WO2019082138A1

WO2019082138A1 - Process for preparation of liraglutide using recombinant saccharomyces cerevisiae

Info

Publication number: WO2019082138A1
Application number: PCT/IB2018/058369
Authority: WO
Inventors: M Lavanya Puppala; Venkataramana Mudili
Original assignee: Lorven Biologics Private Limited
Priority date: 2017-10-27
Filing date: 2018-10-26
Publication date: 2019-05-02

Abstract

The present invention provides for a process for preparation of liraglutide using recombinant saccharomyces cerevisiae. The invention represents an advancement in the field of genetic engineering and discloses a modified nucleic acid for achieving optimum expression of liraglutide in a heterologous host. The invention also discloses vectors carrying the modified nucleic acid and recombinant host cells carrying the vectors. The invention also discloses the process for producing a recombinant host cell, process for production of the recombinant protein and an improved down streaming process for having high yield of liraglutide.

Description

PROCESS FOR PREPARATION OF LIRA GLUTIDE USING RECOMBINANT

SACCHAROMYCES CEREVISIAE CROSS REFERENCE TO RELATED APPLICATION AND CLAIM OF PRIORITY

The present application claims priority from Indian Patent Application No. 201741038302 filed on October 27, 2017, the entire contents of which are hereby incorporated by reference. FIELD OF INVENTION

The present invention is directed to the field of genetic engineering. Particularly, the invention relates to biologically active liraglutide and methods for the producing the same.

BACKGROUND OF THE INVENTION

Diabetes is a metabolic disorder characterized by hyperglycemia, a condition in which an excessive amount of glucose circulates in the blood plasma. Diabetes is associated with a high risk of cardiovascular and other serious health-related consequences. People with diabetes are also at very high risk of developing serious microvascular complications such as nephropathy, renal failure, retinal diseases, blindness, autonomic and peripheral neuropathy etc.

The majority of people with diabetes have type-2 diabetes. Type-2 diabetes is characterized by insulin resistance and eventually impaired insulin secretion. Optimal glycemic control is the treatment goal for people with type-2 diabetes. Despite the availability of several oral anti-diabetic drugs and insulin, these drugs are not able to control the blood sugar level in a significant proportion of people. With the increasing incidence and prevalence of type-2 diabetes, there is an unmet medical need for alternative treatments.

Incretin mimetics are a relatively new group of injectable drugs for treatment of type-2 diabetes.

The incretin mimetics are based on GLP- 1 , an incretin hormone secreted from L-cells in the lower gastrointestinal tract and is known to lower blood glucose by stimulating glucose - dependent insulin secretion. Native GLP-1 has a very short elimination half-life (less than 1.5 minutes after intravenous administration) due to rapid degradation by endogenous dipeptidyl peptidase-4 (DPP-4).

Because of its short life, GLP- 1 is not suitable as a therapeutic drug. Therefore, it has to be modified in order to improve its stability against degradation. Modified GLP-1 class of drugs with improved stability are known as glucagon-like peptide 1 (GLP-1) receptor agonists or GLP-1 analogues. The glucagon-like peptide 1 (GLP-1) receptor agonists or GLP-1 analogues are normally prescribed for patients who have not been able to control their condition with tablet medication. They work by copying, or mimicking, the functions of the natural incretin hormones in your body that help lower post-meal blood sugar levels. The functions of these analogues include:

· Stimulating the release of insulin by the pancreas after eating, even before blood sugars start to rise.

• Inhibiting the release of glucagon by the pancreas. Glucagon is a hormone that causes the liver to release its stored sugar into the bloodstream.

• Slowing down glucose absorption into the bloodstream by reducing the speed at which the stomach empties after eating, thus making you feel more satisfied after a meal.

Liraglutide sold under the brand name VICTOZA^® by Novo Nordisk, is an intentionally modified

GLP-1 analogue molecule with 24-hour durations of blood levels and pharmacologic action.

Liraglutide is expected to improve or normalize glycemic control without increasing the risk of hypoglycemia by improving glucose-dependent insulin secretion.

Liraglutide precursor peptide has been produced by using recombinant DNA technology with subsequent addition of a palmitic acid to the ε-amino group of lysine in position 26.

Synthetic and E coli based host expression systems have been tried to produce liraglutide precursor proteins. However, all approaches have been failed in terms of meeting critical quality attributes (solubility, efficacy and impurities) as well as high production costs associated with the E coli and synthetic processes.

Yeast host expression systems have been used to express and secrete heterologous proteins.

Such approaches have involved modifications to the various molecular components that are involved in expression and secretion of proteins in yeast.

However, these approaches have often failed as the amount of peptide secreted is unacceptably low or incorrect processing leads to inactive forms of the peptide. This is particularly common for peptides that are initially expressed as a precursor polypeptide sequence.

Due to these reasons, the yield of biologically active liraglutide obtained by heterologous expression are extremely low due to degradation, improper folding, inefficient translocation and high downstream processing costs.

Therefore, the present invention contemplates to overcome the challenges of the prior art by expressing an engineered nucleic acid encoding liraglutide precursor peptide in a heterologous host and providing methods for producing large amounts of liraglutide in a biologically active form.

SUMMARY OF THE INVENTION

The present invention relates to a modified nucleic acid encoding a fusion protein in which liraglutide precursor peptide is operably fused to a signal peptide selected from a group comprising alpha-amylase signal peptide of Aspergillus niger, glucoamylase signal peptide of Aspergillus awamori and inulinase signal peptide of Kluyveromyces maxianus.

The present invention also relates to a vector comprising the modified nucleic acid encoding the fusion protein. The invention also discloses a recombinant host cell comprising the modified vector.

Further, the invention relates to a modified liraglutide polypeptide fused to a signal peptide, wherein the signal peptide is selected from a group comprising alpha-amylase signal peptide of Aspergillus niger, glucoamylase signal peptide of Aspergillus awamori and inulinase signal peptide of Kluyveromyces maxianus.

Further, the invention discloses a process for recombinant production of biologically active liraglutide.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the present disclosure will become fully apparent from the following description taken in conjunction with the accompanying figures. With the understanding that the figures depict only several embodiments in accordance with the disclosure and are not to be considered limiting of its scope, the disclosure will be described further through use of the accompanying figures.

Figure 1 is the vector map of recombinant plasmid pD1204 which shows the gene construction for expression of recombinant fusion protein.

Figure 2 depicts SDS-PAGE of cell fractions obtained from the recombinant strains and control strains.

Figure 3 depicts the results mass spectroscopy for identification of the protein obtained from recombinant strain.

Figure 4 depicts the results of N-terminal sequence analysis of the recombinant protein. Figure 5 depicts the results mass spectroscopy for identification of the protein obtained from recombinant strain. Figure 6 shows overlay FTIR spectroscopy of recombinant liraglutide precursor peptide obtained in different batches compared to the standard.

Figure 7 shows the results of HPLC analysis for calculating the purity of the recombinant peptide.

Figure 8 depicts the results of calculating molar mass in comparison to retention time of the recombinant liraglutide precursor peptide with standard.

Figure 9 depicts the results of cAMP Assay with GLP- 1 performed for calculation of half maximal effective concentration (EC50) and compared with GLP-1 (Sigma).

Figure 10 depicts the results of cAMP Assay with GLP-1 performed for calculation of half maximal effective concentration (EC50) and compared with commercially available liraglutide (VICTOZA^®).

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses a modified nucleic acid for expression of recombinant liraglutide precursor peptide in Saccharomyces cerevisiae.

The invention contemplates that the modified nucleic acid would have better expression in a heterologous host leading to better yield of the protein.

The invention also contemplates an improved downstream processing method for recovery of biologically active liraglutide.

The invention contemplates a multidimensional approach for achieving a high rate of expression in a heterologous host. Primarily, the invention contemplates that fusing the nucleotide sequence encoding a signal peptide with the sequence encoding liraglutide precursor peptide leads to more efficient translocation of liraglutide precursor peptide across the cell membrane. This approach coupled with engineering the nucleotide sequence of the native gene encoding liraglutide precursor peptide to match the preferred codon system of the host cell gives a greater efficiency in protein expression.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular as is considered appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for the sake of clarity. Generally, nomenclatures used in connection with, and techniques of biotechnology, fermentation technology, genetic engineering and recombinant DNA technology described herein are those well-known and commonly used in the art. Certain references and other documents cited are expressly incorporated herein by reference. In case of conflict, the present specification, including definitions, will control. The materials, methods, figures and examples are illustrative only and not intended to be limiting.

Furthermore, the methods, preparation and use of the modified nucleic acid encoding the liraglutide precursor peptide employ, unless otherwise indicated, conventional techniques in recombinant DNA technology, fermentation technology and related fields. These techniques, their principles, and requirements are explained in the literature and known to a person skilled in the art.

Before the method of generating the modified nucleic acid encoding the liraglutide precursor peptide, vectors, recombinant hosts, methods of downstream processing and other embodiments of the present disclosure are disclosed and described, it is to be understood that the terminologies used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "comprises" or "comprising" is generally used in the sense to include, that is to say permitting the presence of one or more features or components.

As used herein, the term "disclosure" or "present disclosure" as used herein is a non- limiting term and is not intended to refer to any single embodiment of the particular disclosure but encompasses all possible embodiments as described in the specification and the claims.

As used herein, the term "liraglutide precursor peptide" or "lirapeptide" refers to the peptide precursor of liraglutide, produced by a process that includes expression of recombinant DNA in recombinant host. The liraglutide precursor peptide is modified to add palmitic acid to the ε-amino group of lysine in position 26 in order to produce liraglutide.

As used herein, the term "gene" refers to a nucleic acid fragment corresponding to specific amino acid sequence that expresses a specific protein with regulatory sequences. "Native gene" or "wild type gene" refers to a gene as found in nature with its own regulatory sequences.

As used herein, the term "promoter" refers to a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA capable of controlling the expression of a coding sequence or functional RNA which can be native, derived or synthetic. Some promoters are called constitutive as they are active in all circumstances in the cell, while others are called inducible as they are regulated and become active in response to specific stimuli.

The term "inducible promoter" refers the promoters that are induced by the presence or absence of biotic or abiotic and chemical or physical factors. Inducible promoters are a very powerful tool in genetic engineering because the expression of genes operably linked to them can be turned on or off at certain stages of development or growth of an organism or in a particular tissue or cells.

As used herein, the term "gene expression", refers to the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.

As used herein, the term "transformation" as used herein, refers to the transfer of a nucleic acid fragment into a host organism either in the form of plasmid or integrated stably to the chromosome of the host organisms resulting in genetically stable inheritance. A cloning vector is a small piece of DNA, mostly a plasmid, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning or transformation purposes.

The term "host cell" includes an individual cell or cell culture which can be, or has been, a recipient for the subject of expression constructs. Host cells include progeny of a single host cell. Host cell can be any expression host including prokaryotic cell such as but not limited to Bacillus subtilis, Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum or eukaryotic system, such as, but not limited to Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha.

The term "recombinant strain" refers to a host cell which has been transfected or transformed with the expression constructs or vectors of this invention.

The term "expression cassette" denotes a gene sequence used for cloning in expression vectors or in to integration vectors or integrated in to coding or noncoding regions of chromosome of the host cell in a single or multiple copy numbers, where the expression cassette directs the host cell's machinery to make RNA and protein encoded by the expression cassette. The term "expression construct" or "plasmid" is used here to refer to a functional unit that is built in a vector for the purpose of expressing recombinant proteins/peptides, when introduced into an appropriate host cell, can be transcribed and translated into a biologically active protein.

The term "modified nucleic acid" or "modified nucleotide sequence" is used to refer to an artificially synthesized nucleic acid in which the gene encoding Hraglutide precursor peptide operably fused to a signal peptide.

Although disclosure and exemplification has been provided by way of illustrations and examples for the purpose of clarity and understanding, it is apparent to a person skilled in the art that various changes and modifications can be practiced without departing from the spirit or scope of the disclosure. Accordingly, the foregoing descriptions and examples should not be construed as limiting the scope of the present disclosure.

The present invention discloses a modified nucleic acid encoding Hraglutide precursor peptide in which the gene encoding hraglutide precursor peptide has been operably fused to a signal peptide and having optimal expression levels in heterologous hosts. In a preferred embodiment, the nucleic acid is represented by SEQ ID NO: 5, SEQ ID NO:6 and SEQ ID NO:7.

The present disclosure also relates to a polypeptide encoded by the nucleic acid sequence as in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 or any variant thereof, wherein the polypeptide is hraglutide precursor peptide fused to a signal peptide.

The signal peptide is selected from a group comprising alpha-amylase signal peptide of Aspergillus niger represented by SEQ ID NO: 2, glucoamylase signal peptide of Aspergillus awamori represented by SEQ ID NO: 3 and inulinase signal peptide of Kluyveromyces maxianus represented by SEQ ID NO:4.

In another aspect, the present disclosure discloses suitable vectors comprising the modified nucleic acid for optimal expression of hraglutide precursor peptide in a heterologous host. In yet another aspect, the vector of the disclosure is an expression vector which can be conveniently subjected to recombinant DNA procedures. The choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector could be an autonomously replicating vector, i.e. a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector could be one which, when introduced into a host cell, is integrated into the host cell genome, in part or in its entirety, and replicated together with the chromosomes into which it has been integrated. In another aspect, the vector is preferably an expression vector in which the DNA sequence encoding the liraglutide precursor peptide fused to a signal peptide is operably linked to additional segments required for transcription of the DNA. The term, "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in some promoter and proceeds through the DNA sequence coding for the enzyme.

Preferably, the gene can be cloned into any Saccharomyces cerevisiae expression vectors known in the art. In a preferred embodiment, the vector is a pD1204.

Any suitable promoter can be used. In a preferred embodiment, an inducible promoter GAL1 is used. The cloned gene sequences can be confirmed by restriction digestion or nucleotide sequencing. In a preferred embodiment, the vector pD1204 having GAL1 promoter has been used.

In another embodiment, the host cell into which the DNA construct or the recombinant vector of the disclosure is introduced may be any cell which can produce the present enzyme and includes bacteria, yeast, any other microorganism, a mammalian cell, plant cell or any cell culture of said category.

The host-cell is a bacterial cell selected from a group comprising Bacillus subtilis,

Escherichia coli, Lactococcus lactis, Bacillus megaterium, Pseudomonas putida and Corynebacterium glutamicum or the host cell is a eukaryotic cell selected from a group comprising Saccharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha or any host known in the art for expression of heterologous proteins using T7 promoter-based vectors for expression.

In a preferred embodiment, the host-cell is Saccharomyces cerevisiae INVScl (Invitrogen,

USA). Commercially available Saccharomyces cerevisiae INVScl was used in the preferred embodiment of the invention. In the preferred embodiment, the antibiotic marker in the vector has been made non-functional by in vitro modification before transformation of the Saccharomyces cerevisiae host cell.

In another embodiment, the expression level of the gene was measured by quantifying the amount of recombinant protein. Bradford protein assay was performed for quantifying the protein present in the sample. The disclosure provides enhanced expression of the recombinant liraglutide using the recombinant Saccharomyces cerevisiae which may range upto 11 g/L.

In another embodiment, the process for production of liraglutide protein is provided. In a preferred embodiment, the process of production includes the steps of culturing host cells transformed with a vector comprising a modified nucleic acid in a suitable culture medium, converting Hraglutide precursor peptide to liraglutide and purifying recombinant liraglutide.

In another embodiment, the process of culturing host cells transformed with a vector comprising the modified nucleic acid comprises of adding continuously a carbon source, adding continuously galactose to the culture till A575 of 40 to 200, maintaining the pH of the culture between 5 to 5.4, maintaining the temperature of the culture at between 20°C and 35°C and harvesting the recombinant liraglutide precursor peptide by centrifugation from the culture after about 72 hrs after commencement of inducer addition.

In a preferable embodiment, the carbon source is glucose and concentration of galactose is in the range of 0.02 to 0.4 mM. Further, the ratio of concentration of galactose and glucose is maintained in the range of 4: 1 to 1: 1.

In another embodiment, the method for converting recombinant liraglutide precursor peptide to liraglutide comprises the steps of preparing a reaction mixture comprising liraglutide precursor peptide, EDPA, acetonitrile and water. Further, a solution of Palm-L-Glu(OSu)-OMe linker in acetonitrile is added to the reaction mixture.The reaction mixture is shaken while maintaining the temperature of about 20°C. The pH of the reaction mixture is maintained between 10 and 12. Finally, a solution of glycine in ethanol to the reaction mixture.

In another embodiment, the method for purifying liraglutide is provided which comprises the steps of subjecting the reaction mixture to centrifugation, dissolving the supernatant in TAE buffer and subjecting the dissolved supernatant to column chromatography comprising two mobile phases, wherein the first mobile phase is tris and the second mobile phase is acetonitrile.

In other embodiments, the recombinant protein has been thoroughly characterized. Characterization has been done by N-terminal sequence analysis of recombinant liraglutide precursor peptide, Overlay spectroscopic analysis of liraglutide precursor peptide, Purity profile of liraglutide precursor peptide, Gel -permeation chromatography and GLP-1 receptor assay in CHO Cell Lines. The results of these experiments exhibit that the characteristics of the recombinant liraglutide precursor peptide matches that of the commercially available liraglutide.

Source and geographical origin of biological material

The native nucleotide sequences for the following peptides are publicly available in the prior art: alpha-amylase signal peptide of Aspergillus niger (SEQ ID NO:2), glucoamylase signal peptide of Aspergillus awamori (SEQ ID NO:3) and inulinase signal peptide of Kluyveromyces maxianus (SEQ ID NO:4). Engineered nucleic acids (SEQ ID NO: l, SEQ ID NO:5, SEQ ID N0:6 and SEQ ID N0:7) were synthesized artificially by Genscript, USA, a commercial gene synthesis service provider. SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO: 10 are the polypeptides encoded by SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively. The vector pBR322 was obtained from Atum, USA (Example 1). The host cell Saccharomyces cerevisiae was obtained from Invitrogen, USA (Example 3).

EXAMPLES

The following examples particularly describe the manner in which the invention is to be performed. But the embodiments disclosed herein do not limit the scope of the invention in any manner.

Example 1: Modified nucleic acid for expression of recombinant liraglutide precursor peptide in Saccharomyces cerevisiae

An expression cassette encoding for liraglutide precursor peptide was modified for optimum expression in Saccharomyces cerevisiae. The modified open reading frame contains the nucleotide sequence encoding liraglutide precursor peptide fused to a signal peptide. The signal peptides used in the present invention are alpha-amylase signal peptide of Aspergillus niger, glucoamylase signal peptide of Aspergillus awamori and inulinase signal peptide of Kluyveromyces maxianus. The preferred codons for expression in Saccharomyces cerevisiae has been used in place of rare codons.

The nucleotide sequence of the liraglutide precursor peptide is represented by SEQ ID

NO: l. The nucleotide sequence of the alpha-amylase signal peptide of Aspergillus niger is represented by SEQ ID NO:2. The nucleotide sequence of the glucoamylase signal peptide of Aspergillus awamori is represented by SEQ ID NO:3 and the nucleotide sequence of the inulinase signal peptide of Kluyveromyces maxianus is represented by SEQ ID NO:4.

The nucleotide sequence of the modified open reading frame encoding for liraglutide precursor peptide fused with alpha-amylase signal peptide of Aspergillus niger is represented by SEQ ID NO: 5.

The nucleotide sequence of the modified open reading frame encoding for liraglutide precursor peptide fused with glucoamylase signal peptide of Aspergillus awamori is represented by SEQ ID NO: 6. The nucleotide sequence of the modified open reading frame encoding for liraglutide precursor peptide fused with inulinase signal peptide of Kluyveromyces maxianus is represented by SEQ ID NO: 7.

This modified open reading frame has been artificially synthesized by using the sequence for liraglutide precursor peptide and the signal peptides.

The plasmid used in the process was pD1204 (Atum, USA). The recombinant plasmid contains the open reading frame and an inducible GAL1 promoter.

The modified sequence encoding for the recombinant protein was cloned in to pD1204 expression vector. Further, the antibiotic resistance gene in the expression vector has been made non-functional by in vitro modification before transformation of the Saccharomyces cerevisiae host cell.

The vector map of pD1204 is represented in Figure 1.

Example 2: Polypeptide sequences of liraglutide precursor peptides fused to signal peptides

The recombinant protein obtained by translating the gene encoding for liraglutide precursor peptide fused with alpha-amylase signal peptide of Aspergillus niger is represented by SEQ ID NO: 8.

The recombinant protein obtained by translating the gene encoding for liraglutide precursor peptide fused with glucoamylase signal peptide of Aspergillus awamori is represented by SEQ ID NO: 9.

The recombinant protein obtained by translating the gene encoding for liraglutide precursor peptide fused with inulinase signal peptide of Kluyveromyces maxianus is represented by SEQ ID NO: 10.

Example 3: Development of recombinant host cell by transformation with recombinant plasmids

Recombinant pD1204 plasmids as described in foregoing example carrying the gene for liraglutide precursor peptide fused to signal peptides were used.

Saccharomyces cerevisiae INVScl host cells (Invitrogen, USA) were electroporated with the plasmids as described in foregoing example and resuspended in growth medium with trace minerals. The cultures were incubated at 30°C with shaking for 48 hours. 10 Ε of each of the seed cultures were transferred into triplicate test tubes, each tube containing 5 ml of growth medium supplemented with trace elements, and incubated as before for 24 hours. Galactose was added to each well to a final concentration of 0.05 mM (1%) to induce the expression of target proteins. The temperature was maintained at 25°C. After induction, at every 12 hours, supernatant samples were frozen for later processing.

The recombinant host cell carrying the nucleic acid encoding liraglutide precursor peptide fused to alpha-amylase signal peptide of Aspergillus niger was labelled as Saccharomyces cerevisiae OSM1233.

Example 4: SDS-PAGE for confirmation of insertion of 2-micron plasmids containing liraglutide precursor peptide

Analysis of recombinant host cells was done by SDS-PAGE. The gel was stained by silver stain to visualize the bands.

An aliquot of cell culture was collected at different time points and the cell lysates were subjected to SDS-PAGE. The results of SDS-PAGE are depicted in Figure 2.

For SDS-PAGE, the liraglutide precursor peptide produced by recombinant Saccharomyces cerevisiae host cells were collected. The recombinant protein was subjected to SDS-PAGE using standard protocols.

Protein ladder and a standard was put in the first two lanes and in rest of the lanes recombinant protein purified by standard aromatic hydrophobic interaction chromatography (HIC) medium were placed.

It was confirmed that 2-micron plasmids were integrated into the host cell.

Example 4: Large-scale expression of liraglutide precursor peptide

Recombinant liraglutide precursor peptide was produced in Saccharomyces cerevisiae OSM 1233 in a 650 litre fermenter. Cultures were grown in 650 litre fermenter containing semi synthetic medium.

Culture conditions were maintained at 37°C and pH 7.0 through the addition of sodium hydroxide. Dissolved oxygen was maintained in excess through increases in agitation and flow of sparged air and oxygen into the fermenter. Glucose was delivered to the culture throughout the fermentation to maintain excess levels. These conditions were maintained until a target culture cell density at 575nm (A575) for induction is reached, at which time sucrose is added to initiate liraglutide production. Cell density at induction could be varied from A575 of 40 to 200 absorbance units (AU). Galactose concentrations could be varied in the range from 0.02 to 0.4 mM. The pH of the culture has to be maintained in the range of 5 to 5.4 and temperature should be maintained between 20°C to 35 °C. After 72 hours of induction, the culture from each bioreactor was harvested by centrifugation and the culture supernatant frozen at -80 °C. Samples were analysed by Reverse Phase HPLC and Western blot analysis for product formation.

Multiple fermentation conditions were evaluated resulting in top liraglutide expression as determined by Reverse Phase HPLC. The identities of the induced proteins were confirmed by CHO based GLP 1 receptor assay. The results of the assay are provided in Figure 9 and 10.

Example 5: Conversion of liraglutide precursor peptide to liraglutide

For conversion of liraglutide precursor peptide to liraglutide, a mixture of liraglutide precursor peptide (6.7 mg), EDPA (7.2 mg), acetonitrile (470 μΐ) and water (470 μΐ) was gently shaken for 6 min. at 20°C. To the resulting mixture, a solution of Palm-L-Glu(OSu)-OMe linker (3.3 mg) in acetonitrile (80 ml) was added, and the reaction mixture was gently shaken for 75 min at 20°C.

The reaction was quenched by the addition of a solution of glycine (3.6 mg) in 50% aqueous ethanol (360 ml). A 0.5 % aqueous solution of ammonium-acetate (22 ml) and NMP (540 ml) were added, and the resulting mixture eluted onto a C8 cartridge. Liraglutide (3 mg) was isolated, and the product was analysed by Liquid chromatography-mass spectrometry (LCMS). The m/z value for the protonated molecular ion was found to be 3752.63. The resulting molecular weight was found to be 3751.63 amu (theoretical value 3751 amu). The results of liquid chromatography-mass spectroscopy are depicted in Figure 3 and Figure 5.

Upon comparison with VICTOZA^® (Novo Nordisk), a commercially available injectable liraglutide, it was confirmed that the properties of the recombinantly produced liraglutide was similar to the commercially available pharmaceutical.

Example 5: Purification of Liraglutide using Isoelectric point based Precipitation High Pressure Reverse Phase Purification System

For purification, the liraglutide chemical reaction mixture was adjusted to pH 5.4±0.1 at a concentration of 5 g/L and incubated at room temperature for 6 hours. The crude precipitated was filtered out using centrifugation. The crude load was re-dissolved in TEA buffer and subjected to column chromatography. The column was packed with DASIOGEL 20 μπι C8 100A and 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 temperature of 65°C.

The peak fractions whose purity was greater than 99.00% by analytical HPLC were pooled (4.2 L). Purity of the elution pool was 99.25% with a recovery of 82%. Finally carried forward to the next step of lyophilisation.

Example 6: N-terminal sequence analysis of recombinant liraglutide precursor peptide

Amino-terminal (N-terminal) sequence analysis is used to identify the order of amino acids of proteins or peptides, starting at their N-terminal end. N-terminal sequence analysis was performed by a with a sequencer by standard Edman degradation protocol.

The results are depicted in Figure 4. The results show that the characteristics of the recombinant liraglutide precursor peptide matches that of standard liraglutide.

Example 7: Overlay spectroscopic analysis of liraglutide precursor peptide

Fourier-transform infrared spectroscopy was performed on recombinant protein obtained in different batches and it was compared with VICTOZA^® (Novo Nordisk), a commercially available injectable liraglutide in order to compare the properties.

Figure 6 shows overlay FTIR spectroscopy of recombinant liraglutide precursor peptide obtained in different batches compared to VICTOZA^® (Novo Nordisk).

The results show that the characteristics of the recombinant liraglutide precursor peptide matches that of the commercially available liraglutide.

Example 8: Purity profile of liraglutide precursor peptide

For checking the purity of the recombinant liraglutide precursor peptide, the Retention Time, Relative Retention Time and Area% was calculated by HPLC Analysis.

The analysis of HPLC are depicted in Table 1. The results of HPLC Analysis are provided in Figure 7.

4 15.984 0.928 0.28

5 17.223 1.000 99.22

6 20.809 1.208 0.08

7 25.599 1.486 0.14

8 26.182 1.520 0.04

Table 1: Purity Analysis

The results indicate that at the retention time of 17.223 minutes, the purity of liraglutide precursor peptide is almost 100%, indicating a high level of purity.

Example 9: GPC HPLC

Gel-permeation chromatography was performed on the liraglutide precursor peptide. Molar mass in comparison to retention time was calculated. Liraglutide precursor peptide from different batches were compared to VICTOZA^® (Novo Nordisk), a commercially available injectable liraglutide. The results are depicted in Figure 8.

Example 10: Confirmation of lirapeptide by GLP-1 receptor assay in CHO Cell Lines

A commercially available human GLP-1 receptor-expressing cell line made in the CHO host, which is commonly used for screening for agonists and antagonists at the GLP-1 receptor was used for confirmation of recombinantly produced liraglutide precursor peptide.

The results of the experiments are depicted in Figure 9 and Figure 10.

In the first experiment, cAMP Assay with GLP-1 agonist was performed and the Half maximal effective concentration (EC50) was calculated and compared with that of GLP-1 obtained from SIGMA. The results as calculated from Figure 9 are depicted in the following table:

Table 2: Half maximal effective concentration determination In the second experiment, cAMP Assay with GLP- 1 agonist was performed and the Half maximal effective concentration (EC50) was calculated and compared with VICTOZA^®, a commercially available liraglutide. The results as calculated from Figure 10 are depicted in the following table:

Table 3: Half maximal effective concentration determination The results show that the characteristics of the recombinant liraglutide precursor peptide matches that of the commercially available GLP-1 and liraglutide.

Example 11: Yield Determination

The amount of recombinant protein was consequently quantified. Quantification was done by employing standard Bradford assay protocol. The results of protein quantification assays reveal that the recombinant protein in sample is present in the ranged between 0.1 mg/L to 11 g/L.

A wide range of dilutions starting from 1, 1 : 10, 1: 100, 1: 1000 were prepared and standard Bradford assay protocol was followed. The absorbance was measured at A595 of the samples and standards against the reagent blank between 2 min and 1 h after mixing. The 100^g standard should give an A595 value was also measured.

In a wide range of experiments conducted for yield determination, the yield of recombinant liraglutide precursor protein was found to be about 0.1 mg/L, about 0.6 g/L, about 0.7 g/L, about 0.8 g/L, about 0.9 g/L, about 1 g/L, about 1.5 g/L, about 2 g/L, about 2.5 g/L, about 3 g/L, about 3.5 g/L, about 4 g/L, about 4.5 g/L, about 5 g/L, about 5.5g/L, about 6 g/L, about 6.5 g/L, about 7 g/L, about 7.5 g/L, about 8 g/L, about 8.5 g/L, about 9 g/L, about 9.5 g/L, about 10 g/L, about 10.5 g/L, and about 11 g/L.

The optimum production of recombinant protein was found to be 11 g/L. This result exhibits that the recombinant cells are capable of expressing a very high amount of protein.

Claims

The claim:

1. A modified nucleic acid encoding Hraglutide precursor peptide operably fused to a signal peptide selected from a group comprising alpha-amylase signal peptide of Aspergillus niger, glucoamylase signal peptide of Aspergillus awamori and inulinase signal peptide of Kluyveromyces maxianus, wherein the Hraglutide precursor peptide fused to the alpha-amylase signal peptide of Aspergillus niger comprises the nucleotide sequence of SEQ ID NO:5, the Hraglutide precursor peptide fused to the glucoamylase signal peptide of Aspergillus awamori comprises the nucleotide sequence of SEQ ID NO: 6 and the Hraglutide precursor peptide fused to the inulinase signal peptide of Kluyveromyces maxianus comprises the nucleotide sequence of SEQ ID NO:7.

2. A vector comprising the modified nucleic acid as claimed in 1, wherein the nucleic acid is operably linked to an inducible promoter.

3. The vector as claimed in claim 2, wherein the vector is pD1204 and the inducible promoter is GALL

4. A recombinant host cell comprising the vector as claimed in claim 3.

5. The recombinant host cell as claimed in claim 4, wherein the recombinant host cell is a Saccharomyces cerevisiae host cell.

6. A modified Hraglutide precursor peptide fused to a signal peptide selected from a group comprising alpha-amylase signal peptide of Aspergillus niger, glucoamylase signal peptide of Aspergillus awamori and inulinase signal peptide of Kluyveromyces maxianus, wherein the

Hraglutide precursor peptide fused to the alpha-amylase signal peptide of Aspergillus niger comprises the amino-acid sequence of SEQ ID NO: 8, the Hraglutide precursor peptide fused to the glucoamylase signal peptide of Aspergillus awamori comprises the amino-acid sequence of SEQ ID NO:9 and the Hraglutide precursor peptide fused to the inulinase signal peptide of Kluyveromyces maxianus comprises the amino-acid sequence of SEQ ID NO: 10.

7. A process for producing a recombinant host cell capable of expressing Hraglutide precursor peptide fused to a signal peptide, said process comprising the steps of:

a. synthesizing a modified nucleic acid as claimed in claim 1;

b. constructing a recombinant pD1204 vector harboring the modified nucleic acid, wherein the nucleic acid is operably linked to an GAL1 promoter; and c. transforming a Saccharomyces cerevisiae host cell with the recombinant pD1204 vector to obtain a recombinant host cell.

8. A process for production of hraglutide, said process comprising the steps of:

a. culturing the recombinant host cell as claimed in claim 5 in a suitable culture medium; b. converting hraglutide precursor peptide to Hraglutide, wherein the method for converting recombinant Hraglutide precursor peptide to Hraglutide comprises the steps of: i. preparing a reaction mixture comprising hraglutide precursor peptide, EDPA, acetonitrile and water;

ii. adding a solution of Palm-L-Glu(OSu)-OMe linker in acetonitrile to the reaction mixture;

Hi. shaking the reaction mixture while maintaining the temperature of about 20°C; iv. maintaining the pH of the reaction mixture between 10 and 12;

v. adding a solution of glycine in ethanol to the reaction mixture. ; and c. purifying hraglutide.

9. The process for production of Hraglutide as claimed in claim 8, wherein the method for culturing host cells comprises the steps of:

a. adding continuously a carbon source, wherein the carbon source is glucose;

b. adding continuously galactose to the culture till A575 of 40 to 200 is reached, wherein the concentration of galactose is in the range of 0.02 to 0.4 mM;

c. maintaining the pH of the culture between 5 to 5.4;

d. maintaining the temperature of the culture at between 20 °C and 35°C; and

e. harvesting the recombinant hraglutide precursor peptide by centrifugation from the culture after about 72 hrs after commencement of inducer addition.

10. The process for production of hraglutide as claimed in claim 9, wherein the ratio of concentration of galactose and glucose is maintained in the range of 4: 1 to 1 : 1.

11. The process for production of Hraglutide as claimed in claim 8, wherein the method for purifying Hraglutide comprises the steps of:

a. subjecting the reaction mixture to centrifugation;

b. dissolving the supernatant in TAE buffer; and subjecting the dissolved supernatant to column chromatography comprising two mobile phases, wherein the first mobile phase is tris and the second mobile phase is acetonitrile.