# Patent

## Title
Method of downstream processing for conversion of human insulin precursor molecule into a functional human insulin protein molecule

## Abstract

In one aspect, methods relate to the field of recombinant DNA therapeutics. Methods may involve bio-informatics design, synthesis of artificial genes for a human insulin precursor having a leader peptide coding sequence, cloning artificial genes into an expression vector, and expression in an organism such as one selected from the genus Pichia. In another aspect, methods may include downstream processing for obtaining protein precursor molecules and subsequent conversion of protein precursor molecules to functional proteins.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
The present application is a divisional application of U.S. patent application Ser. No. 13/256,028 filed on Sep. 12, 2011, which is a National Stage Application of PCT Application No. PCT/IN2010/000144 filed on Mar. 12, 2010, which claims priority rights based on the India Patent Application 567/CHE/2009, filed in India on Mar. 12, 2009.


TECHNICAL FIELD
The present disclosure relates to a field of recombinant DNA therapeutics. It involves the bio-informatics design, synthesis of artificial gene for human insulin precursor including leader peptide coding sequence, cloning in an expression vector and expression in an organism, preferably Pichia pastoris. The present disclosure also relates to methods of downstream processing for obtaining protein precursor molecules and subsequent conversion of precursor molecules to functional proteins.
BACKGROUND OF THE DISCLOSURE
Human Insulin is a polypeptide hormone involved in regulation of glucose in blood and body fluids. Production deficiency leads to type 1 or type 2 diabetes. Type 1 is especially Insulin dependent diabetes. Earlier, Insulin used to be supplemented from animal sources (bovine and pig), which often results in undesirable allergic immune response or hypersensitive reaction on continual administration for longer periods. The next generation of Humanized Insulin was produced in E. coli by recombinant DNA technology and is being successfully used for the past several years. Although recombinant human Insulin is being expressed in different hosts through patented processes to meet the diabetes therapeutic requirements, the demand is growing and forcing the man kind to explore new and modified methods to produce commercially viable quantities.
Recombinant human Insulin currently available in the market is produced from at least three different expression systems i.e. E. coli, Pichia pastoris and Hansenula polymorpha. Over expression in E. coli results in proteins accumulating as insoluble inclusion bodies. Solubilization and refolding of the recombinant Insulin from the inclusion bodies requires use of chaotropic chemicals such as guanidine hydrochloride, urea, etc. and presence of traces of these chemicals in the final product even after extensive purification could be hazardous. Alternatively, proteins can be expressed in yeast system and secreted out into the medium at much higher levels in soluble form. However, levels of expression obtained in each yeast system differed from protein to protein for unknown reasons.
The two chains of Human Insulin are also being expressed separately using two different vectors and assembled together in-vitro after purification. Disulphide linkages between two chains is facilitated by chemical methods
STATEMENT OF THE DISCLOSURE
Accordingly, the present disclosure relates to a polynucleotide sequence as set forth in SEQ ID NO: 2; a polypeptide sequence as set forth in SEQ ID NO: 1; a method for obtaining recombinant insulin precursor molecule having polypeptide sequence as set forth in SEQ ID NO: 1, said method comprising steps of: a) synthesizing a polynucleotide sequence set forth in SEQ ID NO: 2 by combining 26 oligonucleotides of SEQ ID NOS: 3 to 28 by assembly PCR, and inserting the synthesized sequence in a vector, b) transforming a host cell with said vector followed by antibiotic screening host selection, and c) fermenting the selected transformed host cell and in-situ capturing of the insulin precursor molecule to obtain said precursor having polypeptide sequence as set forth in SEQ ID NO: 1; a method of downstream processing for in-situ capturing of protein precursor molecule during fermentation process, said method comprising steps of: a) simultaneous pumping of fermentation product obtained during fermentation into a hollow fibre harvesting system to obtain permeate and retentate, b) recycling of the retentate into the fermentor, and c) subjecting the permeate through ion-exchange chromatographic column followed by washing with TRIS elution buffer to obtain said protein precursor molecule; a method of downstream processing for in-situ conversion of protein precursor molecule into functional protein molecule, said method comprising step of: a) concentrating the precursor molecule through TFF Cassette and mixing the concentrate with organic solution to obtain retentate reaction mixture, b) subjecting the reaction mixture to incubation through TPCK trypsin immobilized column to obtain protein ester, and c) subjecting the ester to deblocking buffer followed by hydrophobic interaction chromatographic column to obtain said functional protein molecule; a method for obtaining recombinant insulin molecule from a precursor molecule having polypeptide sequence as set forth in SEQ ID NO: 1, said method comprising steps of: a) synthesizing a polynucleotide sequence set forth in SEQ ID NO: 2 by combining 26 oligonucleotides of SEQ ID NOS: 3 to 28 by assembly PCR, b) inserting the synthesized sequence in a vector and transforming a host cell with said vector followed by antibiotic screening host selection, c) fermenting the selected transformed host cell followed by downstream processing for in-situ capturing of the insulin precursor molecule, and d) in-situ conversion of insulin precursor molecule having polypeptide sequence as set forth in SEQ ID NO: 1 into said recombinant insulin molecule; a recombinant vector comprising the polynucleotide sequence set forth in SEQ ID NO: 2; and a recombinant host cell, transformed by introduction of a vector comprising polynucleotide sequence set forth in SEQ ID NO: 2;



BRIEF DESCRIPTION OF ACCOMPANYING FIGURES
FIG. 01: Flow chart of cloning process—construction of insert, preparation of vector for ligation, ligation of insert with vector, preparation of vector for Pichia transformation
FIG. 02: Agarose gel image of gene construct from Oligos by Assembly PCR.
The product of PCR is checked on 1% agarose gel in TAE buffer 1% agarose gel casting Loading−1.5 μl sucrose dye+5 μl-10 μl PCR product Marker−100 hp marker 0.8-1.0 μI+2 μl milliQ+1.5 dye PCR amplicon size is 483 and is seen on the gel around 500 bp.
FIG. 03: DNA sequencing profile with designed and sequenced codons and amino acid details showing the sequence comparison of bigtec4_AOX_1 (SEQ ID NO: 29) and IN_BIGTEC (SEQ ID NO: 30).
FIG. 04: SDS-PAGE (15%) image of fermentation samples during different stages of induction showing continuous increase of expression and secretion of Insulin precursor into the fermentation medium.
Lane 1: Standard PIP;
Lane 2: Protein molecular weight marker;
Lane 3: Fermentation sample at 0 hour of induction;
Lane 4: Fermentation sample at 6 hour of induction;
Lane 5: Fermentation sample at 12 hour of induction;
Lane 6: Fermentation sample at 24 hour of induction;
Lane 7: Fermentation sample at 30 hour of induction;
Lane 8: Fermentation sample at 36 hour of induction; and
Lane 9: Fermentation sample at 42 hour of induction.
FIG. 05: HPLC profile of Fermentation samples at different periods of induction. [quantity of insulin precursor Vs Time course of fermentation induction phase]. There is a progressive increase in the quantity of precursor from time to time of 20 induction period. The increase in quantity is correlated with size of peaks expressed as milli volts.
FIG. 06: Comparative HPLC profile of standard precursor and precursor in fermentation (final) sample, showing selective capturing of Insulin Precursor. The peak corresponding to standard obtained from protein concentration of 1 mg/mL. The peak corresponding to fermentation sample is obtained from 1:1 dilution of broth. 50% diluted FMN broth peak is more than that of standard precursor. It corresponds ≧2 g/L.
FIG. 07: HPLC profile of enzymatic conversion of Insulin Precursor (PIP) to Human Insulin butyl ester (Transpeptidation Product). Before Transpeptidation reaction, said PIP was loaded. After trypsin digestion and transpeptidation, the product (HI ester) is also loaded onto HPLC to confirm completion of the reaction. The mass balance is almost matched between precursor and its Transpeptidation product.
FIG. 08: The TP product is deblocked to obtain Human insulin and loaded onto HPLC to know the purity and profile. In terms of mass balance it is matching with PIP (precursor). Both PIP in FIG. 09 and HI in this figure are loaded at the same 5 concentration (1 mg/mL. (Purified Human Insulin HPLC profile as per British Pharmacopeia 2007).
FIG. 09: Diagram showing the flow chart of fermentation, in-situ harvesting and clarification of fermentation broth by a hollow fibre harvesting system with 0.2 μM 1 0 cassette. The cells retained after harvesting are directed back into fermenter along with fresh medium.
FIG. 10: Diagram shows the flowchart of concentration and in-situ capturing of human. insulin precursor in cation exchange chromatography column followed by in situ digestion and transeptidation in TPCK trypsin immobilized column for conversion into insulin butyl ester. Human insulin ester is deblocked and passed through HIC column to get purified human insulin.
FIG. 11: Codon Pair Optimization: Oligo assembled sequence (product of assembly PCR) having sense strand as set forth in SEQ ID NO: 31 and antisense strand as set forth in SEQ ID NO: 32.



DETAILED DESCRIPTION OF THE DISCLOSURE
The present disclosure relates to a polynucleotide sequence as set forth in SEQ ID NO: 2.
In an embodiment of the present disclosure, the polynucleotide encodes a fusion polypeptide comprising recombinant Human Insulin Precursor and signal peptide.
The present disclosure relates to a polypeptide sequence as set forth in SEQ ID NO: 1.
In an embodiment of the present disclosure, the polypeptide is a fusion polypeptide comprising recombinant Human Insulin Precursor and signal peptide.
In another embodiment of the present disclosure, the polypeptide sequence corresponds to polynucleotide sequence set forth in SEQ ID NO: 2, wherein the polynucleotide is subjected to post-transcriptional modification and codon optimization to obtain corresponding polypeptide of SEQ ID NO: 1.
The present disclosure relates to a method for obtaining recombinant insulin precursor molecule having polypeptide sequence as set forth in SEQ ID NO: 1, said method comprising steps of:

    
    
        a) synthesizing a polynucleotide sequence set forth in SEQ ID NO: 2 by combining 26 oligonucleotides of SEQ ID NOS: 3 to 28 by assembly PCR, and inserting the synthesized sequence in a vector,
        b) transforming a host cell with said vector followed by antibiotic screening host selection, and
        c) fermenting the selected transformed host cell and in-situ capturing of the insulin precursor molecule to obtain said precursor having polypeptide sequence as set forth in SEQ ID NO: 1.
    
    


In an embodiment of the present disclosure, the polypeptide is a fusion polypeptide comprising recombinant Human Insulin Precursor and signal peptide.
In another embodiment of the present disclosure, the synthesized polynucleotide and the vector are subjected to restriction enzyme digestion for insertion of the polynucleotide into the expression vector and wherein restriction enzymes are selected from a group comprising BamHI, NotI, SacI, BgIII and SacII or any combination thereof.
In yet another embodiment of the present disclosure, the vector is selected from a group comprising pPIC9K and pPICZα, preferably pPIC9K and wherein the host is selected from a group comprising Pichia pastoris, Pichia methanolica; Pichia guilliermondii and Pichia caribbica, preferably Pichia pastoris. 
In still another embodiment of the present disclosure, the in-situ capturing of the precursor molecule is carried out by hollow fibre harvesting system and ion-exchange chromatographic column to obtain said precursor.
The present disclosure relates to a method of downstream processing for in-situ capturing of protein precursor molecule during fermentation process, said method comprising steps of:

    
    
        a) simultaneous pumping of fermentation product obtained during fermentation into a hollow fibre harvesting system to obtain permeate and retentate,
        b) recycling of the retentate into the fermentor, and
        c) subjecting the permeate through ion-exchange chromatographic column followed by washing with TRIS elution buffer to obtain said protein precursor molecule.
    
    


In an embodiment of the present disclosure, the permeate comprise of clarified cell free broth and the retentate comprise of concentrated cells.
In another embodiment of the present disclosure, the wherein the retentate is recycled back to fermentor vessel along with fresh medium in the fermentor and the permeate is passed through the ion-exchange chromatographic column for capturing the protein precursor.
In yet another embodiment of the present disclosure, the protein precursor selectively binds to polymer matrix of the ion-exchange chromatographic column and is eluted with the elution buffer.
The present disclosure relates to a method of downstream processing for in-situ conversion of protein precursor molecule into functional protein molecule, said method comprising step of:

    
    
        a) concentrating the precursor molecule through TFF Cassette and mixing the concentrate with organic solution to obtain retentate reaction mixture,
        b) subjecting the reaction mixture to incubation through TPCK trypsin immobilized column to obtain protein ester, and
        c) subjecting the ester to deblocking buffer followed by hydrophobic interaction chromatographic column to obtain said functional protein molecule.
    
    


In an embodiment of the present disclosure, the precursor molecule is concentrated to a range of about 100 mg/ml to about 200 mg/ml.
In another embodiment of the present disclosure, the organic solution comprise of O-tert-Butyl-L-theronine tert-butyl ester acetate dissolved in 1:1 v/v dimethyl sulfoxide (DMSO): Methanol and the deblocking buffer comprise a combination of tryptophan and trifluoroacetic acid.
In another embodiment of the present disclosure, the TPCK column is equilibrated with a combination of CaCl2 and Acetic acid and the hydrophobic interaction chromatographic column is equilibrated with a combination of Acetic acid and Ammonium sulphate.
In another embodiment of the present disclosure, time for the incubation ranges from about 1.5 hrs to about 3.5 hrs and temperature for the incubation ranges from about 15° C. to about 25° C.
The present disclosure relates to a method for obtaining recombinant insulin molecule from a precursor molecule having polypeptide sequence as set forth in SEQ ID NO: 1, said method comprising steps of:

    
    
        a) synthesizing a polynucleotide sequence set forth in SEQ ID NO: 2 by combining 26 oligonucleotides of SEQ ID NOS: 3 to 28 by assembly PCR,
        b) inserting the synthesized sequence in a vector and transforming a host cell with said vector followed by antibiotic screening host selection,
        c) fermenting the selected transformed host cell followed by downstream processing for in-situ capturing of the insulin precursor molecule, and
        d) in-situ conversion of insulin precursor molecule having polypeptide sequence as set forth in SEQ ID NO: 1 into said recombinant insulin molecule.
    
    


In an embodiment of the present disclosure, the polypeptide is a fusion polypeptide comprising recombinant Human Insulin Precursor and signal peptide.
In another embodiment of the present disclosure, the synthesized polynucleotide and the vector are subjected to restriction enzyme digestion for insertion of the polynucleotide into the expression vector and wherein the restriction enzymes are selected from a group comprising BamHI, NotI, SacI, BgIII and SacII or any combination thereof.
In yet another embodiment of the present disclosure, the vector is selected from a group comprising pPIC9K and pPICZα, preferably pPIC9K and wherein the host is selected from a group comprising Pichia pastoris, Pichia methanolica, Pichia guilliermondii and Pichia caribbica, preferably Pichia pastoris. 
In still another embodiment of the present disclosure, the in-situ capturing of the precursor molecule is carried out by hollow fibre harvesting system and ion-exchange chromatographic column to obtain said precursor.
In still another embodiment of the present disclosure, the in-situ conversion of the precursor molecule is carried out by subjecting the precursor molecule to TFF Cassette and TPCK trypsin immobilized column to obtain protein ester.
In still another embodiment of the present disclosure, the protein ester is subjected to deblocking buffer followed by hydrophobic interaction chromatographic column to obtain said recombinant insulin molecule.
The present disclosure relates to a recombinant vector comprising polynucleotide sequence set forth in SEQ ID NO: 2.
In an embodiment of the present disclosure, the vector is selected from a group comprising pPIC9K and pPICZα, preferably pPIC9K.
The present disclosure relates to a recombinant host cell, transformed by introduction of a vector comprising polynucleotide sequence set forth in SEQ ID NO: 2.
In an embodiment of the present disclosure, the host is selected from a group comprising Pichia pastoris, Pichia methanolica, Pichia guilliermondii and Pichia caribbica, preferably Pichia pastoris and wherein the vector is selected from a group comprising pPIC9K and pPICZα, preferably pPIC9K.
The main object of the present disclosure is to de novo design and express the gene coding for “secretion signal and recombinant Insulin Precursor fusion protein” comprising the amino acid sequence as set forth in SEQ ID NO: 1.
The present disclosure relates to a method for obtaining recombinant human insulin, said method comprising the steps of:

    
    
        a) Designing and constructing an insulin precursor-signal peptide fusion protein coding gene;
        b) Ligating the precursor-signal peptide fusion protein coding gene to a vector;
        c) Obtaining multiple copies of the precursor-signal peptide fusion protein coding gene by transformation (electroporation) into a host.
        d) Fermentation of transformed host cell lines to obtain recombinant human insulin precursor; and
        e) Obtaining the recombinant human insulin by efficient downstream processing of the human insulin precursor,
    
    


In the present disclosure optimization of nucleotide sequence was done for enhanced expression and secretion of target protein into fermentation medium.
Optimization at multiple steps during clone construction, cloning, transformation, fermentation, downstream processing resulted in overall increase in yield, scalability and biological efficacy. Down-stream processing is improvised with in-situ capturing and in-situ conversion of precursor to final product to minimize the processing time, cost, manpower and conserve reagents.
In another embodiment of the present disclosure, said SEQ ID No. 2 is obtained by multiple stages in-silico optimization of nucleotide sequence based on “Codon-Pair Frequency” of highly expressed proteins in Pichia pastoris. The sequence was further tuned to enhance protein synthesis by mRNA secondary structure prediction and removing high melting stem loop structures, which enables un-restricted ribosome movement and high speed protein synthesis.
Codon Pair Optimization
Codon optimisation is a method of gene optimisation, where in the synthetic gene sequence is modified to match the “codon usage pattern” for a particular organism. Here, for a particular amino acid sequence, select “most frequently used codons” (from a list of degenarate codons for an aminoacid), by that organism. So that the aminoacid sequence remains same but with a different DNA sequence, matched for that organism.
However this does not consider the fact that codons are read by ribosomes in “pairs”, during protein synthesis. There are 2 codon binding site in ribosome, on adjacent places. Extensive analysis was done and a particular pattern was observed in which the “codon-pairs” are used by Pichia pastoris. So the construct DNA sequence was modified to match to this “codon-pair usage frequency”. This methodology is novel and never reported for gene expression optimisation. A proprietary in-house developed software was used for this exercise. By doing this gene optimization (FIG. 11), it was found that the expression level could be increased by approx 30% using the synthetic DNA construct shown in FIG. 11 (having sense strand SEQ ID NO: 31 and antisense strand SEQ ID NO: 32) over that of the non-optimized gene sequence.
In another embodiment of the present disclosure, the whole gene sequence i.e. Insulin Precursor and secretion signal was subjected to optimization together, as it is expressed as a single chain protein in the expression host.
In yet another embodiment of the present disclosure, said precursor is constructed with about 26 oligonucleotides coding for Insulin precursor-Signal peptide fusion protein.
In yet another embodiment of the present disclosure, said vector is selected from a group comprising pPIC9K and pPICZ α, preferably pPIC9K
In still another embodiment of the present disclosure, said cloning is carried out at downstream of AOX1 promoter in pPIC9K vector.
In still another embodiment of the present disclosure, said host is selected from a group comprising Pichia pastoris, Pichia methanolica, Pichia guilliermondii and Pichia caribbica, preferably Pichia pastoris. 
In still another embodiment of the present disclosure, said cloning was carried out by simultaneous multiple gene insertions and direct selection using an antibiotic to get high copy number of gene into the host
In still another embodiment of the present disclosure, said fermentation is carried out in a modified low salt minimal medium at optimal temperature range, aeration, cell densities and feeding, which enables high level expression and easy downstream processing.
In still another embodiment of the present disclosure, fermentation process and harvesting process are coupled. It involves a hollow fibre harvesting module is connected to fermenter for in-situ filtration of broth during harvesting. The culture from fermenter is pumped to a hollow fibre cassette to separate cell free broth from the cells. The cells after filtration are recycled back to fermenter vessel along with medium to maintain culture volume and promote normal growth of culture.
In still another embodiment of the present disclosure capturing of human insulin precursor is coupled with trypsin digestion and transpeptidation in an immobilized trypsin column. It involves binding of insulin precursor in cell free broth from hollow fibre filtration system to a chromatography column packed with high binding capacity synthetic resin. The unbound is again channeled back into fermenter along with fresh medium.
The bound protein is eluted and further channeled into a column packed with TPCK trypsin immobilized to matrix. On the way to Trypsin column the eluted precursor is mixed with necessary buffers and desired PH. The precursor is converted into insulin ester by tryptic digestion and transpeptidation in the column. Then the insulin ester is eluted from column and deblocked to convert into human insulin and lyophilized. Finally the human insulin is polished to highest purity by reverse phase chromatography.
In still another embodiment of the present disclosure, said fermentation medium has a pH ranging from about 4.0-5.0, preferably about 4.75 during initial phase of fermentation; about 4.0-5.0, preferably about 4.80 during glycerol phase and about 4.0-5.0, preferably about 4.95 during induction phase.
In still another embodiment of the present disclosure, said temperature at fermentation ranges from about 29-30° C., preferably about 30.0° C. for batch phase; about 29-30° C., preferably about 29.5° C. for glycerol fed batch; and about 27-29° C., preferably about 28.0° C. for induction phase with methanol.
In still another embodiment of the present disclosure, said aeration at fermentation ranges from about 0.5-1.5 VVM pure air, preferably 1 VVM pure air for batch phase; about 0.5-1.5 VVM air:oxygen, preferably about 1.0 VVM air:oxygen (about 90:10) for glycerol batch; and about 1.5 VVM air:oxygen ratio begins at about 85:15 and ends at about 40:60 with an increment/decrement of about 5 at about every 5 hours for methanol batch (induction phase).
In still another embodiment of the present disclosure, during fermentation glycerol feeding is carried out to promote high cell density growth before induction and is continued until cell density (OD600) reaches about 500. Then methanol is fed exponentially to promote increased expression of target protein.
In the present disclosure, a synthetic gene having modified nucleotide sequences and coding for a gene comprising the Mat-α secretion signal, spacer, and the insulin precursor was designed de novo. Extensive bioinformatics analysis was used to arrive at a novel coding sequence, based on nucleotide patterns from highly expressed proteins in Pichia pastoris. The synthetic gene (482 bp) was constructed by synthesizing 26 oligonucleotides and combining them by assembly PCR.
Pichia expression system is known for its very high levels of expression, using a methanol inducible promoter. Proteins can be expressed as secretory proteins and therefore purification of the same becomes simple. The doubling time of the strain, ease of handling, minimal growth requirements, availability of convenient vectors, host systems and selection methodologies make Pichia pastoris an ideal and attractive system for study. High cell densities are achievable in minimal mineral media and the ease of induced expression of proteins adds to the convenience of using this system for recombinant protein expression.
The insulin precursor fusion protein gene obtained by assembly PCR was confirmed by DNA sequencing (FIG. 03), where bigtec4 AOX (SEQ ID NO: 29) represents the sequencing data and IN_BIGTEC w (SEQ ID NO: 30) represents the expected insulin precursor fusion protein sequence. The insulin precursor fusion protein was then cloned into Pichia pastoris expression vector pPIC9K. The vector after linearization transformed into GS 115 strain of Pichia by electroporation. The expression cassette was integrated into the Pichia host system by homologous recombination. Clones harboring high copy number inserts were picked by antibiotic screening. Clones showing maximum resistance to the antibiotic genticin (G418) were picked and screened for their ability to express and secrete the Insulin precursor into the culture medium. Promising clones were further evaluated by 7 liter capacity fermenter. The fermentation yield of insulin precursor is around 1.5 gm/liter. This can be further increased through additional optimization of the process.
The secreted insulin precursor was captured from the broth, purified and enzymatically modified to obtain Human Insulin. Biological activity of the final product in terms of regulating blood glucose has been established in mice and rats and found to be comparable with commercially available therapeutic recombinant Human Insulin formulations.
Thus, the process has been optimized at multiple steps, which has cumulative effects and resulted in increased yields. To name some of the major parameters optimized in this system, the “codon-pair sequence” of the entire coding region, stability of mRNA, multiple copy insertions, optimized media components and growth and induction parameters.
Integration of the expression cassette into the host genome ensures performance and stability of the recombinant strain after repeated sub-culturing. The possibility of multi-copy gene expression in the Pichia system makes it feasible to exploit the expression, folding and secretory capacities of the cells to the maximum. Expression of Human Insulin as a single chain protein enables proper disulphide bridge formation resulting in proper folding leading to a molecule that is biologically active. Further, the process of the present disclosure in which in-vitro processing and use of hazardous chemicals are kept to a minimum is ideally suited for scale-up and commercial production of recombinant Human Insulin. The fermentation yields are significantly better than reported literature and unreported market figures.
In still another embodiment of the present disclosure, fermentation yields are high.
In still another embodiment of the present disclosure, use of high efficiency synthetic polymeric resins for capturing and purification process resulted in enhanced recovery and purity with minimal unit operations, as depicted in examples given below. Use of synthetic resins enhanced the robustness of the process, stringent sanitation protocols and ease of scale-up & overall techno-economic feasibility of the process.
The disclosure is further elaborated with the help of following examples. However, these examples should not be construed to limit the scope of disclosure.
EXAMPLES
Example 1
Gene Construction and Clone Generation
26 Oligonucleotides [as given in SEQ 3] coding for the fusion protein “Mat-α.-Insulin Precursor” fusion protein were designed and custom synthesized. These oligonucleotides were assembled by assembly PCR. The PCR product was double digested with restriction enzymes BamHI and NotI and ligated into similarly processed vector pPIC9K using T4 DNA ligase.
Assembly PCR
Master stocks of oligos resuspended in water and stored in original vials of Bioserve and kept in −20° C. Resuspension of oligos result in 100 pm/μl concentration of each oligo (1 μM=1 p mole/μl). Assembly PCR require 0.1 μM concentration of each oligo. 10 μl of each oligo is diluted to 200 μl (20 times) to give 5 μM solution. 1 μl of each diluted oligo is added to PCR master mix before assembly PCR.







TABLE 1





Reaction Mix; using Phusion High Fidelity DNA Polymerase (NEB) Kit


PCR MIX (50 μl)


















5X rxn buffer (Hi Fidelity)
10.0 μl



10 mM dNTPs
 1.0 μl



Oligos
  26 μl



Taq
0.5 μl (1 unit/μl)



MilliQ
12.5 μl



Total volume
  50 μl

















TABLE 2





PCR Program for 1st Assembly PCR


PCR MIX (50 μl)





















Step 1
Initial
98° C.
30 sec
1 cycle




denaturation



Step 2
Denaturation
98° C.
10 sec
30 cycles 



Step 3
Annealing
57° C.
30 sec



Step 4
Extension
72° C.
30 sec



Repeat
30 times



2, 3 & 4



Step 5
Final
72° C.
  7 min
1 cycle




extension



Step 6
Final Hold
 4° C.
α










The product of assembly PCR is used as template for 2nd PCR. Product quantity is not increased in exponential way, hence it is not checked on gel electrophoresis. The product from assembly PCR is directly used as template for second PCR where the assembled gene is amplified by using AOX1 primers.







TABLE 3





Reaction Mix for amplification of Clone (2nd PCR)


PCR MIX (NEB) (20 μl)


















5X rxn buffer (Hi Fidelity)
4.0 μl



10 mM dNTP mix
0.4 μl



AOX1 Primer F (100 μM)
0.2 μl



AOX1 Primer R (100 μM)
0.2 μl



Template (Assembly PCR mix)
1.2 μl



Phusion Taq (NEB)
0.2 μl



MilliQ
13.8 μl 



Total volume
 20 μl

















TABLE 4





PCR Program for 2nd PCR


PCR MIX (50 μl)





















Step 1
Initial
95° C.
30 sec
1 cycle




denaturation



Step 2
Denaturation
98° C.
10 sec
30 cycles 



Step 3
Annealing
57° C.
30 sec



Step 4
Extension
72° C.
30 sec



Repeat
30 times



2, 3 & 4



Step 5
Final
72° C.
  7 min
1 cycle




extension



Step 6
Final Hold
 4° C.
α










Resultant Sequence
Amplicon obtained from second PCR and size of the amplicon is matching with the size  ̃500 bp (482 bp) as shown in FIG. 02. The product of second PCR is extracted from agarose gel for sequencing.
The ligation mix i.e. pPIC9K vector containing the ligated gene of interest (insulin precursor+mat-α secretion signal) is used for transformation into chemically competent TOP 10 E. coli strain (FIG. 01). CaCl2 was used for competent cell preparation. Transformation was done by heat shock method. Transformation mix was plated on LB medium containing Ampicillin in order to select transformed colonies. Colonies were obtained after incubation of plates at 37° C. for 12-14 h. Glycerol stocks of transformed cells were prepared and stored at −70° C. Plasmid from E. coli is prepared by the protocol from Promega Kit (Wizard plus SV minipreps DNA purification system). Recombinant pPIC9K plasmid vector is linearized with restriction enzymes SacI/BglII/salI, purified, quantified and used for transformation into Pichia pastoris. Approximately 10 μg of the linearized plasmid DNA with insert were used for electroporation of electrocompetent host cells. The specifications used for electroporation are 760 Volts/5 milli seconds in 2 mm cuvette.
The transformation mixture was incubated in 1M sorbitol for 30 min for cells to recover and further incubated in liquid regeneration media for 4 hours at 30° C. with shaking. Cells were then plated on to minimal media lacking histidine and containing antibiotic G418. The His+ colonies that grew on these plates were screened by PCR by using AOX1 primers PCR positive cell lines are plated on fresh RD medium 20 plates for further screening of high copy number lines.
Clones containing multiple copies of the gene inserted into the genome were further screened using higher concentrations of antibiotic G418. Colonies resistant to more than 4 mg G418 are considered to contain more than twelve copies of the gene. Such colonies were selected, grown on YPD medium and maintained as glycerol stock at −70° C.
Example 2
Expression Screening
Transformed colonies growing on RD plates with 4 mg G418 were screened for expression by shake flask cultures according to the Invitrogen's Pichia expression protocols. More than 100 such colonies were screened to identify few promising clones.
Each colony to be screened was grown in 5 ml YPD in a culture tube by incubating at 30° C./230 rpm/24 hrs. The seed (1 ml) is inoculated to 50 ml BMG (buffered minimum glycerol medium) in 250 ml Erlenmeyer flask and incubated at 30° C./220 rpm/24 hrs. Cells were harvested by centrifuging at 2000 g/5 minutes at room temperature. Supernatant was decanted and the cell pellet was resuspended in 25 ml BMM (buffered minimum methanol medium) in 150 ml baffled flasks and then allowed to grow at 30° C./200 rpm for 3 days. The culture was induced with methanol to a final concentration of 1.0% at every 24 hrs. Samples were taken at 24 hr intervals and analyzed by HPLC and SDS-PAGE.
Colonies showing good expression were made into glycerol stocks for further evaluation at fermentation level.
Example 3
High Cell Density Fermentation in Low Salt Minimal Media [LSMM]
Fermentation was carried in in-situ autoclavable automated vessel (BioFlo 415, NBS) of 7 liter capacity. All parameters like agitation, gas flow rates, feeding, pH adjustments, antifoam were controlled by PID controller.
The fermentation medium used is a Low Salt Minimal Medium (LSMM) supplemented with trace metal salts solution (PTM4) and Biotin, as follows:

    
    
        Phosphoric acid=26.7 ml
        CaSO4.2H2O=0.465 gm
        K2SO4=9.1 gm
        MgSO4.7H2O=7.45 gm
        KOH=4.13 gm
        Glycerol=50 ml
        [All quantities per Liter of medium]
    
    


To promote rapid growth and high cell density yield in fermenter, glycerol stock is inoculated and grown in YPD medium by shake flask culture for 18-20 hrs at 220 rpm/30° C. till OD600 reaches 10-12. The first seed is again inoculated onto YPG medium and grown at above mentioned conditions. When culture reached log phase (around 20 hrs) with OD600 around 25-30, the cells are harvested at 1500 g/5 min and suspended in autoclaved milliQ water. Then cells are inoculated into basal salt medium in fermenter upto OD600 of 5.0.
Batch Phase:
The fermenter medium pH adjusted to 4.75 before inoculation to avoid precipitation of medium if any. Dissolved oxygen (DO) probe is also calibrated before inoculation. Trace metal solution of 8% added to the vessel before and after inoculation at fixed intervals. Temperature of the culture is maintained at 30° C. Vessel aeration was maintained as 1.0 VVM pure air. Initial batch phase last for 18 hrs until OD600 reaches 120-150 with an indication of DO shoot up.
Glycerol Fed Batch:
The glycerol fed batch started with feeding of 50% glycerol containing 12% trace metal solution on exponential feed rate to achieve high cell density before induction. Temperature and pH were maintained at 29.5° C. and 4.80. Vessel aeration was maintained as 1.0 VVM air and oxygen in a ratio of 9:1.
Methanol Batch:
Induction of Insulin Precursor (IP) was started by feeding 100% methanol containing 12% PTM4 trace metal solution. Initial methanol feed was given as spikes until culture gets adapted, subsequently switched on to exponential feed. The DO spike method was used to determine ramp of methanol feed. Methanol feed for Mut+ and Muts clones were based on Stratton et al., (Pichia protocols, Methods in Molecular Biology, Vol. 103). Residual methanol in the vessel is continuously monitored using an in-house designed methanol probe and sensor connected to the vessel. Consumption of methanol signals increase in vessel temperature which is maintained at 28.5° C. through out methanol fed batch. Medium pH was maintained at 4.95. Vessel aeration was maintained as 1.5 VVM due to high density with air and oxygen in a ratio begins at 85:15 and ends at 40:60 with an increment/decrement of 5 at every 5 hrs. During induction phase samples were analyzed at 6-hours interval to check growth, induction and contamination if any. Induced protein secreted into broth is analyzed by HPLC using 0.1% TFA/Acetonitrile solvents in C18 column. HPLC samples at 6 hour intervals showed progressive increase in protein level (FIG. 05). Fermentation samples were also analyzed by electrophoresis (SDS-PAGE) to assess the expression of insulin precursor and its increase with induction time (FIG. 04).
Fermentation samples during induction phase are periodically checked to know any protease activity by azocasein assay. Fermentation conditions were optimized for high level expression of insulin precursor which is more than 65% of total proteins present in the final sample. Results showed that the total protein present in the final sample is ranging from 2.3 g/L with insulin precursor being 1.5 g/L.
Example 4
Harvesting of Culture and In-Situ Capturing Insulin Precursor
When the induced culture is more than 36 hrs old, it is pumped from fermenter into hollow fiber harvesting system with 0.2μ cartridge via a peristaltic pump. The permeate contains clarified cell free broth and retentate contains concentrated cells. The retentate with cells is recycled back to fermenter vessel along with fresh medium to maintain normal growth and volume (FIG. 09).
The permeate containing clarified cell free broth is passed through column packed with strong cation exchanger resin, SP sepharose (methacrylic polymer with sulphopropyl functional derivatization—GigaCap S 650, Toyopearl) at pH 3.0. for protein capturing.
The column after protein binding is washed with 2 column volumes of pH 3.0 Tris buffer. The Insulin precursor selectively binds to the polymer matrix and is eluted with Tris buffer at pH 8.0. The chromatographic purity of insulin precursor is around 75% as checked on HPLC (FIG. 06) and step yield is around 90% w/w.
Example 5
In-Situ Conversion of Insulin Precursor to Human Insulin
The PIP obtained in Example 4 is converted to Human Insulin via trypsin mediated digestion and transpeptidation followed by deblocking. Insulin precursor eluted from ion exchange column is passed through 1 kda MWCO TFF cassette and concentrated to 100-200 mg/ml and its pH is adjusted to 7.3 with 1 N HCl. The concentrated PIP is mixed with O-tert-Butyl-L-theronine tert-butyl ester acetate dissolved in 1:1 v/v dimethyl sulfoxide (DMSO):Methanol. The reaction mixture is passed through TPCK-treated trypsin immobilized column (25 ml XK column with cooling jacket) equilibrated with 50 mM CaCl2 and 0.5% acetic acid. When reaction mixture is completely loaded into the column, the column is closed for 2-3 hrs to permit incubation of reaction contents and column temperature is maintained at 20° C. Then the insulin precursor converted to Insulin butyl ester is eluted and checked by HPLC (FIG. 07).
After the completion of reaction in-situ, the eluted from TPCK trypsin column is mixed with deblocking buffer i.e. 0.1% tryptophan in trifluoroacetic acid (TFA), incubated for 20 min at room temperature and passed into into hydrophobic interaction chromatography column (PPG 650 M Toyopearl) which is equilibrated with 100 mM Acetic acid having 0.8 M Ammonium sulphate for binding. The column is then washed with 100 mM Acetic acid with 0.4 M Ammonium sulphate, for 2 column volumes. Finally the bound Insulin was eluted with 100 mM acetic acid and lyophilized.
Step yield=75%
Chromatographic purity=85%
Example 6
Final Polishing of Human Insulin
Human Insulin obtained in example 5 is further purified from small molecular weight impurities and salts by size exclusion chromatography column packed with sepahdex G25 matrix and checked by HPLC and lyophilized to powder form.
Step yield=85%
Chromatographic purity=98.5%
The purified Human Insulin meets the quality norms as per monograph of recombinant Human Insulin under British Pharmacopoeia 2007, by HPLC analysis (FIG. 08). In addition, the purity and activity of the purified human insulin obtained from methods in accordance with embodiments disclosed herein was verified by comparison to SDS PAGE (15%) and western blot of a commercial human insulin preparation (results not shown).

## Claims

1. A method of downstream processing for in-situ conversion of a protein precursor molecule having the amino acid sequence set forth in SEQ ID NO: 1 into a functional protein molecule, said method comprising:
a) concentrating the protein precursor molecule through a tangential flow filtration cassette to obtain a concentrate of the protein precursor molecule and mixing the concentrate with an organic solution to obtain a retentate reaction mixture,
b) incubating the retentate reaction mixture in a tosyl phenylalanyl chloromethyl ketone trypsin immobilized column for a period of time, and then passing the retentate reaction mixture through the tosyl phenylalanyl chloromethyl ketone trypsin immobilized column to obtain a protein ester, and
c) mixing the protein ester with a deblocking buffer and purifying the protein ester using a hydrophobic interaction chromatographic column and a size exclusion chromatographic column to obtain the functional protein molecule having purity of at least 98.5%.

2. The method as claimed in claim 1, wherein the protein precursor molecule is concentrated to a range of about 100 mg/ml to about 200 mg/ml.

3. The method as claimed in claim 1, wherein the organic solution comprises O-tert-Butyl-L-threonine tert-butyl ester acetate dissolved in 1:1 v/v dimethyl sulfoxide:Methanol, and wherein the deblocking buffer comprises a combination of tryptophan and trifluoroacetic acid.

4. The method as claimed in claim 1, wherein the tosyl phenylalanyl chloromethyl ketone trypsin immobilized column is equilibrated with a combination of CaCl2 and Acetic acid, and wherein the hydrophobic interaction chromatographic column is equilibrated with a combination of Acetic acid and Ammonium sulphate.

5. The method as claimed in claim 1, wherein the period of time for the incubation ranges from about 1.5 hrs to about 3.5 hrs and temperature for the incubation ranges from about 15° C. to about 25° C.