codon-optimized polynucleotide sequence encoding IL-IRa, said sequence comprising SEQ ID NO. 2 or structural variants thereof having at least 70% homology to SEQ ID NO.2.
2. The codon-optimized polynucleotide sequence as claimed in claim 1, wherein structural variants are selected from a group consisting of SEQ ID NO. 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 and 16.
3. An expression vector construct comprising
(a) codon-optimized polynucleotide sequence encoding IL-IRa, said sequence comprising SEQ ID NO. 2 or structural variants thereof having at least 70% homology to SEQ ID NO.2 as claimed in claim 1; and
(b) one or more regulatory sequences selected from the group consisting of a promoter, origin of replication, ribosomal binding site, transcription termination sequence, selectable markers and multiple cloning sites.
4. The expression vector construct as claimed in claim 3, wherein the expression vector construct is a plasmid selected from the group consisting of pUC57, pET9a, pET3a, pET3b, pET3c, pET5a, pET5b, pET5c, pET9b, pET9c, pET12a, pET28a, pET28b, pET28c, pET30a, pET30b, pET30c, pTWIN 1, pTWIN2, pET12b, pET12c, pET17b, pRSET A, pRSET B, pRSET C, pTYBl, pTYB2, pTYB3, pTYB4, pL, rhaBAD, and rhaT.
5. A process for producing soluble form of interleukin 1-receptor antagonist (IL-IRa), comprising the steps of: a) providing an expression vector construct as claimed in claim 3 comprising a codon-optimized polynucleotide sequence encoding IL-IRa comprising SEQ ID NO.
2 or structural variants thereof having at least 70% homology to SEQ ID NO.2 as claimed in claim 1 ; b) transforming a host cell with the expression vector construct of step a); c) selecting the transformed host cells of step b); d) culturing the transformed host cell of step d) in a culture medium followed by inducing the expression of IL-IRa; e) maintaining an induction temperature between 10 to 40°C in the culture medium of step d) to produce IL-IRa; f) harvesting the transformed host cells from the culture medium of step e), followed by resuspension in lysis buffer followed by cell lysis and centrifugation at 6000-9000 rpm to obtain soluble fraction of IL-IRa in homogenate cell supernatant solution; g) filtering the homogenate cell supernatant solution of step f) containing the soluble fraction of IL-IRa; and h) purifying the folded and soluble IL-IRa from the homogenate cell supernatant solution of step g) to obtain the folded and soluble IL-IRa polypeptide with >70% yield using multimodal chromatography.
6. The process as claimed in claim 5, wherein the host cell is selected from the group consisting of Escherichia coli strains BL21 (DE3), BL21 Al, HMS174 (DE3), DH5a, W3110, B834, Lemo21 (DE3), T7, ER2566 and C43 (DE3).
7. The process as claimed in claim 5, wherein the codon-optimized polynucleotide sequence encoding IL-IRa as claimed in claims 1 is cloned adjacent to T7 lac promoter, wherein the T7 lac promoter is induced by the presence of IPTG, galactose, lactose or auto-induction medium.
8. The process as claimed in claim 5, wherein the purification of the folded IL-IRa comprises:
(i) subjecting the supernatant of step (g) to multimodal anion exchange chromatography phase I in a flow-through mode, by feeding the supernatant having pH in the range of 3.0 to 5.0 to multimodal anion exchange chromatography resin, with a buffer comprising 10 - 50 mM sodium acetate and 0.1 - 1.0 M sodium chloride and eluting in the presence of an elution buffer using a step salt gradient having a salt concentration gradient of 5-10 CV; and
(ii) subjecting the folded and soluble IL-IRa obtained from step (i) having pH of 3.0 to 5.0 to multimodal anion exchange chromatography phase II in a flow-through mode, and eluting in the presence of an elution buffer using a step salt gradient having a salt concentration gradient of 5-20 CV to obtain folded and solubilized IL-IRa polypeptide.
9. The process as claimed in claim 8, wherein the multimodal chromatography is in batch, integrated continuous or pseudo -continuous mode; and wherein the anion exchange resin is selected from a group consisting of sulfonate group, sulfopropyl group, and sulphonic acid linked to bead-based, membrane-based, hydrogel-based, and fiber-based chromatography matrix.
10. The process as claimed in claims 8, wherein the folded and solubilized IL-IRa polypeptide contains no more than 0.3 % soluble aggregate and no more than 10 ng/mL of DNA.
PCT/IN2024/050358 2024-04-05 2024-04-05 Interleukin 1-receptor antagonist with increased solubility and process of preparation thereof Pending WO2025210641A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140011728A1 (en) * 2011-02-15 2014-01-09 Novo Nordisk A/S Long-acting il-1 receptor antagonists
WO2022235551A2 (en) * 2021-05-03 2022-11-10 President And Fellows Of Harvard College Fc-fusion protein therapeutic for the treatment of pancreatitis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140011728A1 (en) * 2011-02-15 2014-01-09 Novo Nordisk A/S Long-acting il-1 receptor antagonists
WO2022235551A2 (en) * 2021-05-03 2022-11-10 President And Fellows Of Harvard College Fc-fusion protein therapeutic for the treatment of pancreatitis

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