WO2017021819A1 - Procédé de préparation de protéines ou de peptides - Google Patents

Procédé de préparation de protéines ou de peptides Download PDF

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Publication number
WO2017021819A1
WO2017021819A1 PCT/IB2016/054470 IB2016054470W WO2017021819A1 WO 2017021819 A1 WO2017021819 A1 WO 2017021819A1 IB 2016054470 W IB2016054470 W IB 2016054470W WO 2017021819 A1 WO2017021819 A1 WO 2017021819A1
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Prior art keywords
peptide
process according
protein
ubiquitin
lirapeptide
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PCT/IB2016/054470
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English (en)
Inventor
Jagan Mohan Reddy VELURI
Sairam MUSTOORI
Srinivasulu Reddy YERUVA
Tapas Barui
Jyothishwaran JYOTHISHWARAN
Venkata Sada Siva Rao KANDURI
Tajamul FURKHAN
Krishnan MALOLANARASIMHAN
Narayanasamy Murugan RAVICHANDRAN
Chaitanyakumar KEDARI
Rakesh Goud VASKER
Sahejad DESAI
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Dr. Reddy’S Laboratories Limited
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Publication of WO2017021819A1 publication Critical patent/WO2017021819A1/fr

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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the present application relates to a process for the preparation of peptides or proteins or derivatives thereof by expression of synthetic oligonucleotide encoding desired protein or peptide in prokaryotic cell as ubiquitin fusion construct.
  • Synthetic peptides are valuable research tools in a variety of biological disciplines. Small peptides are widely used to generate antibodies. Immunologists have found peptides useful for assessing antigenic variation and for studying antigen presentation. Cell biologists employ small peptides to disrupt cell-substrate adhesion and to target proteins to specific cellular compartments. Peptides have long served as model systems in studies on the structure, folding or associations of proteins. Peptides also possess useful therapeutic or pharmacological properties.
  • CEP carboxyl extension proteins
  • Ecker et al. J. Biol. Chem. 264, 7715-7719 (May 5, 1989) discloses the expression of cloned eukaryotic genes in microorganisms to allow for the isolation of large quantities of naturally occurring protein products which are present in only trace amounts from natural sources.
  • Butt et al. Proc. Natl. Acad. Sci. 86, 2540-2544 (April 1989) discloses an expression system for cloning ubiquitin-fusion proteins using E. coli and discloses that fusion of ubiquitin by its carboxyl terminal end to the N-terminus of these proteins increases the yield of unstable or poorly expressed proteins such as those referred to by Ecker et al, supra. Butt et al. conclude that ubiquitin fusion technology has the potential for general application in augmenting the yield of cloned gene products in both prokaryotes and eukaryotes.
  • ubiquitin may be helpful in preparing [beta]-galactosidase fusion proteins having any N- terminal amino acid when expressed in both bacteria and yeast.
  • the emphasis is on the use of both eukaryotic and prokaryotic cell expressions utilizing ubiquitin fusion protein or peptide for the cloning and production of natural intracellular proteins. These natural proteins are often larger than ubiquitin.
  • the objective of the present application is to provide an improved recombinant process for the preparation of peptides or proteins involving the use of ubiquitin fusion tag which addresses the problems associated with the processes reported in prior art discussed above.
  • present invention provides a process for producing a protein or peptide or a derivative thereof comprising:
  • the process for producing a protein or peptide or a derivative thereof further comprises:
  • the present invention provides a synthetic oligonucleotide construct of ubiquitin fusion tag along with an affinity tag and a GLP-1 analogue.
  • the present invention provides a synthetic oligonucleotide construct of ubiquitin fusion tag along with linker and a GLP-1 analogue.
  • the present invention provides a synthetic oligonucleotide construct of ubiquitin fusion tag with GLP-1 analogue along with combination of affinity tag and linker.
  • the present invention provides a synthetic oligonucleotide construct of ubiquitin with an affinity tag and Lirapeptide.
  • the present invention provides a synthetic oligonucleotide construct of ubiquitin fusion tag along with linker and Lirapeptide.
  • the present invention provides a synthetic oligonucleotide construct of ubiquitin fusion tag with Lirapeptide along with combination of affinity tag and linker.
  • the present invention provides a process for expression of fusion protein or peptide in high yield.
  • the present invention uses multiple copies of ubiquitin fusion construct cloned together for the expression.
  • the multiple copies of ubiquitin fusion construct can be cloned together as a single construct for expression.
  • the present invention provides processes to obtain pure synthetic peptides, by expressing ubiquitin fusion construct in prokaryotic cells, enzymatically cleaving the peptide from ubiquitin and purification of obtained peptide.
  • the present invention provides a process for producing protein or peptide comprising the steps of:
  • the present invention provides a purification of protein or peptide by precipitation at its isoelectric (pi) point.
  • present invention provides a process for preparing Liraglutide or a derivative thereof comprising the steps of:
  • R 2 is selected from C3_39-alkyl, C3_39-alkenyl or €3.39 alkadienyl;
  • R 3 is selected from hydroxy or a reactive ester thereof such as N-hydroxy imide ester
  • the present invention provides a process for preparing Liraglutide or a derivative thereof comprising the steps of:
  • Rl is selected from hydrogen or Ci- 6 -alkyl
  • R3 is selected from hydroxy or a reactive ester thereof such as N-hydroxy imide ester
  • step (b) optionally, hydrolyzing the acylated Lirapeptide obtained in step (b) when Ri is Ci-6 alkyl to obtain Liraglutide.
  • Figure 1 is Restriction analysis of recombinant constructs.
  • the recombinant plasmids (pUC57-Ubi-Lirapeptide with multiple copies-pentamer) were analyzed by restriction digestion and resolved on 1.2% agarose gel as per loading pattern subsequently stained with EtBr and image captured using UV light.
  • Figure 2 is SDS-PAGE analysis of Lirapeptide with ubiquitin fusion construct prepared according to Example 1.
  • Ubiquitin is a highly conserved, 76-residue protein having a C-terminus composed of Arg-Gly-Gly and is found in all eukaryotic cells both free and covalently conjugated to a variety of cellular proteins. Ubiquitin is found in cells as diverse as mammals, yeast and celery. Ubiquitin is attached by its carboxyl terminus to amino groups of other proteins. When ubiquitin is attached to the alpha-amino terminus, such products are referred to in the literature as ubiquitin carboxyl extension proteins.
  • the extension proteins are cleaved from the ubiquitin molecule by hydrolases (peptidases). It has been postulated that attachment of ubiquitin to a protein is a signal for the latter's degradation by proteolysis.
  • Ubiquitin has a neutral isoelectric point of 6.7 and a molecular weight of 8565.
  • Ubiquitin is extremely stable to heat and extremes of pH which are essential properties for its use as a substrate to facilitate preparation of peptides of the desired amino acid sequence and allow for cleavage by an appropriate enzyme.
  • a variety of peptides having any desired amino acid sequence can be prepared by utilizing ubiquitin as a fusion tag in which synthetic oligonucleotide encoding desired protein or peptide amino -terminal is fused to carboxyl terminal of ubiquitin gene.
  • the eukaryotic enzymes that cleave peptides from the ubiquitin-peptide fusion products are necessary components in the production of pure peptides.
  • Ubiquitin and the cleavage enzymes are either not present in prokaryotes such as E. coli or are present in such small amounts as not cleavable at the ubiquitin-extension peptide bond. Therefore, cleavage of ubiquitin fusion protein or peptide extended at its carboxyl terminus by synthetic peptides will not occur without the addition of an appropriate cleavage enzyme.
  • amino acid sequence of Lirapeptide has Seq. ID no. l as follows: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
  • amino acid sequence of Liraglutide has Seq. ID no.2 as follows:
  • the present invention provides a process for producing a protein or peptide or a derivatives thereof comprising the steps of:
  • present invention involves the preparation of ubiquitin fusion construct comprising fusion of amino terminal of synthetic oligonucleotide encoded for desired protein or peptide with carboxyl terminal of ubiquitin.
  • the ubiquitin fusion construct may further fuse with affinity tag or linker or combination of affinity tag and linker.
  • the synthetic oligonucleotide constructs of desired protein or peptide fused with ubiquitin, optionally along with affinity tag or linker or combination of affinity tag and linker may be cloned in cloning vector.
  • the synthetic construct cloned in cloning vector transformed into cloning prokaryotic cell strains and transformants can be selected by antibiotic selection marker present in cloning vector.
  • the ubiquitin fusion construct isolated by restriction digestion of the recombinant plasmid with the restriction enzymes.
  • the process for producing a protein or peptide or a derivative thereof further comprises:
  • the expression vector can be ligated with obtained ubiquitin fusion construct.
  • the expression vector may already have desired restriction sites for ligation or can be introduced into expression vector by using restriction enzyme.
  • Ligated ubiquitin fusion construct optionally linked to nucleotide encoding affinity tag or linker or combination of affinity tag and linker and expression vector can be transformed into host cell for inducing the expression.
  • the ligated ubiquitin fusion construct and expression vector optionally can be transformed into cloning prokaryotic strains and transformants can be selected by antibiotic selection marker present in expression vector.
  • the ubiquitin synthetic construct optionally can be purified by gel electrolysis or any other technique well known in the art.
  • synthetic construct can be purified by gel electrophoresis or any other technique well known in the art.
  • the ubiquitin fusion construct is transformed into expression host cell and transformants can be selected by antibiotic selection marker present in expression vector.
  • protein expression in recombinant expression host is induced by chemical agent. After expression the positive colonies of expression host having fusion protein or peptide can be selected with the help of selection marker and further used for fermentation process.
  • the suitable affinity tag can be selected from Polyarginine-tag (Arg-tag), Polyhistidine-tag (His-tag), S-tag, SBP-tag (streptavidin-binding peptide), Maltose binding protein, chitin binding domain (CBD) and the like.
  • the suitable linker is a peptide chain of acidic amino acids or basic amino acid, wherein chain length is of 1-10 acidic or basic amino acids.
  • the said linker is a peptide chain comprising one or more acidic amino acids selected from Glutamate (Glu), Aspartate (Asp) or derivatives thereof; wherein chain length is of 1-10 acidic amino acids.
  • the said linker is a peptide chain comprising one or more basic amino acids selected from Lysine (Lys), Arginine (Arg) or derivatives thereof; wherein chain length is of 1-10 basic amino acids.
  • the suitable vector used in cloning can be selected from pUC57, pTZ, pBR322 and the like.
  • the suitable prokaryotic host cell used in cloning can be selected from E. coil, Pseudomonas flurescence, Bacillus subtitis and the like.
  • the suitable E. coli strains used in cloning can be selected from E. coli DH5a, E. coli Top 10 and the like.
  • the suitable restriction enzymes used in cloning can be selected from Nde l and Xhol, Bam HI, Sapl, EcoRi, Hind III, Kpnl and the like.
  • the suitable prokaryotic expression host cells used in expression can be selected from E. coli, Pseudomonas flurescence, Bacillus subtitis and the like.
  • the suitable E. coli strains used in expression can be selected from E. coli BL21(DE3) pLysS, E. coli JM109, E. coli JM109 (DE3), Rosetta, Origami and the like.
  • the suitable chemical agent used for induction can be selected from IPTG (isopropylthiogalactoside), tryptophan, nalidexic acid, oxalinic acid, nitrogen or sugars analogs; wherein sugar analogs can be selected from lactose, maltose, arabinose and the like.
  • the transformation method can be selected from heat shock method, electroporation and the like.
  • heat shock method involves heat shock to cells at about 42°C for about 30 sec to 120 sec and subsequently kept on ice for about 2-10 minutes.
  • expression vector that is used in the process of the present invention can be commercially available expression vectors or custom designed vectors selected from pET vectors, pd451sR and the like.
  • pET vectors may be selected from pET24a, pET28a or any other pET vector known to person skill in the art.
  • expression can be carried out at a pH of about 5.0 to about 7.5 and/or at a temperature of about 25 °C to about 42°C.
  • the expression vector according to present invention comprises of promoters, selection marker, multiple cloning region, origin of replication & operator /repressor system.
  • the suitable promoter can be selected from T7, TRC, TRP, BAD, LacUV5 or their derivatives or combination thereof.
  • the suitable selection marker may be selected from kanamycin, ampicillin, chloramphenicol or tetracycline or their combinations in their wild or mutated forms.
  • the suitable origin of replication can be selected from pUC ORI, pPR322 and the like in their wild type or mutated form.
  • the suitable Operator/repressor systems can be selected from Lac operon system (see Miller et al. "The operon”, Cold Spring Harbor Laboratory, 1980 and Hillen et al, J. Mol. Biol. 172, 185-201 [1984]).
  • the present invention provides a synthetic oligonucleotide construct of ubiquitin fusion tag along with an affinity tag and a GLP-1 analogue.
  • the present invention provides a synthetic oligonucleotide construct of ubiquitin fusion tag along with linker and a GLP-1 analogue.
  • the present invention provides a synthetic oligonucleotide construct of ubiquitin fusion tag with GLP-1 analogue along with combination of affinity tag and linker.
  • the present invention provides a synthetic oligonucleotide construct of ubiquitin with an affinity tag and Lirapeptide.
  • the present invention provides a synthetic oligonucleotide construct of ubiquitin fusion tag with Lirapeptide in combination with an affinity tag and linker.
  • the present invention provides a process for expression of fusion protein or peptide in high yield.
  • the present invention uses multiple copies of ubiquitin fusion construct cloned together for the expression.
  • the multiple copies of ubiquitin fusion construct can be cloned together as a single construct for expression.
  • the present invention uses two copies of ubiquitin fusion construct cloned together for the expression.
  • the present invention provides processes to obtain pure synthetic peptides, by expressing ubiquitin fusion construct in prokaryotic cells, enzymatically cleaving the peptide from ubiquitin and purification of obtained peptide.
  • the present invention provides a process for producing protein or peptide comprises:
  • present invention involves fermentation process for increase in accumulation of resulting ubiquitin fusion protein or peptide by inducing transformant prokaryotic cells having expression vector and ubiquitin fusion construct in culture medium with chemical agent.
  • the fermentation may be carried out in fed-batch or fed-mode to produce ubiquitin fusion protein or peptide.
  • Improved expression of the proteins using the ubiquitin fusion construct of the present invention depends on various parameters of the fermentation process. Some of the suitable parameters are fermentation media, concentration of the inducer, feed media and nutrient feed rate.
  • the fermentation medium is the medium required for the growth and expression of transformant prokaryotic cells at fermenter scale.
  • the fermentation medium comprises of suitable salts, vitamins, carbon source and nitrogen source.
  • the suitable salts can be selected from ammonium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, calcium chloride, magnesium chloride, EDTA sodium salt, sodium molybdate, zinc sulphate, ferrous sulphate, copper sulphate, monopotassium phosphate, dipotassium phosphate, magnesium sulphate and the like or combination thereof.
  • the carbon source may comprise glucose, glycerol, maltose, sucrose, dextrose, fructose or mannitol and the like or combination thereof.
  • the nitrogen source may comprise ammonia, nitrate, peptone, soya peptone, yeast extract, tryptone and the like or combination thereof.
  • the suitable vitamin can be selected from Thiamine (vitamin B or its related compounds) and the like or combination thereof.
  • the fermentation medium further comprises acids selected from citric acid, boric acid and the like or combination thereof.
  • An aspect of present invention involves preparation of inclusion body.
  • the inclusion body preparation involves resuspending of cell pellet in non-denaturing lysis buffer (Tris-HCl + NaCl+ EDTA, pH 8.0) by stirring and treating with lysozyme at room temperature.
  • the resuspended cells can be homogenized under chilled conditions and centrifuged (Sorvall, Thermofisher). The unbroken cells, large cellular debris, and the inclusion body will be pelleted down and supernatant can be transfer from the pellet.
  • the proteinaceous and non- proteinaceous contaminants present with inclusion body can be removed by washings.
  • the pellet can be resuspended in wash buffer containing detergents selected from but not limited to sodium deoxycholate, Triton and Tween.
  • the suspension can be centrifuged (Sorvall, Thermofisher).
  • the supernatant containing contaminants can be transfer from the pellet.
  • the inclusion body pellet was resuspended in wash buffer (Tris-HCl + NaCl + EDTA, pH 8.0) and centrifuged.
  • the supernatant containing contaminants was transferred carefully from the inclusion body pellet.
  • the obtained inclusion body pellet can be solubilized in denaturing buffer (Tris-HCl + Urea, pH 8.0) and centrifuged.
  • the supernatant can be transfer from the pellet.
  • the obtained supernatant may be optionally clarified by Tangential flow filtration (0.45 or 0.65 ⁇ ) to give ubiquitin fusion protein or peptide.
  • clarification of lysate by Tangential Flow Filtration involves subjecting of above obtained supernatant to TFF using 0.65 ⁇ hollow fiber (Asahi Kasei) system in order to remove insoluble debris and improve clarity.
  • the cell lysis can be performed without inclusion body preparation.
  • the cell pellets can be directly resuspended in denaturing buffer (Tris-HCl + Urea, pH 8.0), homogenized and centrifuged.
  • the supernatant can be transfer from the pellet.
  • the obtained supernatant may be optionally clarified by Tangential flow filtration (0.45 or 0.65 ⁇ ) to give clarified supernatant containing ubiquitin fusion protein or peptide.
  • ubiquitin fusion protein or peptide may be purified by the purification techniques selected from affinity chromatography i.e Ni NTA chromatography, ion exchange chromatography (cation or anion), reverse phase chromatography or any other technique well known in the art.
  • ion exchange chromatography can be used for purification of fusion protein or peptide by modulating the isoelectric (pi) point of fusion protein.
  • the suitable linker can be added to the N-terminal of ubiquitin fusion construct.
  • cation exchange chromatography can be used for purification when basic amino acid linker is used with ubiquitin fusion construct for protein expression.
  • anion exchange chromatography can be used for purification when acidic amino acid linker is used with ubiquitin fusion construct for protein expression.
  • cation exchange chromatography can be used for purification when combination of basic amino acid linker and affinity tag with ubiquitin fusion construct is used for protein expression.
  • anion exchange chromatography can be used for purification when combination of acidic amino acid linker and affinity tag with ubiquitin fusion construct is used for protein expression.
  • cells were harvested and lysed to release the expressed protein in a buffer containing chaotropic agents like urea or guanidine hydrochloride.
  • the purification process generally includes one or two steps to produce a recombinant protein product from crude cell lysates.
  • Another unique feature of preparation of peptides using ubiquitin fusion protein or peptide is that the production is intracellular.
  • the protein of interest is expressed in insoluble inclusion body, it is easy to separate the inclusion body from soluble materials derived from E. coli such as the proteins of host cell, DNA, polysaccharides in the early stage of purification.
  • the desired peptide is obtained as partly as soluble and partly insoluble form.
  • the protein of interest can be recovered from intracellular proteins by using the difference in charge, solubility, size, hydrophobicity, etc.
  • the ubiquitin fusion protein or peptide can be cleaved enzymatically in vivo or in vitro (using either pure or partially purified fusion specific protease) by cleavage enzyme which cleaves at the junction between ubiquitin fusion tag and the protein or peptide of interest to generate the protein or peptide of interest having the desired amino acid at its amino-terminus.
  • the cleavage enzyme for ubiquitin fusion protein or peptide cleavage can be selected from Yeast ubiquitin hydrolase (YUH1) and the like.
  • expression during fermentation can be carried out at a pH of about 5.0 to about 7.5 and/or at a temperature of about 25°C to about 42°C.
  • purification of protein or peptide is carried out by precipitation at its isoelectric (pi) point.
  • purification of protein or peptide comprises adjusting the pH of the reaction mixture comprising protein or peptide to its isoelectric (pi) point and isolating the pure protein or peptide.
  • Lirapeptide isolated at isoelectric point has a purity of about 80% or about 90% or about 95% as measured by High Performance Liquid Chromatography (HPLC).
  • Lirapeptide isolated at isoelectric point has purity of about 80% as measured by High Performance Liquid Chromatography (HPLC).
  • the desired protein or peptide can be further purified using purification method which can be selected from ion exchange chromatography, affinity chromatography, reversed phase chromatography or any other well-known method in the art.
  • Ubiquitin fusion systems are employed for the preparation of peptides. They are extremely stable and are expressed at high levels in soluble form or insoluble form. E. coli has been used as a model for ubiquitin-peptide fusion systems in 20-liter batch cultures. Ubiquitin fusion protein or peptide is produced at very high levels in E. coli and related hosts. The specific yield is defined as the percentage, taken as a ratio, of recombinant protein product to total cellular protein, as measured by densitometry of SDS-PAGE gels run on whole cell samples lysed in loading buffer and loaded directly. Specific yields in the E. coli ubiquitin fusion system, grown and induced as described, exceed 20% and approach 30%.
  • the highest reported accumulation of a recombinant protein in E. coli is 50% of the total cellular protein, i.e., 50% specific yield.
  • Another E. coli expression system claims 40% specific yields for some recombinant proteins.
  • the yield of recovered fusion protein or peptide is one gram to four gram of fusion protein or peptide per liter of bacterial culture. This protein or peptide is both soluble and recoverable.
  • the majority of protein or peptide in supernatants of lysed cells is the product.
  • the fusion can be further purified from host proteins with an 85 °C heat step, in which most of the host proteins precipitate while the ubiquitin fusion stays in solution.
  • converting protein or peptide to a derivative thereof comprises converting protein or peptide selected from GLP-1 analogue to a derivative thereof.
  • the said derivative is Liraglutide.
  • present invention provides a process for preparing Liraglutide or derivatives thereof comprising the steps of:
  • R 2 is selected from C3_39-alkyl, C3_39-alkenyl or €3.39 alkadienyl;
  • Liraglutide or derivatives thereof Liraglutide or derivatives thereof.
  • the compound of formula I is selected from a compound of formula II or a compound of formula III:
  • the present invention provides a process for preparing Liraglutide or derivatives thereof comprising the steps of:
  • Rl is selected from hydrogen or Ci-6-alkyl
  • R3 is selected from hydroxy or a reactive ester thereof such as N-hydroxy imide ester
  • step (b) optionally, hydrolyzing the acylated Lirapeptide obtained in step (b) when Ri is Ci-6 alkyl to obtain Liraglutide or derivatives thereof.
  • the compound of formula IV is selected from a
  • the suitable halide for palmitoyl halide can be selected from chloride, bromide or iodide.
  • the palmitate ester may contain alkyl group selected from C ⁇ - alkyl, e.g. methyl, ethyl, propyl, prop-2-yl, butyl, but-2-yl, 2-methylprop-l-yl, 2-methyl-prop-2-yl (tert-butyl), hexyl and the like.
  • acylation of Lirapeptide or derivatives thereof further comprises the steps of:
  • acylating the transition metal complex of protein or peptide with acylating agent selected from compound of formula I or formula II or formula III or formula IV or formula V or formula VI in the presence of coupling reagent and solvent.
  • suitable transition metal agent comprising transition metal hydroxide, transition metal carbonate, transition metal chloride, transition metal sulfate, transition metal acetate and the like.
  • the transition metal can be selected from Scandium (Sc), Titanium(Ti), Vanadium(V), Chromium (Cr), Manganese (Mn), Iron(Fe), Cobalt(Co), Nickel (Ni), Copper (Cu) and the like or combination thereof.
  • transition metal agent can be selected from a group comprising copper sulfate, nickel sulfate, nickel acetate, copper acetate and cobalt acetate, cobalt sulfate and the like or combination thereof.
  • the coupling agent used for the coupling of the amino acids can be selected from HATU, HBTU, EDC, DCC, DIC, BOP and the like or combinations thereof.
  • additive may be added with coupling reagent which can be selected from HOBt, HOSu, HO At, and the like or combinations thereof.
  • the solvent used for the coupling reaction can be selected from dichloromethane, tetrahydrofuran(THF), dimethylformamide (DMF), N-methylpyrolidone, acetonitrile, dimethylsulfoxide (DMSO), and the like or combinations thereof.
  • the said acylated Lirapeptide obtained in the present invention may contain functional groups such as esters can be selected from Ci-6 alkyl, e.g. methyl, ethyl, propyl, prop-2-yl, butyl, but-2-yl, 2-methylprop-l-yl, 2-methyl-prop-2-yl (tert-butyl), hexyl and the like.
  • esters can be selected from Ci-6 alkyl, e.g. methyl, ethyl, propyl, prop-2-yl, butyl, but-2-yl, 2-methylprop-l-yl, 2-methyl-prop-2-yl (tert-butyl), hexyl and the like.
  • ester can be hydrolyzed by basic hydrolysis or acidic hydrolysis.
  • Basic hydrolysis can be carried out using bases such as alkali metal hydroxides including sodium hydroxide, potassium hydroxide, lithium hydroxide and the like; alkali metal carbonates including sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate or the like; ammonia, sodium t-butoxide, potassium t-butoxide, sodium methoxide, and the like.
  • the acidic hydrolysis can be carried out using inorganic or organic acids, but are not carboxylic acids.
  • Suitable inorganic acids are those having pKa values below about 4.0 at room temperature in aqueous solution (see Moeller, Inorganic Chemistry, John Wiley & Sons (1952) at pages 314 and 315). Specific examples of such acids are sulfuric acid which is a preferred strong acid catalyst and hydrochloric acid, perchloric acid, nitric acid, phosphoric acid, and hydrofluoric acid.
  • Organic acids suitable for strong acid catalysts herein are noncarboxylic acids having pKa values below 2.0 in water at room temperature (see Handbook of Chemistry and Physics, 58th edition, Chemical Rubber Publishing Company at pages D-150 et seq.).
  • Suitable organic acids are methane sulfonic acid, naphthalene sulfonic acid, trifluoromethyl sulfonic acid, and p-toluene sulfonic acid. Mixtures of strong acid catalysts can also be used.
  • the reaction time for ester hydrolysis should be sufficient to complete the reaction which depends on scale and mixing procedures, as is commonly known to one skilled in the art. Typically, the reaction time can vary from about few minutes to several hours. For example the reaction time can be from about 10 minutes to about 24 hours, or any other suitable time period. Suitable temperatures for the said hydrolysis may be less than 120°C, less than 100°C, less than 80°C, less than 60°C, less than 40°C, less than 20°C, less than 10°C, or any other suitable temperatures.
  • the Liraglutide or derivative thereof can be isolated by techniques known in the art.
  • isolation can be done by removal of solvent from the solution containing the product.
  • Suitable techniques which can be used for the removal of solvent include but not limited to evaporation, flash evaporation, simple evaporation, rotational drying, spray drying, agitated thin-film drying, agitated nutsche filter drying, pressure nutsche filter drying, freeze drying or lyophilization or any other technique known in the art.
  • the reaction product of a given step can be carried forward to the next step without the isolation of the product from the previous step i.e., one or more reactions in a given process can be carried out in-situ as one pot process optionally in the presence of the same reagent/s used in a previous step wherever appropriate to do so, to make the process of the present invention economical and commercially more viable.
  • the resulting compounds may be optionally further dried. Drying can be carried out in a tray dryer, vacuum oven, air oven, cone vacuum dryer, rotary vacuum dryer, fluidized bed dryer, spin flash dryer, flash dryer, lyophilizer, or the like.
  • the drying can be carried out at temperatures of less than about 60°C, less than about 50°C, less than about 40°C, less than about 30°C, less than about 20°C, or any other suitable temperatures; at atmospheric pressure or under a reduced pressure; as long as the Liraglutide is not degraded in its quality.
  • the drying can be carried out for any desired time until the required product quality is achieved. Suitable time for drying can vary from few minutes to several hours for example from about 30 minutes to about 24 or more hours.
  • the reaction product of a given step can be isolated and purified by the methods described herein or the methods known to a person skilled in the art before using in a subsequent step of the process.
  • purification of protein or peptide can be performed one to five times.
  • the purification processes include but not limited to preparative reverse phase HPLC, ion exchange chromatography, size exclusion chromatography, affinity chromatography or any other well-known technique in the art.
  • the purification of protein or peptide comprising the steps of:
  • the sample can be prepared by dissolving the crude protein or peptide in suitable buffer.
  • the suitable buffer that can be used in step (a) can be acidic or basic.
  • the suitable buffer can be selected, but are not limited to Tris (Tris(hydroxymethyl)aminomethane), ammonium acetate, ammonium hydrogen carbonate and the like.
  • the sample of protein or peptide in suitable buffer can be filtered through a filter of about 0. ⁇ to about 1 ⁇ .
  • Suitable silica gel column types that can be used in above step (c) can be selected from, but are not limited to the following silica gel sorbents: DaisogelTM, KromasilTM C18 100-16, KromasilTM C18 100-10, KromasilTM C8 100-16, KromasilTM C4 100-16, KromasilTM Phenyl 100-10, KromasilTM C18 Eternity 100-5, KromasilTM C4 Eternity 100-5, ChromatorexTM C18 SMB 100-15 HE, ChromatorexTM C8 SMB 100-15 HE, ChromatorexTM C4 SMB 100-15 HE, DaisopakTM SP 120-15 ODS-AP, DaisopakTM SP 120-10-C4-Bio, DaisopakTM SP 200-10-C4-Bio, ZeosphereTM C18 100-15, ZeosphereTM C8 100-15, ZeosphereTM C4 100-15, SepTech ST 150-10 C18, Luna C18 100-10, Gemini C18 110-10, YMC Triart C18
  • elution of the protein or peptide from silica gel column can be performed by eluent.
  • elution can be performed by gradient method or isocratic method.
  • the eluent used in reverse phase High performance Liquid chromatography (HPLC) can be selected from polar solvent, water or suitable mixtures thereof.
  • polar solvent and water can be used at different/independent run time of eluent through the silica gel column of reverse phase High performance Liquid chromatography (HPLC).
  • the polar solvent can be selected from acetonitrile, tetrhydrofuran, acetone, methanol, ethanol, propanol, isopropanol or suitable mixture thereof and the like.
  • modifier can be added to the eluent before elution, wherein modifier is trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • trifluoroacetic acid of about 0.1% to about 0.001% by volume relative to the total volume of the water solution can be used during elution.
  • pure protein or peptide is obtained from pure fractions as collected from reverse phase High performance Liquid chromatography (HPLC) by removing polar solvent and optionally lyophilizing.
  • HPLC High performance Liquid chromatography
  • pure protein or peptide is obtained from pure fractions as collected from reverse phase High performance Liquid chromatography (HPLC) by removing polar solvent from pure fractions and precipitating at isoelectric (pi) point and optionally lyophilizing the obtained pellet.
  • HPLC High performance Liquid chromatography
  • step (d) pure protein or peptide is obtained from pure fractions as collected from reverse phase High performance Liquid chromatography (HPLC) by removing polar solvent from pure fractions and lyophilizing.
  • HPLC High performance Liquid chromatography
  • acid can be used for isoelectric (pi) point precipitation.
  • the suitable acid can be selected from an organic acid or an inorganic acid.
  • the suitable organic acid can be selected from formic acid, acetic acid and propionic acid, halogenated acetic acids such as chloroacetic acid, dichloroacetic acid, trifluoroacetic acid and the like or combinations thereof.
  • the suitable inorganic acid can be selected from hydrohalides such as hydrochloric acid, hydrobromic acids, hydrofluoric acid, sulfuric acid, nitric acid or boric acid and the like or combinations thereof.
  • the isoelectric (pi) point precipitation can be performed one or more times e.g., five times for obtaining pure protein or peptide.
  • pure fraction collected from reverse phase High performance Liquid chromatography can be neutralized by using alkali carbonates selected from sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate or the like.
  • protein or peptide purified by reverse phase High Performance Liquid Chromatography has purity at least of about 85%.
  • protein or peptide purified by reverse phase High Performance Liquid Chromatography has purity at least of about 95%.
  • Lirapeptide purified by reverse phase High Performance Liquid Chromatography has purity at least of about 85%.
  • Lirapeptide purified by reverse phase High Performance Liquid Chromatography has purity at least of about 95%.
  • amino acid refers to an organic compound comprising at least one amino group and at least one acidic group.
  • the amino acid may be a naturally occurring amino acid or be of synthetic origin, or an amino acid derivative or amino acid analog.
  • amplification refers to the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction (PCR) technologies well known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold 25 Spring Harbor Press, Plainview, N.Y.).
  • PCR polymerase chain reaction
  • peptide refers to any peptide comprising two or more amino acid residues connected by peptide linkage.
  • protein refers to large molecule composed of one or more chains of amino acids in a specific order.
  • protein or peptide refers to GLP-1 analogues or any other protein or peptide, which contain two or more terminal and/or side chain amino groups.
  • GLP-1 analogues refers to GLP-1 selected from GLP-1 (1- 35), GLP-1 (1-36), GLP-l(l-36)amide, GLP-l(l-37), GLP-l(l-38), GLP-l(l-39), GLP-1(1- 40), GLP-1(1-41) and the like.
  • Preferred GLP-1 include but not limited to Arg 26 -GLP-l(l- 37); Arg 34 -GLP-l(7-37); Arg 34 Lys 26 -GLP-l(7-37); Lys 36 -GLP-l-(7-37); Arg 26 34 Lys 36 -GLP- 1(7-37); Arg 26 ' 34 Lys 38 GLP-l(7-38); Arg 26 ' 34 Lys 39 -GLP-l(7-39); Arg 26 ' 34 Lys 40 -GLP- 1(7-40); Arg 26 Lys 36 GLP- 1(7-37); Arg 34 Lys 36 -GLP-l(7-37); Arg 26 Lys 39 -GLP-l(7-39);
  • Arg 34 Lys 40 GLP- 1(7-40); Arg 26 ' 34 Lys 36 ' 39 -GLP-l(7-39); Arg 26 ' 34 Lys 36 ' 40 -GLP- 1(7-40); Gly 8 Arg 26 -GLP- 1(7-37); Gly 8 Arg 34 -GLP-l(7-37); Gly 8 Lys 36 -GLP-l(7-37);
  • derivative is chemically modified protein or peptide or an analogue thereof, wherein at least one substituent is not present in the unmodified protein or peptide or an analogue thereof, i.e. a peptide which has been covalently modified.
  • Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters and the like.
  • acylating refers to the introduction of one or more acyl groups covalently bonded to the free amino groups of the protein or peptide.
  • acylation means the acylation of the amino group of the protein or peptide.
  • Lirapeptide is Arg 34 -GLP-l(7-37) which is liraglutide before acylation.
  • Ultra-Lirapeptide refers to synthetic oligonucleotide construct of ubiquitin fusion tag and Lirapeptide.
  • Palm is palmitoyl.
  • HATU is 2-(7-aza-lH-benzotriazole-l-yl)-l, l,3,3-tetramethyl uranium hexafluorophosphate .
  • EDC is l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide.
  • DCC is Dicyclohexylcarbodiimide.
  • DIC is Diisopropylcarbodiimide.
  • BOP is Benzotriazol-l-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate .
  • HOBt is 1-Hydroxybenzotriazole.
  • HSu N-Hydroxysuccinimide
  • HOAt is l-Hydroxy-7-aza-lH-benzotriazole.
  • room temperature refers to the temperatures of the thing close to or same as that of the space, e.g., the room or fume hood, in which the thing is located'. Typically, room temperature can be from about 20°C to about 30°C, or about 22°C to about 27°C, or about 25 °C.
  • reactions of the processes described herein can be carried out in air or under an inert atmosphere.
  • reactions containing reagents or products that are substantially reactive with air can be carried out using air sensitive synthetic techniques that are well known to the person skilled in art.
  • Example 1 Cloning and expression of Lirapeptide-Ubiquitin fusion construct in E. coli. Step a. Cloning of synthetic gene construct of Lirapeptide with Ubiquitin fusion tag
  • the synthetic gene construct of Lirapeptide with Ubiquitin fusion tag and 6XHis affinity tag was prepared as a synthetic construct and cloned in pUC57.
  • the cloned synthetic construct was transformed by heat shock method into E. coli DH5a and incubated at 37°C for lhr. After incubation, the cells were pellet down and re-suspended. The re-suspended cells were spread over ampicillin medium and incubated overnight (18hr) to obtain recombinant colonies of E. coli DH5a.
  • the plasmid containing his tag-ubiquitin-Lirapeptide construct was isolated from overnight grown culture of recombinant colonies of E. coli DH5a using Ndel and Xhol as restriction digestion enzymes.
  • Figure 1 refers to restriction analysis of recombinant plasmids (pUC57-Ubi-Liraglutide with pentamer copies).
  • the recombinant plasmid was analyzed by restriction digestion (Nde l and Xhol) and resolved on 1.2% agarose gel as per loading pattern subsequently stained with EtBr and image captured using UV light.
  • Step b Sub-cloning & Characterization
  • the expression vector pET24a was also digested with Nde l and Xhol for cohesive end ligation.
  • the digested plasmid (pET24a) and the above isolated synthetic construct were ligated with insert: vector in molar ratio of 3: 1 was incubated at 16°C for 18 hr.
  • the ligated product (pET24a: : his tag-ubiquitin-Lirapeptide) was transformed in to competent E. coli DH5a by heat shock method and transformants were selected by antibiotic selection marker (Kanamycin (5C ⁇ g/ml)). Positive colonies were initially screened by PCR amplification using T7 primers.
  • Step c Expression studies of Lirapeptide in E. coli expression hosts
  • the recombinant expression plasmids (pET24a + his tag-ubiquitin-Lirapeptide) were transformed into E. coli BL21 (DE3) cells by heat shock method and incubated at 37°C for lhr. After incubation, the cells were pellet down and re-suspended. The re-suspended cells were spread over kanamycin (5C ⁇ g/ml) and incubated overnight (18hr) to obtain isolated recombinant colonies of E. coli BL21 (DE3).
  • E. coli BL21 (DE3) were induced with ImM IPTG and incubated at 37°C with shaking overnight (18 hr.). Samples were collected at different intervals (3hrs, 18hrs) for checking expression. All induced and un-induced samples were resolved on 1.2% agarose gel. After resolving on gel, the gel was stained with stain (coomassie blue stain) followed by de-staining with de-staining solution (water: methanol: acetic acid). Research cell bank was prepared by inoculating positive colony in 100 ml of culture medium with kanamycin and grown overnight at 37°C and 250 rpm shaking.
  • Example 2 Cloning and expression of Lirapeptide-Ubiquitin fusion construct in E. coli.
  • Step a Cloning of synthetic gene construct of Lirapeptide with Ubiquitin fusion tag: The his tag-ubiquitin-Lirapeptide fusion construct was amplified from pUC57-his tag- Ubiquitine-Lirapeptide5 template by PCR using primers, Forward primer (Electra Lira for Sapl -FP-- 5 -CGC TGA AGC TCT TCT ATG CAC CAT CAC CAT CAC ATG C - 3') Reverse primer (Electra Lira Rav Sapl -RP- 5 -TTG ACG GCT CTT CTA CCG GAT CCT TAG CCA CGA CCA C -3').
  • the single copy, two copies and three copies his tag- Ubiquitin-Lirapeptide fusion constructs were amplified and purified using QIAquick gel extraction kit.
  • the gel purified PCR amplicons, single copy, two copies and three copies his tag-ubiquitin-Lirapeptide fusion construct was restriction digested with Sapl and purified using QIA PCR purification kit.
  • the above example obtained single copy, two copies and three copies his tag-ubiquitin- Lirapeptide fusion construct separately ligated into Sapl site of linear pD451SR expression vector (DNA2.0) and transformed into chemically competent E. coli ToplO by heat shock method and recombinant clones were selected on LB+ Kanamycin plates. The transformed clones were confirmed for the presence of Ubiquitin-Lirapeptide fusion protein encoding gene by PCR and restriction digestion.
  • Step c Expression studies of Lirapeptide in E. coli expression hosts
  • the confirmed recombinant plasmids, pD451SR-hisUbiLiral (single copy) pD451SR- hisUbiLira2 (two copies) and pD451SR-hisUbiLira3 (three copies) were transformed into E.coli expression hosts, JM109DE3 and HMS174DE3 by heat shock method and selected on LB+ Kanamycin plates.
  • the recombinant E. coli expression clones of JM109 (DE3) and HMS174(DE3) were screened for the expression of Ubiquitin-Lirapeptide fusion protein by inducing with ImM IPTG at 37°C for overnight.
  • Pre-seed medium 500 ml is prepared by dissolving 20 gm/L of yeast extract and 10 gm/L of sodium chloride and dispensed in to a 2L flask and sterilized. To the sterilized pre-seed medium, 500 ⁇ of kanamycin stock solution (50 mg/ml) was added. 250 ⁇ of glycerol stock having recombinant clone of E. coli containing Lirapeptide fusion construct was inoculated and incubated at 37°C for 8- 12 hours in an incubator shaker.
  • the following component was used for preparing seed medium in seed fermenter:
  • components (1-4) as per above table no. 1 are weighed and dissolved in DM water(1.5L) and dispensed in to a 3.0 liter fermenter. Further, components (5-6) mentioned as per table no. 1 were weighed and dissolved in DM water and made up to 200ml and dispensed in to a 500 ml bottle.
  • 1.8 ml of Kanamycin stock solution was added in the bottle containing Glucose and Magnesium sulphate under aseptic conditions. Further stock solution of bottle having glucose, magnesium sulphate and Kanamycin was transferred in to the seed fermenter.
  • 90ml of Pre-seed culture medium having recombinant clone of E. coli containing Lirapeptide fusion protein from above step a) was transferred into seed fermenter and seed fermenter was maintained at pH 7, temperature of about 37°C, dissolved oxygen about 25%, and agitation at rate about 300-600 rpm.
  • the production tank fermentation medium of 20 liters was prepared according to components mentioned in Table.2 and sterilized.
  • the fermentation medium was poured in production tank fermenter and sterilized.
  • Feed medium of 12 liters for fermentation was prepared according to the components of Table 3 and sterilized.
  • glucose and magnesium sulphate heptahydrate aseptically was added in bio vessel and adjusted pH 7.0 by using liquor ammonia solution. Further, kanamycin was added to tank media to get final concentration of kanamycin 50 ⁇ g/ml and inoculate mature seed (1L) from seed fermenter into production tank and maintained pH 7.0 ⁇ 0.2 by liquor ammonia; DO 2 > 20 - 30% and temperature 37 ⁇ 1° C throughout the batch. Total Batch cycle was used to be 21 ⁇ 3 hrs. Feed was added to fermenter on rise of pH and DO. Further, tank medium was induced by 1M IPTG solution to get final concn ImM IPTG, once OD reaches 70 ⁇ 5. Maintain all other parameters same as earlier and maintained for 6-8 hrs after induction. After completion of fermentation, the temperature of broth was decreased to 10-15°C, centrifuged, collected cell mass and stored at -80°C.
  • lysis buffer containing 20mM tris (242 gm) and 8M Urea (48kg) was prepared having pH 9.4 - 9.6 at room temperature. 4000g of cells were suspended in 30-40 L lysis buffer. Then cells were lysed in a homogenizer and incubated for 2 hours for solubilization. The cell lysate obtained after homogenization was passed through a Hollow fiber tangential flow filtrations (TFF) system and permeate (251) was collected and performed dia-filtration with lysis buffer till permeate wash received around 40 - 50 L. Permeate and wash permeate was adjusted to 8 ⁇ 0.1 with dilute HCL.
  • TMF Hollow fiber tangential flow filtrations
  • Permeate (4L) having ubiquitin fusion protein was loaded in to the affinity chromatography (Ni-NTA) which was equilibrated with urea buffer (1M urea+20mM tris, pH 8.0), at the flow rate of 10CV/HR. After loading, matrix washed with same urea buffer and started eluting the impurities with elution buffer containing 20mM Imidazole followed by 200mM Imidazole to give elute having Ubiquitin fusion protein (8L) . Purity of the fusion protein was 60 % as determined RP-HPLC.
  • Ubiquitin hydrolase enzyme (205ml) was added to 200mM elute (Ubiquitin fusion protein of 81) from the affinity chromatography in the ratio of 1 :20 (20 parts of fusion protein add 1 part of enzyme) for enzymatic digestion and incubated for 4-8 hrs at 30°C under stirring.
  • Example 4 3.5M Sodium chloride was added to the digestion mixture as obtained in Example 4, and stirred for 30 minutes for dissolution. After dissolution the pH adjusted to 4.7 - 4.8 with diluted HC1 and sample was incubated for 2-10hrs at 2-8°C. After incubation, sample was centrifuged and collected pellet was washed with acidified water of pH 4.7 ⁇ 0.2 to get lirapeptide pellet (142gm).
  • the Lirapeptide was then eluted with 9 CVs of 28-50% of mobile phase B when fractions (500mL) were collected.
  • the peak fractions whose purity was greater than 90.00% by analytical HPLC were pooled (4.2 L).
  • Purity of the Elution pool was 94.25% with a recovery of 82%.
  • acetonitrile present in Elution pool was 30 to 45% (v/v) that was evaporated using rota- vapour at 22 °C and finally carried forward to the next step.
  • palmitic acid (63.8 g, 248 mmol) was dissolved in 700 mL of dichloromethane at room temperature under argon atmosphere.
  • Triethylamine (42.5 mL, 303 mmol) was then added drop wise and the mixture was stirred for 5 min.
  • 2-(7-aza- lH-benzotriazole-l-yl)-l,l,3,3-tetramethyl uranium hexafluorophosphate (HATU) (115.59 g, 303 mmol) was added and allowed to stir for additional 10 min.
  • Trifluoroacetic acid (139 mL, 1800 mmol) was added to a solution of 5-(t-butyl)-l- methyl palmityl glutamate (82 g, 147 mmol) in dichloromethane (450 mL) and allowed to be stirred at room temperature for 2h. The resulting solution was quenched with water and extracted using dichloromethane. The solvent was removed under reduced pressure to give 1- methyl palmityl glutamic acid as a dry white solid in quantitative yield.
  • Diisopropylcarbodiimide (DIC, 29 mL, 186 mmol) was added to a solution of 1- methyl palmitoyl glutamic acid (74.5 g, 186 mmol) in tetrahydrofuran (630 mL) at room temperature and stirred for 15 minutes. Further, N-hydroxy succinimide (21.4 g, 186 mmol) was added to the above solution and stirred at room temperature overnight. The suspension was quenched with water (500 mL) extracted with dichloromethane (2000 mL) and dried over anhydrous sodium sulfate.
  • NiS04 6H20 (3.11 mg, 0.01182 mmol, 1 equiv.) was added directly to the lirapeptide (40 mg, 0.01182 mmol) and azeotropically dried using toluene and suspended in dimethylformamide (2 mL) at room temperature. DMAP (5.8 mg, 0.04728 mmol) was added and the suspension was stirred for 5 min.
  • Ni(ac)2 4H20 (2.94 mg, 0.01182 mmol, 1 equiv.) was added directly to the lirapeptide (40 mg, 0.01182 mmol) and azeotropically dried using toluene and suspended in dimethylformamide (2 mL) at room temperature. DMAP (5.8 mg, 0.04728 mmol) was added and the suspension was stirred for 5 min.
  • Example 15 Conjugation of lirapeptide with palmityl glutamate derivatives in the presence of Cobalt acetate
  • Co(ac)2 (1.05 mg, 0.00591 mmol, 0.5 equiv.) was added directly to the lirapeptide (40 mg, 0.01182 mmol) and azeotropically dried using toluene and dissolved in dimethylformamide (2 mL) at room temperature.
  • DMAP (5.8 mg, 0.04728 mmol) was added and the suspension was stirred for 5 min.
  • Example 16 Conjugation of lirapeptide with N-hydroxy succinimide ester of palmitoyl glutamic acid in an organic medium
  • Triethylamine (0.8 ⁇ , 5.9 ⁇ ) was added to a solution of Lirapeptide (10 mg, 2.95 ⁇ ) in dimethylformamide (1 mL) at room temperature and the suspension was stirred for 5 min. Then, 1.2 M CuS04 5H20 solution (2.4 ul, 5.9 umol) was added and the mixture was stirred for 10 minutes. Next, tert-Butyldimethyl silyl chloride (2.2 mg, 14.74 ⁇ ) was added to the above reaction mixture and stirred for another 10 min.
  • Example 17 Conjugation of lirapeptide with N-hydroxy succinimide ester of palmitoyl glutamic acid in an aqueous medium (Process I)
  • Triethylamine (40 ⁇ , 0.27 mmol) followed by 1.2 M CuS04 5H20 solution (493 ⁇ , 0.59 mmol) were added to a solution of lirapeptide ( ⁇ 2 g, 0.59 mmol) in water (400 mL) at 0°C and stirred for 5 min. Further, additional triethylamine (83 ⁇ , 0.60 mmol) was added to the above reaction mixture in order to maintain the pH ⁇ 9.5.
  • Liraglutide crude product (14.2 grams of wet pellet) was dissolved in lOOmM Tris pH 8.5 ⁇ 0.1 (913 ml) at a concentration of 1.95g/L.
  • the crude load was filtered through 1.2 ⁇ followed 0.8 ⁇ and 0.45 ⁇ PES filters.
  • the column packed with Dasiogel 10 ⁇ C8 100 A (Dimensions 50x250mm, 491ml CV) was equilibrated with (10% of mobile phase B (Mobile Phase A: 10 mm Tris pH 8.5 ⁇ 0.1; Mobile phase B: 100% Acetonitrile) at linear flow rate of 230cm/hr before loading.
  • the peak fractions whose purity was greater than 90.00% by analytical HPLC were pooled (4.2 L).
  • the product was loaded onto the resin at a ratio of 4.36 grams of product/litre of resin or 0.73% gram of product /gram of resin.
  • the column was washed with 3 column volume (CV) of 10% mobile phase B and then with 3 CV of 25% of mobile phases B at a linear flow rate of 230cm/hr.
  • the Liraglutide was eluted with 15 CVs of 28-42 % of mobile phase B at a linear flow rate of 220cm/hr when fractions (23ml) were collected.
  • the peak fractions whose purity was greater than 98% by HPLC were pooled (380ml).
  • acetonitrile present in elution pool was 31 to 34% (v/v) and was evaporated using rotary evaporator at 22 °C, pi precipitated using acetic acid ( ⁇ ) and centrifuged at 8500 rpm for 20 min at 5 ⁇ 2°C. Finally, the pellet was lyophilized at 0.16mbar for 24hours and 1.08 grams of 98.46% pure white powder obtained.
  • Liraglutide crude product 140 grams of wet pellet was dissolved in lOOmM Tris pH 8.5 ⁇ 0.1 (4000 ml) at a concentration of 2.34g/L.. The crude load was filtered through 1.2 ⁇ followed 0.8 ⁇ and 0.45 ⁇ PES filters. The column packed with Dasiogel 10 ⁇ C8 100 A (Dimensions 100x268mm, 2104ml CV) was equilibrated with (10% of mobile phase B (Mobile Phase A: 10 mm Tris pH 8.5 ⁇ 0.1; Mobile phase B: 100% Acetonitrile) at a linear flow rate of 220cm/hr before loading.
  • Dasiogel 10 ⁇ C8 100 A (Dimensions 100x268mm, 2104ml CV) was equilibrated with (10% of mobile phase B (Mobile Phase A: 10 mm Tris pH 8.5 ⁇ 0.1; Mobile phase B: 100% Acetonitrile) at a linear flow rate of 220cm/hr before loading.
  • the product was then loaded onto the resin at a ratio of 4.45 grams of product/litre of resin or 0.74% gram of product /gram of resin at a linear flow rate of 191 cm/hr.
  • the column was washed with 4 column volumes (CV) of 10% mobile phase B and then with 3 CV of 25% of mobile phases B at a linear flow rate of 220cm/hr.
  • the Liraglutide was eluted with 18 CVs of 28-40 % of mobile phase B at a linear flow rate of 220cm/hr when fractions ( ⁇ 220mL) were collected.
  • the peak fractions whose purity was greater than 98% by HPLC were pooled (2000mL).
  • acetonitrile present in Elution pool was 32 to 34% (v/v) and was evaporated using rotary evaporator at 22 °C, pi precipitated using HC1 (5.5 N, 4.8 mL) and centrifuged at 8000 rpm for 30 min at 5 ⁇ 2°C. Finally, the pellet was lyophilized at 0.04mbar for 24hours and 5.25 grams of 99.1% pure white powder obtained.

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Abstract

La présente invention concerne un procédé de préparation de peptides ou de protéines ou de dérivés de ceux-ci au moyen de l'expression d'oligonucléotide synthétique codant pour la protéine ou le peptide désiré dans une cellule procaryote comme produit de synthèse de fusion d'ubiquitine.
PCT/IB2016/054470 2015-07-31 2016-07-27 Procédé de préparation de protéines ou de peptides WO2017021819A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2018020417A1 (fr) * 2016-07-27 2018-02-01 Dr. Reddy's Laboratories Limited Procédé de préparation de protéines ou de peptides
WO2020053683A1 (fr) * 2018-09-13 2020-03-19 Sajjala Bio Labs Private Limited Procédé de production de peptides recombinants solubles
WO2022064517A1 (fr) * 2020-09-23 2022-03-31 Dr. Reddy's Laboratories Limited Procédé de préparation de sémaglutide et de sémapeptide
WO2022208554A2 (fr) 2021-03-31 2022-10-06 Biological E Limited Constructions et procédés pour une expression accrue de polypeptides
WO2023049357A3 (fr) * 2021-09-24 2023-05-11 The Uab Research Foundation Régulation de la stœchiométrie de sous-unités dans des nanopores msp à chaîne unique

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US6458924B2 (en) * 1996-08-30 2002-10-01 Novo Nordisk A/S Derivatives of GLP-1 analogs
US20110112990A1 (en) * 2009-11-09 2011-05-12 The Regents Of The University Of Colorado, A Body Corporate Efficient Production of Peptides
CN104745597A (zh) * 2015-03-27 2015-07-01 杭州北斗生物技术有限公司 一种高效表达重组利拉鲁肽的方法
US9089538B2 (en) * 2010-04-27 2015-07-28 Zealand Pharma A/S Peptide conjugates of GLP-1 receptor agonists and gastrin and their use
WO2016005931A1 (fr) * 2014-07-09 2016-01-14 Lupin Limited Système d'expression bactérienne duelle du cistron

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US6458924B2 (en) * 1996-08-30 2002-10-01 Novo Nordisk A/S Derivatives of GLP-1 analogs
US20110112990A1 (en) * 2009-11-09 2011-05-12 The Regents Of The University Of Colorado, A Body Corporate Efficient Production of Peptides
US9089538B2 (en) * 2010-04-27 2015-07-28 Zealand Pharma A/S Peptide conjugates of GLP-1 receptor agonists and gastrin and their use
WO2016005931A1 (fr) * 2014-07-09 2016-01-14 Lupin Limited Système d'expression bactérienne duelle du cistron
CN104745597A (zh) * 2015-03-27 2015-07-01 杭州北斗生物技术有限公司 一种高效表达重组利拉鲁肽的方法

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018020417A1 (fr) * 2016-07-27 2018-02-01 Dr. Reddy's Laboratories Limited Procédé de préparation de protéines ou de peptides
WO2020053683A1 (fr) * 2018-09-13 2020-03-19 Sajjala Bio Labs Private Limited Procédé de production de peptides recombinants solubles
WO2022064517A1 (fr) * 2020-09-23 2022-03-31 Dr. Reddy's Laboratories Limited Procédé de préparation de sémaglutide et de sémapeptide
WO2022208554A2 (fr) 2021-03-31 2022-10-06 Biological E Limited Constructions et procédés pour une expression accrue de polypeptides
WO2023049357A3 (fr) * 2021-09-24 2023-05-11 The Uab Research Foundation Régulation de la stœchiométrie de sous-unités dans des nanopores msp à chaîne unique

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