FIELD OF THE INVENTION:
The present invention relates to a field of Recombinant DNA therapeutics. It involves the production of recombinant human insulin in pichia pastoris by optimization of codons in pichia expression system and the process parameters. Optimization at multiple steps of the process resulted in increased yield.
BACKGROUND OF THE INVENTION:
Earlier, insulin was purified from animal sources (bovine and porcine), which often resulted in some undesirable immune and hypersensitive reactions in diabetics when administered continuously for long 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 human insulin was expressed in yeast systems like Saccharomyces cerevisiae and patented, attempts to produce a commercially viable therapeutic product met with little success.
Human Insulin is currently available in the market from at least three different systems viz. E. coli, Pichia pastoris and Hansenuela polymorpha. Over expression in E. coli results in proteins accumulating as inclusion bodies in an insoluble form. 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 reasons still not well understood.
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
Pichia expression system is known for its very high levels of expression. Proteins can be expressed as secretory proteins and therefore purification of the same becomes more easy especially when the cells are grown in minimal mineral media. 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 add to the convenience of using this system for recombinant protein expression.
In the present invention, Synthetic Porcine Insulin Precursor (PiP) gene was constructed using eight different oligonucleotides. The gene was cloned into a Pichia Pastoris expression vector and the DNA sequence of the insert and its fusion were confirmed by sequencing. 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 G418 were picked and screened for its performance in terms of its ability to express and secrete the insulin precursor into the culture medium. Promising clones were further evaluated by fermentation at 4L bench scale. The precursor was captured from the broth, purified and enzymatically modified to obtain human insulin with yields up to 1.1 gm/litre which can be further increased through additional optimization of the process. 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 that of commercially available therapeutic human insulin.
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 GC content of the coding region, the N-terminal spacer sequence, the C-peptide region connecting the B and A chains, 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, our process 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.
OBJECTS OF THE INVENTION:
The main object of the present invention is to obtain recombinant human insulin comprising the amino acid sequence as set forth in SEQ ID NO: 1.
Another main object of the present invention is to obtain recombinant human insulin comprising the amino acid sequence as set forth in SEQ ID NO: 2.
Yet another object of the present invention is to develop a method for obtaining recombinant human insulin comprising the amino acid sequence as set forth in SEQ ID Nos. 1 and 2.
Still another object of the present invention is to produce recombinant human insulin in pichia pastoris for therapeutic use.
Still another object of the present invention is to obtain recombinant human insulin by optimization of codons in Pichia expression system.
Still another object of the present invention is to obtain recombinant human insulin by optimization of parameters during fermentation of Pichia.
Still another object of the present invention is to obtain high fermentation yields.
STATEMENT OF THE INVENTION:
Accordingly, the present invention relate to recombinant human insulin comprising the amino acid sequence as set forth in SEQ ID NO: 1; recombinant human insulin comprising the amino acid sequence as set forth in SEQ ID NO: 2 and a method for obtaining recombinant human insulin comprising the amino acid sequence as set forth in SEQ ID Nos. 1 and 2, said method comprising steps of: a) constructing an insulin precursor gene; b) ligating the precursor gene into a vector; and c) cloning the ligated precursor gene into a host, followed by fermentation to obtain recombinant human insulin.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS:
Fig. 1: 15% SDS PAGE showing insulin intermediates
Lane l:HiOMe
Lane2:DesB30
Lane 3: PIP
Lane 4: Standard Insulin
Fig. 2: HPLC Data for insulin intermediates
DETAILED DESCRIPTION OF THE INVENTION;
The present invention relates to recombinant human insulin comprising the amino acid sequence as set forth in SEQ ID NO: 1.
In another embodiment of the present invention, said SEQ ID No. 1 is obtained by optimization of codons in Pichia species.
The present invention also relates to recombinant human insulin comprising the amino acid sequence as set forth in SEQ ID NO: 2.
In another embodiment of the present invention, said SEQ ID No. 2 is obtained by optimization of codons in Pichia species.
The present invention also relates to a method for obtaining recombinant human insulin comprising the amino acid sequence as set forth in SEQ ID Nos. 1 and 2, said method comprising steps of:
a) constructing an insulin precursor gene;
b) ligating the precursor gene into a vector; and
c) cloning the ligated precursor gene into a host, followed by fermentation to obtain recombinant human insulin.
In another embodiment of the present invention, said insulin precursor is selected from a group comprising porcine insulin precursor and bovine insulin precursor, preferably porcine insulin precursor.
In yet another embodiment of the present invention, said precursor is constructed with about eight oligonucleotides coding for porcine insulin precursor.

In still another embodiment of the present invention, said vector is selected from a group comprising pPIC9K and pPICZoc, preferably pPIC9K.
In still another embodiment of the present invention, said cloning is carried out at downstream of AOX1 promoter and Mating Factor Alpha (MFa) signal sequences.
In still another embodiment of the present invention, 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 invention, said fermentation is carried out in a suitable medium contained in a modified vessel at a suitable temperature range, aeration, cell densities and feeding.
In still another embodiment of the present invention, said 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 invention, said modified vessel comprises methanol probe and sensor to monitor residual methanol and methanol feeding rate; modified air sparger to increase efficiency of aeration and oxygen transfer rate (OTR), aeration delivery pores in sparger are relocated to top side and air bubbles are made to hit impeller directly.
In still another embodiment of the present invention, said temperature ranges from about 29 - 31°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 invention, said aeration ranges from about 0.5 -1.5 VVM pure air, preferably 1VVM 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 invention, glycerol feeding is carried out to promote high cell density before induction and is continued until cell density (OD600) reaches about 500; methanol is fed exponentially to promote increased expression of target protein.
In still another embodiment of the present invention, said method is optimized with respect to various parameters selected from a group comprising increasing GC content of coding region, N-terminal spacer sequence, C-peptide region connecting B and A chains of insulin, multiple copy insertions, optimized media components and growth and induction parameters, or a combination thereof.
In still another embodiment of the present invention, said N-terminal spacer sequence is selected from a group comprising a di-peptide KR and long spacers KREAEA, KREEAEAEAEPK or a combination thereof.
In still another embodiment of the present invention, said C-peptide region is selected from a group comprising KR and AAK or a combination thereof.
In still another embodiment of the present invention, incorporation of an aromatic amino acid in C-peptide EWK improves folding stability of insulin precursor.
The present invention relates to production of recombinant human insulin in pichia pas tor is for therapeutic use.
In still another embodiment of the present invention, recombinant human insulin is produced by optimization of codons in Pichia expression system. In still another embodiment of the present invention, recombinant human insulin is produced by optimization of parameters during fermentation of Pichia.
In still another embodiment of the present invention, fermentation yields are high.
In the present invention, Synthetic Porcine Insulin Precursor (PiP) gene was constructed using eight different oligonucleotides. The gene was cloned into a Pichia

Pastoris expression vector and the DNA sequence of the insert and its fusion were confirmed by sequencing. 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 G418 were picked and screened for its performance in terms of its ability to express and secrete the insulin precursor into the culture medium. Promising clones were further evaluated by fermentation at 4L bench scale. The precursor was captured from the broth, purified and enzymatically modified to obtain human insulin with yields up to 1.1 gm/litre which can be further increased through additional optimization of the process. 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 that of commercially available therapeutic human insulin.
The present invention relates to production of recombinant human insulin in Pichia pastoris by optimization of codons in pichia expression system and the process parameters. The parameters optimized include GC content of the coding region, the N-terminal spacer sequence, the C-peptide region connecting the B and A chains, multiple copy insertions, optimized media components and growth and induction parameters. Thus, the process has been optimized at multiple steps, which has cumulative effects and resulted in increased yields.
The invention is further elaborated with the help of following examples. However, these examples should not be construed to limit the scope of invention.
Examples:
Example 1: Gene construction & clone generation
Eight Oligonucleotides coding for Porcine Insulin Precursor (PIP) were designed and custom synthesized. These Oligos were annealed and ligated into a cloning vector and the sequence of the insert was confirmed by sequencing. The insert was then excised and cloned into a Pichia pastoris secretory expression vector pPIC9K at the downstream of the AOX1 promoter and Mating Factor Alpha (MFa) signal sequences, which enabled the secretion of PIP into the culture medium. The coding region contained optimized codons for Pichia pastoris with increased GC content. The

recombinant plasmid was isolated and purified from E. coli transformants and the insert in the vector was confirmed by sequencing.
Other parameters optimized apart from increasing GC-content include N-terminal spacer sequence, the C-peptide region connecting the B and A chains, multiple copy insertions, optimized media components and growth and induction parameters. Thus, the process has been optimized at multiple steps, which has cumulative effects and resulted in increased yields.
In some of the clones, the N-terminal spacer sequence was kept as short as a di-peptide, KR. While in some, long spacers KREAEA, and KREEAEAEAEPK rich in hydrophilic amino acids were incorporated. A short C-peptide KR or AAK connected B & A chains. Folding stability of the precursor was improved by incorporating an aromatic amino acid in the C-peptide viz. EWK. Comparative analyses were done for these clones both in shake-flasks as well as in fermentor scale. The effects of having a single copy of these genes as in pPICZa vector and multiple copies using pPIC9K vectors were compared both in KM71 and GS115 strains oiPichiapastoris.
Recombinant pPIC9K plasmid was linearized with Sacl/Bglll, cleaned, estimated and used for transformation into Pichia pastoris. Approximately 20 ug of the cut DNA were used for each transformation. Spheroplast method of transformation was preferred over the lithium chloride method in order to obtain multiple insertions. Expression cassette was inserted into the host genome by homologous recombination thereby stably maintaining the insert in the clones. Copy numbers of the gene in each transformant depends upon the number of insertions that had taken place during the transformation. The transformants were plated on minimal media lacking histidine and the His+ colonies that grew on these plates were picked and grown on fresh plates for further screening of the colonies.
Clones containing multiple copies of the gene inserted into the genome were screened using antibiotic G418. Colonies resistant to more than 6mg G418 are considered to contain more than twelve copies of the gene. Such colonies were selected, grown and maintained at -70°C as glycerol stock.

Example 2: Shake flask experiments
Transformed colonies after G418 selection are plated onto YPD plates. These colonies were then screened for expression cloned gene by shake flask cultures according to the Invitrogen's Pichia Expression Kit. More than 3000 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 / 230rpm / 24hrs. The seed is transferred to 20 ml YPD in 100ml Erlenmeyer flask and incubated at 30°C / 230rpm / 24hrs. Cells were harvested by centrifuging at 8000rpm for 10 minutes at room temperature. Supernatant was decanted and the cell pellet was resuspended in 75ml BMG (buffered minimum glycerol medium) to a final OD6oonm of 2.0. Cells were then allowed to grow at 30°C / 230rpm. After the depletion of glycerol in the culture medium (i.e.after 18-20hrs), the culture was induced with 1.0% methanol at every 24hrs to maintain a fed-batch induction for 72hrs. Growth and induction was monitored every 24hrs. Total protein content in the supernatant was quantified by Bradford colorimetric method, RP-HPLC and/or by gel electrophoresis (SDS-PAGE).
Colonies showing good expression were then picked, streaked and stored separately for further evaluation at fermentation scales.
Example 3: High cell density fermentation
Fermentation was carried in in-situ autoclavable automated vessel (BioFlow 415, NBS) of 7 litre capacity. The vessel is controlled by touch screen monitor for calibration and adjusting all parameters like agitation, gas flow rates, feeding, pH adjustments, antifoam. The fermentation process is optimized by making some changes in the vessel like modification of air sprger, modification of impeller, attachment of methanol probe along with methanol sensor to the vessel.
The fermentation medium used is Basal Salt Medium (BSM) supplemented with tracemetal salts solution (6%) and biotin. 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 1500g/5min and suspended in autoclaved milliQ water. Then cells are inoculated into basal salt medium in fermenter upto ODtoo of 3.0.
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. Glycerol batch last for 4 - 6 hours until OD600 reaches 500.
Methanol batch: Induction of PIP was started by feeding 100% methanol containing 12% 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 methanol 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. 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 which is analyzed by HPLC using acetonitrile / ammonium sulphate solvents in CI8 column. Samples were also analyzed by SDS-PAGE to assess the expression of PIP and content of host proteins. Induction was

carried for 48-50 hrs until cell OD60o reaches 1200 - 1500. Then the batch is terminated and cells are harvested by centrifugation. The cell free broth is clarified by filtration through 0.22u membrane to remove suspended particles and then used for purification of secreted PIP.
Fermentation conditions were optimized for high level expression of PIP which is more than 85% of total proteins present in the terminated sample. Results showed that the total protein present in the final sample is ranging from 1.5 to 1.75g/L with PIP being 1.25 to 1.5g/L. Sequence data for different constructs, gel pictures and HPLC profile of fermentation batches are included.
Example 4: Down stream processing
The clarified broth from fermentation batch contain high molecular weight host proteins and PIP bound pigment. The broth is diluted thrice with MilliQ water and passed through Millipore TFF system (Pellicon) fixed with lOkda , 0.1m2 NMWC membrane using peristaltic pump. The protein in the broth is concentrated from 1.5 mg/mL to 7.5mg/mL. The retentate and permeate were checked by HPLC as well as SDS-PAGE. The lOkda permeate is showing traces of PIP which was again concentrated using 3kda membrane. The retentate of 3kda showing around 4-5mg/mL PIP with elimination of most of the host proteins. The retentate of lOkda is further involved in purification of PIP, where as the retentate of 3kda is lyophilized and used for trypsin digestion and transeptidation.
Purification of PIP: The PIP in 3kda and lOkda retentates is subjected to ion exchange chromatography using CM Sepharose (Fast Flow) in acetate buffer pH 4.5 using gradient with 0.5 M NaCl. The fractions containing PIP are pooled, dialyzed to remove salt and then lyophilized. Thus pure PIP is sequestered from high molecular weight impurities and pigment. The PIP in lyophilized form is >95% pure and showed rhombohedral crystals.
Conversion of PIP to human Insulin: Human insulin from Purified, lyophilized PIP was obtained by a combined reaction called trypsin digestion and transpeptidation. The lyophilized PIP was dissolved in Tris buffer and treated with TPCK treated trypsin

(without chymotrypsin activity) at 37°C for 1 hr. The reaction was stopped by lowering the pH. Crude digestion mixture forms an intermediate namely DES B30 which is mostly converted into methylated human insulin (HIOMe) in presence of threonine methyl ester (TME) by a coupling reaction in presence of Tris buffered DMSO : 1,4 Butanediol (1:2). The reaction mixture is then precipitated using ten volumes of ice-cold acetone acidified with 0.1% HC1. The precipitate collected by centrifugation was washed once with acetone, dried and dissolved in 1.0M acetic acid. Trypsin and TME in the mixture were removed by Sephadex G-50 gel filtration in an acidic medium. The fraction corresponding to HIOMe was collected, diluted with water and lyophilized to obtain the pure form.
Human Insulin was obtained from HIOMe by deblocking at 25°C and pH 10.5. The conversion was confirmed by HPLC and the product was purified by preparative FPLC using acetonitrile : ammonium sulphate buffer mixture. Peak corresponding to HI was collected, acetonitrile was removed by rotavapor at 40°C and then lyophilized. The resultant powder was re-dissolved in 1.0 M acetic acid and subjected to gel filtration to remove the residual buffer. Finally, acetic acid was removed by lyophilization and human insulin (HI) was obtained by crystallization.
Example 5; Animal studies and biological activity:
HI thus obtained in our lab was reconstituted in saline and m-cresol to a concentration of 6.62mg/ml based on the HPLC peak area in comparison with the commercial human insulin. This sample was administered to experimentally induced diabetic mice. Commercially available human insulin from Eli Lilly was used as the reference standard. Our preparation exhibited biological activity very similar to that of the reference sample. However, elaborate evaluation of the same is under way. Results from statistically significant population of mice and rats established the biological activity for our recombinant human insulin.

We claim:
1) Recombinant human insulin comprising the amino acid sequence as set forth in SEQ ID NO: 1.
2) The recombinant human insulin as claimed in claim 1, wherein said SEQ ID No. 1 is obtained by optimization of codons in Pichia species.
3) Recombinant human insulin comprising the amino acid sequence as set forth in SEQIDNO.2.
4) The recombinant human insulin as claimed in claim 3, wherein said SEQ ID No. 2 is obtained by optimization of codons in Pichia species.
5) A method for obtaining recombinant human insulin comprising the amino acid sequence as set forth in SEQ ID Nos. 1 and 2, said method comprising steps of:

a) constructing an insulin precursor gene;
b) ligating the precursor gene into a vector; and
c) cloning the ligated precursor gene into a host, followed by fermentation to obtain recombinant human insulin.

6) The method as claimed in claim 5, wherein said insulin precursor is selected from a group comprising porcine insulin precursor and bovine insulin precursor, preferably porcine insulin precursor.
7) The method as claimed in claim 5, wherein said precursor is constructed with about eight oligonucleotides coding for porcine insulin precursor.
8) The method as claimed in claim 5, wherein said vector is selected from a group comprising pPIC9K and pPICZa, preferably pPIC9K.
9) The method as claimed in claim 5, wherein said cloning is carried out at downstream of AOX1 promoter and Mating Factor Alpha (MFa) signal sequences.

10) The method as claimed in claim 5, wherein said host is selected from a group
comprising Pichia pastoris, Pichia methanolica, Pichia guilliermondii and
Pichia caribbica, preferably Pichia pastoris.
11) The method as claimed in claim 5, wherein said fermentation is carried out in a
suitable medium contained in a modified vessel at a suitable temperature range,
aeration, cell densities and feeding.
12) The method as claimed in claim 11, wherein said 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.
13) The method as claimed in claim 11, wherein said modified vessel comprises methanol probe and sensor to monitor residual methanol and methanol feeding rate; modified air sparger to increase efficiency of aeration and oxygen transfer rate (OTR), aeration delivery pores in sparger are relocated to top side and air bubbles are made to hit impeller directly.
14) The method as claimed in claim 11, wherein said temperature ranges from about 29 - 31°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.
15) The method as claimed in claim 11, wherein said aeration ranges from about 0.5 -1.5 VVM pure air, preferably 1VVM 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).
16) The method as claimed in claim 11, wherein glycerol feeding is carried out to promote high cell density before induction and is continued until cell density (OD600) reaches about 500; methanol is fed exponentially to promote increased expression of target protein.

17) The method as claimed in claim 5, wherein said method is optimized with
respect to various parameters selected from a group comprising increasing GC
content of coding region, N-terminal spacer sequence, C-peptide region
connecting B and A chains of insulin, multiple copy insertions, optimized media
components and growth and induction parameters, or a combination thereof.
18) The method as claimed in claim 11, wherein said N-terminal spacer sequence is
selected from a group comprising a di-peptide KR and long spacers KREAEA,
KREEAEAEAEPK or a combination thereof.
19) The method as claimed in claim 11, wherein said C-peptide region is selected
from a group comprising KR and AAK or a combination thereof.
20) The method as claimed in claim 11, wherein incorporation of an aromatic amino acid in C-peptide EWK. improves folding stability of insulin precursor.
21) The Recombinant human insulin and the method thereof, as substantially herein described with reference to examples and figures.