WO2020053875A1

WO2020053875A1 - Process for producing recombinant peptides

Info

Publication number: WO2020053875A1
Application number: PCT/IN2019/050471
Authority: WO
Inventors: Anurag Singh Rathore; Kathiresan PANDI; Vishwanath Hebbi; James Gomes; Claire Komives
Original assignee: Indian Institute Of Technology, Delhi
Priority date: 2018-09-14
Filing date: 2019-06-21
Publication date: 2020-03-19

Abstract

The present disclosure relates to a process for producing recombinant peptides by culturing recombinant host cell under microaerobic growth conditions, wherein the recombinant peptide is localized in inclusion bodies, and the inclusion bodies are solubilized, and the recombinant peptide is cleaved in a single step. The disclosure also provides with the recombinant peptides obtained from the process.

Description

PROCESS FOR PRODUCING RECOMBINANT PEPTIDES

FIELD OF INVENTION

[001] The present disclosure broadly relates to field of recombinant DNA technology. In particular it relates to a process for producing recombinant peptides.

BACKGROUND OF INVENTION

[002] The recombinant DNA technology has provided a major breakthrough in the field of drug discovery and therapeutics. Gene manipulation has led to expression of recombinant peptides with desired characteristics. These recombinant peptidesare becoming increasingly useful as therapeutic drugs due to high specificity for their target, low non-specific binding and low accumulation.

[003] Upcoming peptide and therapeutic protein drugs are targeting therapeutic areas of big unmet needs and high growth potential, such as, oncology, infectious diseases, diabetes, neurobiological disorders and obesity. Furthermore, peptides are components of vaccines and can be used as targeting devices for other types of drugs or be conjugated to cytotoxics to make them more potent.

[004] There are several technologies that are available for the production of recombinant peptides and proteins, such as, production in cell free expression system (F. Katzen, G. Chang, and W. Kudlicki,“The past, present and future of cell-free protein synthesis,” Trends BiotechnoL, vol. 23, no. 3, pp. 150-156, 2005), production in plants (C. Cunningham and A. J. R. Porter, Recombinant proteins from plants: production and isolation of clinically useful compounds. Springer Science & Business Media, 1998), production by chemical synthesis (V. du Vigneaud, C. Ressler, C. J. M. Swan, C. W. Roberts, P. G. Katsoyannis, and S. Gordon,“The synthesis of an octapeptide amide with the hormonal activity of oxytocin,” J. Am. Chem. Soc., vol. 75, no. 19, pp. 4879-4880, 1953), and production by recombinant DNA technology (I. Gill, R. Lopez-Fandino, X. Jorba, and E. N. Vulfson,“Biologically active peptides and enzymatic approaches to their production,” Enzyme Microb. Technol., vol. 18, no. 3, pp. 162-183, 1996). However, the processes as outlined constitute high cost of production.

[005] Alternatively, chemical synthesis is a viable technology for production of small and medium size peptides ranging from about 5 to 30 residues but uses expensive raw materials, long cycle time, large number of unit operations, generate complex impurities, and environmentally harmful waste. Both liquid and solid phase synthesis would be good options for production of peptides at kg- metric tons. However, for very large scale, economics would be main constraint, primarily due to the high cost of raw materials, including the treatment of waste streams generated during manufacturing.

[006] While advances in the field of peptide science have led to excellent growth in the pharmaceutical market, several barriers remain to be overcome, such as, high production cost, long cycle time, disposal of harmful wastes, and so on. Therefore, there exists a requirement for development of processes that along with being cost effective are also environment friendly and has reduced turn-around time.

SUMMARY OF THE INVENTION

[007] In an aspect of the present invention, there is provided a process for producing recombinant peptides, said process comprising: (a) obtaining a recombinant host cell comprising a recombinant DNA construct, said DNA construct comprising a nucleotide fragment and a promoter to drive expression of the nucleotide fragment encoding a recombinant peptide; (b) culturing the recombinant host cell in a growth medium under microaerobic conditions suitable for expression of the recombinant peptide, wherein the recombinant peptide is localized in inclusion bodies; (c) solubilizing the inclusion bodies with 3-4M urea and treating the recombinant peptide with at least one cleaving agent in a single step; and (d) isolating and purifying the recombinant peptide from the inclusion bodies.

[008] In an aspect of the present invention, there is provided a recombinant peptide produced by a process, said process comprising: (a) obtaining a recombinant host cell comprising a recombinant DNA construct, said DNA construct comprising a nucleotide fragment and a promoter to drive expression of the nucleotide fragment encoding a recombinant peptide; (b) culturing the recombinant host cell in a growth medium under microaerobic conditions suitable for expression of the recombinant peptide, wherein the recombinant peptide is localized in inclusion bodies; (c) solubilizing the inclusion bodies with 3-4M urea and treating the recombinant peptide with at least one cleaving agent in a single step; and (d) isolating and purifying the recombinant peptide from the inclusion bodies.

[009] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0010] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0011] Figure 1 depicts a general scheme of product processing of peptide production by conventional process using recombinant technology, in accordance with an embodiment of the present disclosure.

[0012] Figure 2 depicts a diagram of pET29a vector, in accordance with an embodiment of the present disclosure.

[0013] Figure 3 depicts an agarose gel picture for restriction digestion analysis of clone comprising pramlintide, wherein Lane M is Marker (lKb plus DNA ladder from GeneRuler), Lane 1 is pET29a(+) clonel, Lane 2 is pET29a(+) clone2, Lane 3 is pET29a(+) clonel digested with Ndel and Xhol, pET29a(+) clone2 digested with Ndel and Xhol, in accordance with an embodiment of the present disclosure. [0014] Figure 4 depicts expression profile of recombinant pramlintide, wherein Lane M is Protein marker, Lane 1 is control untransformed BL2l(DE3), Lane 2 is uninduced transformed BL2l(DE3) with pET29a(+) clone 1, Lane 3 is induced transformed BL2l(DE3) with pET29a(+) clonel with 0.1 mM IPTG, Lane 4 is induced transformed BL2l(DE3) with pET29a(+) clonel with 1 mM IPTG, Lane 5 is uninduced transformed BL2l(DE3) with pET29a(+) clone2, Lane 6 is induced transformed BL2l(DE3) with pET29a(+) clone2 with 0.1 mM IPTG, Lane 7 is induced transformed BL2l(DE3) with pET29a(+) clone2 with 1 mM IPTG, in accordance with an embodiment of the present disclosure.

[0015] Figure 5 depicts tricine gel electrophoresis of shake flask samples of recombinant LTNF, wherein Lane 1 is Marker, Lane 2 is uninduced cell lysis supernatant, Lane 3 is induced cell lysis supernatant, Lane 4 is uninduced solubilized inclusion bodies, and lane 5 is induced solubilized inclusion bodies, in accordance with an embodiment of the present disclosure.

[0016] Figure 6 depicts the profile of dissolved oxygen of aerobic and microaerobic process for producing peptides (LTNF), in accordance with an embodiment of the present disclosure.

[0017] Figure 7 depicts profile of product titre (LTNF) during aerobic and microaerobic process, in accordance with an embodiment of the present disclosure.

[0018] Figure 8 depicts profile of the comparative cell viability (LTNF) under aerobic and microaerobic process, in accordance with an embodiment of the present disclosure.

[0019] Figure 9 depicts the effect of urea concentration on solubilization of inclusion bodies (LTNF production), in accordance with an embodiment of the present disclosure.

[0020] Figure 10 depicts the effect of urea concentration on cleavage reaction using alpha-chymotrypsin, wherein II to 15 are impurities and LTNF is the product of interest, in accordance with an embodiment of the present disclosure.

[0021] Figure 11 depicts a preparative chromatogram of RP-HPLC purification of recombinant LTNF, in accordance with an embodiment of the present disclosure. [0022] Figure 12 depicts analytical chromatogram of RP-HPLC confirmation of recombinant pramlintide, in accordance with an embodiment of the present disclosure.

[0023] Figures 13 and 14 depict the LC-MS profiles of recombinant LTNF and recombinant pramlintide respectively, in accordance with an embodiment of the present disclosure.

[0024] Figure 15 depicts a scheme of the claimed process for producing and purifying recombinant peptides, in accordance with an embodiment of the present disclosure.

Sequences used in the present disclosure:

[0025] SEQ ID NO: 1 Amino acid sequence of recombinant lethal toxin- neutralising factor (LTNF)

MWLKAMDPTPPLWLKAMDPTPPLWLKAMDPTPPLWLKAMDPTPPLWL KAMDPTPPLWLKAMDPTPPLWLKAMDPTPPLWLKAMDPTPPLWLKAMD PTPPLWLKAMDPTPPLWLKAMDPTPPLWHHHHHH

[0026] SEQ ID NO: 2 Amino acid sequence of recombinant pramlintide

WKCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTYWKCNTATCATQ RLANFLVHS S NNF GPILPPTN V GS NT YWKCNT AT CAT QRLANFLVHS S NN F GPILPPTN V GS NT YLEHHHHHH

[0027] SEQ ID NO: 3 Nucleotide sequence of recombinant LTNF

ATGTGGCTGAAAGCGATGGATCCCACACCGCCTCTTTGGCTGAAGGCG ATGGATCCGACCCCACCGCTCTGGTTAAAAGCAATGGATCCAACCCCG CCGTTATGGCTCAAAGCCATGGACCCGACCCCACCGTTGTGGCTGAAA GCTATGGATCCGACGCCTCCCCTGTGGCTGAAAGCAATGGACCCTACG CCTCCGCTGTGGCTTAAAGCCATGGATCCGACTCCGCCGCTGTGGTTG AAAGCGATGGACCCCACTCCGCCACTGTGGATTAAGACCGAACTCGAG CACCACCACCACCACCAC

[0028] SEQ ID NO: 4 Nucleotide sequence of recombinant pramlintide

ATGTGGAAATGCAACACCGCGACCTGCGCGACCCAACGTCTGGCGAA CTTCCTGGTTCACAGCAGCAATAACTTTGGCCCGATTCTGCCGCCGACC AATGTTGGTAGCAACACCTACTGGAAATGCAATACTGCTACCTGCGCG

ACCCAGCGTCTGGCGAACTTCCTGGTACACAGCAGCAACAACTTTGGT

CCGATCCTGCCGCCGACCAACGTTGGCAGCAACACCTATTGGAAGTGC

AATACTGCCACTTGTGCTACTCAACGTCTGGCGAACTTCCTGGTGCAC

AGCAGCAATAACTTTGGCCCGATTCTGCCGCCGACCAACGTGGGTAGC

AACACCTATCTCGAGCACCACCACCACCACCAC

DETAILED DESCRIPTION OF THE INVENTION

[0029] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

Definitions

[0030] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0031] The articles“a”,“an” and“the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0032] The terms“comprise” and“comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as“consists of only”.

[0033] Throughout this specification, unless the context requires otherwise the word“comprise”, and variations such as“comprises” and“comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0034] The term“including” is used to mean“including but not limited to”. “Including” and“including but not limited to” are used interchangeably.

[0035] The term“recombinant peptide” as used herein refer to a compound consisting 2 or more amino acids. Said term has been interchangeably used herein with the term“protein”. Therefore, the term“recombinant protein” falls within the scope of the term“recombinant peptide”.

[0036] The term LTNF and lethal toxin-neutralizing factor have been used interchangeably throughout the present disclosure. LTNF is an antivenom peptide and is represented by SEQ ID NO: 1 in the present disclosure.

[0037] Pramlintide is an analogue of amylin, a hormone released into bloodstream. It is represented by SEQ ID NO: 2 in the present disclosure.

[0038] Microaerobic condition for the purposes of the present disclosure refers to conditions with oxygen content in the range of 0-20%, more preferably in the range of 0-10%.

[0039] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0040] As illustrated in the background section, though there are variety of processes that are available for the production of recombinant peptide, said processes find limited application as they suffer from several deficiencies. Recombinant technology is a greener and the preferred choice for production of peptides (G. Walsh,“Therapeutic insulins and their large-scale manufacture,” Appl. Microbiol. Biotechnol., vol. 67, no. 2, pp. 151-159, 2005). The choice of a recombinant expression system for the high level of production of proteins or peptides depends on technical, design and economic considerations. The relative merits and demerits of E.coli, yeast, Pichia, insect are well known in the art (F. R. Schmidt, “Recombinant expression systems in the pharmaceutical industry,” Appl. Microbiol. BiotechnoL, vol. 65, no. 4, pp. 363-372, 2004). However, E.coli remains most attractive due to wide availability of tools for genetic manipulation, high expression levels of product, low cost, easily scalable and short production cycle (K. Terpe,“Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems,” Appl. Microbiol. BiotechnoL, vol. 72, no. 2, p. 211, 2006). Many therapeutic proteins and peptides in the market are successfully produced from bacteria. Microbial processes are generally regarded as eco-friendly process with little or no use of hazardous chemicals and uses less expensive raw materials. However, the process has inherent limitations include high development costs, lower product yield, and multiple unit operations. Existing microbial process for peptide production are energy intensive with low to moderate product yields and use multiple chromatographic unit operations for purification. This leads to higher overall costs per gram of product (S. Wegmuller and S. Schmid,“Recombinant peptide production in microbial cells,” Curr. Org. Chem., vol. 18, no. 8, pp. 1005- 1019, 2014). Often used strategies to improve product yield during fermentation include media optimization, temperature shift, variation of inducer strength, and feeding strategy (A. Shoja, K. Varedi, V. Babaeipour, and A. M. Farnoud, “Recent advances in high cell density cultivation for production of recombinant protein,” 2008). Currently many peptides are produced as concatemers and then processed to obtain a monomeric active peptide in pure form.

[0041] The conventional process uses multiple steps of unit operations for processing the product. Major steps as depicted in Figure 1 include cell separation, cell lysis to recover inclusion bodies (IBs), IB solubilisation, concatemer capture using affinity-based separation, cleavage of partially purified concatemer using cleavage agent such as cyanogen bromide (CNBr), formic acid or specific enzyme. The reaction mixture is purified using reverse phase high performance chromatography (RP-HPLC). The usage of affinity tags like His₆ is well known capture technique which purifies the product up to 90% in a single step. However, it poses many problems during manufacturing including lower binding capacity, nickel leaching, heavy metal contamination and additional analytical methods needed for monitoring. Nickel clearance across unit operations is also a concern. These limitations make the process expensive comparable to synthetic route of peptide production.

[0042] The present invention discloses a novel method for producing the peptide using recombinant DNA technology. The invention is directed to exploiting the oxygen capacity of cells producing recombinant peptide or recombinant protein which in turn improves the overall productivity of process. Moreover, directing the cells to produce large quantity of peptides in inclusion bodies without affecting the metabolic state of cells by growing in micro aerobic environment is also one of the objectives of the present invention.

[0043] The present invention provides a process for improving high expression levels of peptide by modulation of oxygen in the bioreactor and directing the produced peptides towards inclusion bodies. Presence of inclusion bodies permits production of increased concentration of target peptide due to reduced toxicity of peptide to cell upon segregation in inclusion body. Highly homogenous aggregated inclusion bodies containing target peptide results in lower degradation of product and ease for further isolation or purification. Producing peptide in the inclusion body form facilitates for simultaneous solubilization and cleavage of peptide to improve the overall process economics.

[0044] The novelty of the instant process consists of microaerobic fermentation wherein optimal oxygen is maintained compared to that of conventional cascade controls for high oxygen (between agitation and oxygen content). The present disclosure relates to the development of a microaerobic, less energy intensive and easy to control fermentation process for producing peptides using recombinant technology. It also includes minimal step purification (three steps, simultaneous solubilization and enzymatic cleavage, RP-HPLC and lyophilization) of concatemer to obtain purified product.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0046] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.

[0047] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides, said process comprising: (a) obtaining a recombinant host cell comprising a recombinant DNA construct, said DNA construct comprising a nucleotide fragment and a promoter to drive expression of the nucleotide fragment encoding a recombinant peptide; (b) culturing the recombinant host cell in a growth medium under microaerobic conditions suitable for expression of the recombinant peptide, wherein the recombinant peptide is localized in inclusion bodies; (c) solubilizing the inclusion bodies with 3-4M urea and treating the recombinant peptide with at least one cleaving agent in a single step; and (d) isolating and purifying the recombinant peptide from the inclusion bodies.

[0048] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides as described herein, wherein the recombinant host cell comprises a recombinant vector, said recombinant vector comprises a recombinant DNA construct, said recombinant DNA construct comprises a nucleotide fragment and a promoter to drive expression of the nucleotide fragment encoding a recombinant peptide.

[0049] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides, said process comprising: (a) obtaining a recombinant host cell comprising a recombinant vector, said recombinant vector comprising a recombinant DNA construct, said recombinant DNA construct comprising a nucleotide fragment and a promoter to drive expression of the nucleotide fragment encoding a recombinant peptide; (b) culturing the recombinant host cell in a growth medium under microaerobic conditions suitable for expression of the recombinant peptide, wherein the recombinant peptide is localized in inclusion bodies; (c) solubilizing the inclusion bodies with 3-4M urea and treating the recombinant peptide with at least one cleaving agent in a single step; and (d) isolating and purifying the recombinant peptide from the inclusion bodies.

[0050] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides as described herein, wherein the recombinant host cell is a bacterial cell. In another embodiment of the present disclosure, the host cell is E. coli.

[0051] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides as described herein, wherein the recombinant peptide comprises‘n’ number of tandem repeats of the peptide, and‘n’ ranges from 1-20. In another embodiment of the present disclosure,‘n’ ranges from 1-10. In yet another embodiment of the present disclosure,‘n’ is 1.

[0052] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides as described herein, wherein the recombinant peptide is selected from a group consisting of lethal toxin-neutralizing factor (LTNF), pramlintide, exenatide, teriparatide, insulin, liraglutide, granulocyte colony stimulating factor (G-CSF), antibody fragment (Fab), and human growth hormone.. In another embodiment of the present disclosure, the recombinant peptide is lethal toxin-neutralizing factor (LTNF). In yet another embodiment of the present disclosure, the recombinant peptide is pramlintide.

[0053] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides as described herein, wherein the recombinant peptide is lethal toxin-neutralizing factor (LTNF) comprising 8 tandem repeats.

[0054] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides as described herein, wherein the microaerobic condition comprises dissolved oxygen content in a range of 0-10%. In another embodiment of the present disclosure, the microaerobic condition comprises dissolved oxygen content in a range of 1-10%. In yet another embodiment of the present disclosure, the microaerobic condition comprises dissolved oxygen content in a range of 1-5%.

[0055] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides as described herein, wherein culturing the recombinant host cell comprises stirring in a range of 300-1200 rpm for a time period in a range of 12-20 hours at a temperature in a range of 30-39 °C. In another embodiment of the present disclosure, culturing the recombinant host cell comprises stirring in a range of 400-900 rpm for a time period in a range of 12-16 hours at a temperature in a range of 31-37 °C. In yet another embodiment of the present disclosure, culturing the recombinant host cell comprises stirring in a range of 400-900 rpm for a time period of 14 hours at a temperature of 37 °C.

[0056] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides as described herein, wherein solubilizing the inclusion bodies and treating the recombinant peptide comprises stirring in a range of 50-250 rpm, for a time period in a range of 0.5-3 hours, at a temperature in a range of 4-30 °C, and at a pH in a range of 5-9. In another embodiment of the present disclosure, solubilizing the inclusion bodies and treating the recombinant peptide comprises stirring in a range of 50-200 rpm, for a time period in a range of 0.5-2 hours, at a temperature in a range of 4-25 °C, and at a pH in a range of 5- 7.5.

[0057] In an embodiment of the present disclosure, there is provided a process for producing recombinant protein as described herein, wherein solubilizing the inclusion bodies and treating the recombinant protein comprises stirring in a range of 50-250 rpm, for a time period in a range of 0.5-3 hours, at a temperature in a range of 4-30 °C, and at a pH in a range of 5-9. In another embodiment of the present disclosure solubilizing the inclusion bodies and using the said solubilized inclusion bodies are used for further refolding and purification of recombinant protein

[0058] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides as described herein, wherein the at least one cleaving agent is selected from a group consisting of chymotrypsin, Glu C endoproteinase, O-iodosobenzoic acid, cyanogen bromide, N-bromosuccinimide, N-chlorosuccinimide, and combinations thereof. In another embodiment of the present disclosure, the at least one cleaving agent is chymotrypsin.

[0059] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides as described herein, wherein the at least one cleaving agent has a concentration in a range of 1-5 units/mg. In another embodiment of the present disclosure, the at least one cleaving agent has a concentration in a range of 1-3 units/mg.

[0060] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides as described herein, wherein the at least one cleaving agent is selected from a group consisting of chymotrypsin, Glu C endoproteinase, O-iodosobenzoic acid, cyanogen bromide, N-bromosuccinimide, N-chlorosuccinimide, and combinations thereof. In another embodiment of the present disclosure, the at least one cleaving agent is chymotrypsin, and the at least one cleaving agent has a concentration in a range of 1-5 units/mg.

[0061] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides as described herein, wherein the at least one cleaving agent is chymotrypsin, having a concentration in a range of 1-5 units/mg.

[0062] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides, said process comprising: (a) obtaining a recombinant host cell comprising a recombinant DNA construct, said DNA construct comprising a nucleotide fragment and a promoter to drive expression of the nucleotide fragment encoding a recombinant peptide; (b) culturing the recombinant host cell in a growth medium under 0-10% dissolved oxygen, wherein the recombinant peptide is localized in inclusion bodies; (c) solubilizing the inclusion bodies with 3-4M urea and treating the recombinant peptide with chymotrypsin in a single step; and (d) isolating and purifying the recombinant peptide from the inclusion bodies.

[0063] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides as described herein, wherein purifying the recombinant peptide comprises at least one method selected from a group consisting of reverse phase-high performance liquid chromatography (RP-HPLC), low pressure cation exchange chromatography, hydrophobic interaction chromatography, mixed mode chromatography, and combinations thereof. In another embodiment of the present disclosure, the at least one method is reverse phase-high performance liquid chromatography (RP-HPLC).

[0064] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides as described herein, wherein the promoter is selected from a group consisting of lac promoter, T7 promoter, T5 promoter, Tac promoter and LacUV5 promoter. In another embodiment of the present disclosure, the promoter is lac promoter.

[0065] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides as described herein, wherein the process has a batch time in a range of 40-43 hours for a lOkg scale.

[0066] In an embodiment of the present disclosure, there is provided a process for producing recombinant peptides as described herein, wherein the process has a productivity in a range of 0.22-0.26 kg/hour

[0067] In an embodiment of the present disclosure, there is provided a recombinant peptide produced by a process, said process comprising: (a) obtaining a recombinant host cell comprising a recombinant DNA construct, said DNA construct comprising a nucleotide fragment and a promoter to drive expression of the nucleotide fragment encoding a recombinant peptide; (b) culturing the recombinant host cell in a growth medium under microaerobic conditions suitable for expression of the recombinant peptide, wherein the recombinant peptide is localized in inclusion bodies; (c) solubilizing the inclusion bodies with 3-4M urea and treating the recombinant peptide with at least one cleaving agent in a single step; and (d) isolating and purifying the recombinant peptide from the inclusion bodies.

[0068] In an embodiment of the present disclosure, there is provided a recombinant lethal toxin-neutralizing factor (LTNF) produced by a process, said process comprising: (a) obtaining a recombinant host cell comprising a recombinant DNA construct, said DNA construct comprising a nucleotide fragment and a promoter to drive expression of the nucleotide fragment encoding a recombinant LTNF; (b) culturing the recombinant host cell in a growth medium under microaerobic conditions suitable for expression of the recombinant LTNF, wherein the recombinant LTNF is localized in inclusion bodies; (c) solubilizing the inclusion bodies with 3-4M urea and treating the recombinant LTNF with at least one cleaving agent in a single step; and (d) isolating and purifying the recombinant LTNF from the inclusion bodies.

[0069] In an embodiment of the present disclosure, there is provided a recombinant pramlintide produced by a process, said process comprising: (a) obtaining a recombinant host cell comprising a recombinant DNA construct, said DNA construct comprising a nucleotide fragment and a promoter to drive expression of the nucleotide fragment encoding a recombinant pramlintide; (b) culturing the recombinant host cell in a growth medium under microaerobic conditions suitable for expression of the recombinant pramlintide, wherein the recombinant pramlintide is localized in inclusion bodies; (c) solubilizing the inclusion bodies with 3-4M urea and treating the recombinant pramlintide with at least one cleaving agent in a single step; and (d) isolating and purifying the recombinant pramlintide from the inclusion bodies.

[0070] In an embodiment of the present disclosure, there is provided a recombinant lethal toxin-neutralizing factor (LTNF) peptide having an amino acid sequence as set forth in SEQ ID NO: 1.

[0071] In an embodiment of the present disclosure, there is provided a recombinant pramlintide peptide having an amino acid sequence as set forth in SEQ ID NO: 2.

[0072] Although the subject matter has been described in considerable detail with reference to certain examples and implementations thereof, other implementations are possible.

EXAMPLES

[0073] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply.

[0074] The working and non-working examples depicted here assert the inventiveness of the present disclosure. Working of the claimed process has been depicted by production of recombinant LTNF and pramlintide protein. It is further well understood by a person skilled in the art that the claimed process can be employed for production of any recombinant protein.

Example 1

Details of the cells and other materials used in the study

[0075] E. coli BL21 (DE3)

Genotype: E. coli str.

lacUV5-T7p07 indl sam7 hί

- an E. coli B strain with DE3, a l prophage carrying the T7 RNA polymerase gene and lacl^q. Transformed plasmids containing T7 promoter driven expression are repressed until lactose or IPTG induction of T7 RNA polymerase from a lac promoter.

- Derived from B834 (Wood, William B. "Host specificity of DNA produced by Escherichia coli: bacterial mutations affecting the restriction and modification of DNA." Journal of molecular biology 16.1 (1966): 118-IN3) by transducing to Met+.

[0076] Vector: pET 29a was obtained from Novagen Inc.

[0077] Inducer: Lactose procured from Merck Specialities

[0078] Cleaving agent: Alpha-chymotrypsin procured from Sisco Research laboratories Pvt Ltd. [0079] Solubilizing agent: Urea

Example 2

Cloning of gene of interest in pET 29a vector and transformation in bacterial cell

[0080] Using conventional molecular technology tools, nucleotide sequences as represented in SEQ ID NO: 3 and SEQ ID NO:4 capable of expressing the peptide depicted by SEQ ID NO: 1 (LTNF) and SEQ ID NO: 2 (pramlintide) respectively, were prepared and inserted into pET 29a expression vector (Figure 2). The nucleotide sequences coding for pramlintide and LTNF were codon optimized for expression in E. coli and were independently cloned in pET 29a expression vector. The respective sequence was cut and inserted into a pET29a plasmid (Novagen) between Ndel and Xhol (Promega) sites. The positive clone was selected for transformation in E. coli BL2l(DE3). The transformants thus obtained were further used for producing protein of interest. The pET29a plasmid contains a termination sequence downstream of a 6xHIS tag that is immediately following the Xhol cutting site. Both the genes, pramlintide and LTNF were cloned independently by following a similar method. Two pramlintide clones (clone 1 and clone 2) and one LTNF clone were confirmed using restriction digestion analysis. Figure 3 depicts the analysis of clone confirmation for two pramlintide clones, wherein expected band of 369 bp was obtained after restriction digestion analysis. LTNF clone was also confirmed in a similar manner, whereby restriction digestion analysis yielded a band around 306 bp.

[0081] After confirmation of the clone, the recombinant vector comprising the respective nucleic acid was transformed in E. coli BL21 (DE3) cells and their expression profile was checked.

Example 3

Expression profile of Pramlintide clone and LTNF clone

[0082] Expression profile of Clone 1 and Clone 2 of pramlintide: Clone 1 and

Clone 2 was induced using two different concentrations of IPTG, viz., O.lmM and lmM. The expression of the proteins was checked using SDS PAGE and is depicted in Figure 4. As can be appreciated, overexpression of pramlintide protein (13.57 kDa) was observed in the samples. Lactose (lOg/L) was also used to study the expression of pramlintide protein (data not shown).

[0083] Expression profile of LTNF clone: Figure 5 depicts the expression profile of LTNF. IPTG (lmM) was used as an inducer for the expression of LTNF protein. It can be appreciated that Lane 5 corresponds to induced solubilized inclusion bodies depicts the over expression of LTNF protein of 11.43 kDa. Lactose (lOg/L) was also used for studying the expression of LTNF (data not shown).

Example 4

Optimization of growth conditions

[0084] Aerobic vs Microaerobic conditions

1. Profiles of the dissolved oxygen of aerobic and microaerobic process for producing peptide LTNF: Cells were grown at high cell density and induced with lOOg/L of lactose (Figures 6 and 7). E. coli possesses distinct catabolic routes to survive and metabolic active under wide range of oxygen conditions. Growing cells in the aerobic environment leads to oxidative stress due to endogenous production of reactive oxygen species [S. Alexeeva, B. de Kort, G. Sawers, K. J. Hellingwerf, and M. J. T. de Mattos,“Effects of limited aeration and of the ArcAB system on intermediary pyruvate catabolism in Escherichia coli,” J. Bacteriol., vol. 182, no. 17, pp. 4934-4940, 2000]. Comparative analysis of conventional and microaerobic fermentation using dissolved oxygen were performed carefully in the bioreactor as shown in Figure 6. Dissolved oxygen was maintained at 3% in microaerobic and 25% at aerobic condition, respectively.

2. Profiles of product titre during aerobic and microaerobic process for producing peptide: The product titre for the recombinant LTNF polypeptide has been depicted in Figure 7. As can be observed, dissolved oxygen limitation shows increased recombinant peptide expression. 3. Profiles of the comparative cell viability of aerobic and microaerobic product: As can be observed from the graph depicted in Figure 8, microaerobic process improves cell viability compared to cells grown in aerobic process.

[0085] Effect of urea concentration on solubilization of inclusion bodies: Effect of different concentrations of urea ranging from 1M-6M with respect to the peptide production (LTNF) was checked. Figure 9 illustrates the data, wherein it can be deduced that 3-4M of urea is sufficient for solubilization.

[0086] Effect of urea concentration on cleavage reaction: One of the aspects of the claimed process of the present disclosure is solubilization of the inclusion bodies with urea and treatment of the recombinant peptide (LTNF) in a single step. In order to arrive at the suitable concentration of the solubilizing and cleaving agent, effect of urea on cleavage reaction using alpha-chymotrypsin was analyzed and the results have been depicted in Figure 10. As inclusion bodies contain impurities, different concentration of urea might have effect on the impurity generation during cleavage reaction. Figure 10 shows the effect of urea (from 1-4 M) on impurity (11-15) generation. It is clear from the figure that cleavage approaches equilibrium at 3M and impurities II and 13 decrease with increase in urea concentration whereas, 12, 14 and 15 increase with the concentration.

[0087] Although the optimization conditions have been depicted for LTNF, but in view of the disclosed method of production of protein, optimization of pramlintide can also be done before proceeding with production of pramlintide.

Example 5

Preparation of seed culture

[0088] Once the conditions for the production of the peptide was standardized, the BL21 cells obtained from Example 2 were grown in shake flask under aerobic condition using 50-70% dissolved oxygen for 2-3 hours till the O.D.₆oo of 45 was attained. The composition of media for seed culture comprises glucose, magnesium sulphate heptahydrate, Luria Bertini, and kanamycin. The cells were grown in the seed media at temperature in a range of 25-38 °C, and agitation of 180-250 rpm for 8-14 hours. This step was performed to obtain sufficient biomass for inoculation of the production media for peptide and protein production. The method of preparation of seed culture as disclosed herein applies for both LTNF and pramlintide.

Example 6

Production of recombinant peptide employing the claimed process

[0089] The biomass generated in Example 5 (seed culture: 5-20% of the reactor volume) was used to inoculate production media that comprised the below components as depicted in Table 1:

Table 1:

[0090] For production in bioreactor, the cells were cultured at a temperature in a range of 30-39 °C (preferably 35 °C), agitation in a range of 300-1200 rpm (preferably 400-900 rpm), dissolved oxygen in a range of 0-10% (preferably 1- 5%), airflow rate of 0.3-0.7 litre/minute for 12-20 hours (preferably 14 hours) in

0.4-0.8 litre media. The working range of the seed culture was 5-20% of the production medium, and 10% was the operational value. The process for production of recombinant peptide as disclosed in the present section stands true for both the protein LTNF and pramlintide.

[0091] Table 2 depicts the process parameters for production in bioreactor.

Table 2:

Example 7

Post protein production under microaerobic condition

[0092] In case of post protein production under microaerobic conditions for both the proteins (LTNF and pramlintide), the cells were centrifuged and the cell pellet (inclusion bodies or IB) was subjected to simultaneous solubilization and cleavage using 3-4 M urea and 1-5 units/mg of alpha-chymotrypsin. The composition of buffer and the sequence of addition is depicted in Table 3 below.

Table 3:

Example 8

Purification of the recombinant protein

[0093] Post solubilization and cleavage, the recombinant protein (LTNF and pramlintide) was purified by reverse phase-high performance liquid chromatography (RP-HPLC) using Akta Explorer/ Akta Avant. The conditions for the purification has been depicted in Table 4 below.

Table 4:

[0094] Figure 11 and Figure 12 show the preparative chromatogram of RP- HPLC purification of recombinant LTNF and analytical RP-HPLC profile for production of recombinant pramlintide, respectively. RP-HPLC method was developed using 25 mM acetic acid (mobile phase A) and acetonitrile (mobile phase B). Following the process, recombinant LTNF was eluted at 19-20% of mobile phase B. The purified material was directly subjected to lyophilization to get recombinant LTNF and pramlintide in powder form. Figures 13 and 14 depict the LC-MS profile of recombinant LTNF and pramlintide obtained by the claimed process, respectively.

[0095] To summarize, the instant disclosure provides with a scalable and flexible process for producing recombinant peptides. A scheme depicting the steps of the claimed process has been provided in Figure 15. A comparative table differentiating the claimed process from the conventional process is provided in Table 5 below.

Table 5:

Advantages of the present disclosure:

[0096] The present disclosure discloses a process for producing peptides which involves microaerobic fermentation. The process leads to high cell viability, high productivity and also flexibility of running the process for peptide production. In product isolation and purification, the proposed process uses fewer unit operations compared to traditional process. In particular, the solubilization and reaction steps are combined together and performed in a single unit operation. Reverse phase high performance liquid chromatography (RP-HPLC) is employed to purify the protein. The net impact of these changes is a significant reduction in the cost of manufacturing (more than 30%) than the conventional process without compromising product quality. It also offers other advantages pertaining to stability of samples. Inclusion bodies generally have higher stability compared to intermediate process streams. Minimization of process steps enhances the operation flexibility, lower foot print, lowers capital expenditure, reduces the need of buffer tanks, minimizes cleaning and steaming in place and thereby significantly reduces time of operation.

Claims

I/We Claim:

1. A process for producing recombinant peptides, said process comprising: a) obtaining a recombinant host cell comprising a recombinant DNA construct, said DNA construct comprising a nucleotide fragment and a promoter to drive expression of the nucleotide fragment encoding a recombinant peptide;

b) culturing the recombinant host cell in a growth medium under microaerobic conditions suitable for expression of the recombinant peptide, wherein the recombinant peptide is localized in inclusion bodies;

c) solubilizing the inclusion bodies with 3-4M urea and treating the recombinant peptide with at least one cleaving agent in a single step; and

d) isolating and purifying the recombinant peptide from the inclusion bodies.

2. The process as claimed in claim 1, wherein the recombinant host cell comprises a recombinant vector, said recombinant vector comprises a recombinant DNA construct, said recombinant DNA construct comprises a nucleotide fragment and a promoter to drive expression of the nucleotide fragment encoding a recombinant peptide.

3. The process as claimed in any one of the claims 1 and 2, wherein the recombinant peptide comprises ‘n’ number of tandem repeats of the peptide, and‘n’ ranges from 1-20.

4. The process as claimed in any one of the claims 1 and 2, wherein the recombinant peptide is selected from a group consisting of lethal toxin neutralizing factor (LTNF), pramlintide, exenatide, teriparatide, insulin, liraglutide, granulocyte colony stimulating factor (G-CSF), antibody fragment (Fab), and human growth hormone.

5. The process as claimed in claim 1, wherein the microaerobic condition comprises dissolved oxygen content in a range of 0-10%.

6. The process as claimed in claim 1, wherein culturing the recombinant host cell comprises stirring in a range of 300-1200 rpm for a time period in a range of 12-20 hours at a temperature in a range of 30-39 °C.

7. The process as claimed in claim 1, wherein solubilizing the inclusion bodies and treating the recombinant peptide comprises stirring in a range of 50-250 rpm, for a time period in a range of 0.5-3 hours, at a temperature in a range of 4-30 °C, and at a pH in a range of 5-9.

8. The process as claimed in claim 1, wherein the at least one cleaving agent is selected from a group consisting of chymotrypsin,Glu C endoproteinase, O-iodosobenzoic acid, cyanogen bromide, N-bromosuccinimide, N- chlorosuccinimide, and combinations thereof.

9. The process as claimed in claim 1, wherein the at least one cleaving agent has a concentration in a range of 1-5 units/mg.

10. The process as claimed in claim 1, wherein purifying the recombinant peptide comprises at least one method selected from a group consisting of reverse phase-high performance liquid chromatography (RP-HPLC), low pressure cation exchange chromatography, hydrophobic interaction chromatography, mixed mode chromatography, and combinations thereof.

11. The process as claimed in claim 1, wherein the promoter is selected from a group consisting of lac promoter, T7 promoter, T5 promoter, Tac promoter, and LacUV5 promoter.

12. A recombinant LTNF peptide produced by a process as claimed in claim

1.

13. A recombinant pramlintide peptide having an amino acid sequence as set forth in SEQ ID NO: 2.