FIELD OF INVENTION:

The present invention relates to an improved process for preparing Urate oxidase and more particularly an improved process for preparing Urate oxidase using the qualified equipments having calibrated monitoring and measuring devices.

PRIOR ART:

Urate oxidase, is known in the art and is made by Yeast fermentation, by Saccharomyces cerevisiae. It takes about 7 days to complete the process and it will go through a typical growth pattern of Yeast cells.

After the 7 day process, the cells will be washed and taken for cell breakage! Supernatant will be taken to Chromatographic processes.

Chromatographic processes are mainly intended at capturing of protein and to get rid of impurities, if any.

Once the specific protein is captured, it will be eluted and taken for buffer exchange to isolate the active pharmaceutical ingredient and finally, it will be taken for lyophilization.

We give below disadvantages of prior art:

1. Thorough genomic sequencing is done, but limited for industrial application

2. Antibodies against S. cerevisiae are found in 60-70% of patients,' suffering from auto immune diseases, according to research reports whre as the same is not limited for E.coli fermentation. S. cerevisiae is food grade yeast and some people have specifically allergic to it.

3. S.cerevisiae fermentation is a 7 day process and it needs complex nutrients

4. .It needs complex nutrients throughout the process and need to show clearance from all the spent nutrients in washed cells which is difficult

5. Fermentation parameters are more and needs long time intervals up to 7 days

6. Needs to establish cleaning validation and sterility control for at least 10 days (Including 7 day process time)

7. In process verification and compliance with respect to growth pattern has many check points and vary with in house processes

8. Achieving Consistency in Expression levels can be challenging as it is a novel process for this molecule

9. Cell Harvesting by TFF needs specific custom made cartridges

10. Cell Lysis needs dynomill with glass beads and showing clearance from glass beads further in downstream is a difficult task

11. Capturing the specific protein need aerosol binding and desorption and need to show clearance from aerosols at a later stage

12. Further capturing by Chromatography is a sensitive process and can cause non specific aggregation

13. Required to have a gel filtration to get rid of non specific aggregation

14. Need to establish analytical methods in house based on the process required

15. Analytical method validation is a difficult task for in house developed test methods

16. Yeast based products need to go through stringent formulation conditions and stability studies based on their N-terminal positions

It has been therefore long felt need to overcome disadvantages of prior art and we give below interlaid a few advantages of the teaching of present invention:

1. It is a one day process, relatively simplest fermentation.

2. It has minimal nutrients required. Clearance from spent nutrients is well established and generic processes prevailing across all biotech industries

3. Fermentation parameters are simple and can be cascaded to each other and up to only for 24.hours

4. Establishing cleaning validation and sterility control for 3 Day is substantial and relatively easier and proven over the decade

5. In process verification and compliance with respect to growth pattern is well established for E.coli processes irrespective of the end product.

6. Established procedures are used for E.coli fermentations irrespective of the end product

7. Cell Harvesting can be done with readily available cartridges

8. Cell lysis can be done with a cell disruptor on a simple principle based on high pressures through a small orifice
9. Cell lysate can be clarified through a selective TFF cassettes and it is a simple process

10. Capturing can be done by affinity matrices readily available and non specific aggregation is very rare and can be controlled by process parameters.

11. Analytical methods for E.coli based products are well established and available as compendial methods in pharmacopoeia where as the same is very rare for yeast based products for bio therapy.

12. Formulation studies should be definitely stringent, but stability studies can be generic again

OBJECT OF THE INVENTION:

The main object of the invention is to have an effective process in place which would give a stable product with requisite units of activity and at the same to make therapy affordable.

Other object of the invention is unique by itself as the host is very simple in its genomic configuration and well deciphered one.

Still other object of the invention is that Urate oxidase, is made by E.coli fermentation. It is a one day fermentation after which cells will be broken by high pressure cell disruptor.
Supernatant will be collected and taken for two step Chromatographic separation.

Another object of the invention is that after chromatographic separation, sample will be subjected to buffer exchange and taken for lyophilization.

Yet another object of the invention is that Lyophlization involves freezing and annealing , which means protein is allowed to take its active structural form and then sequential drying to the room temperature.

Still another object of the invention is it is a simplified process in terms of the expertise gained in the area of bio similar industry.

Yet object of the invention is host is typically a micro organism whose genetic constitution is well known.

Further object of the invention is fermentation process is a one day process with which we can control the parameters very well during the process and there by the yields of the product of interest.

The still further object of invention is once after the fermentation, cells can be broken by high pressure cell disruption which is again universally acceptable as it does not require additional chemicals / reagents.

Yet object of invention is that two step chromatography would ensure that the impurities are washed out effectively.

Still object of invention is final lyophilization step with the E.coli derived proteins, would be a smooth process resulting in the stable product.

SUMMARY OF THE INVENTION:

According to the present invention there is provided an improved process for preparing urate oxidase comprising:-

(i) passing working cell bank (WCB) to thaw and inoculating the thawed WCB into inoculum medium;

(ii) The inoculated medium is kept for incubation in a rotary shaker till the time optical density measurement at 600 nm reaches between 1.6 to 2.2.

(iii) The above inoculum will be taken for fermentation in a pre-sterilized medium,fermenting the above medium by incubating at temperature 37 to 42°C for 16 to 24 hours;

(iv) The induction is carried out for 10 to 12 hours at optical measurement of 35 to 40, using IPTG for the selective expression of urate oxidase,

(v) cell harvesting the product of step (ii) using a batch centrifuge and taking harvested cell pellet for cell lysis with 35 to 45 fold dilution by tris buffer, at a pH of 8.5 by using sonicator for 35 to 60 minutes and collecting the supernatant product;

(vi) The centrifugation of Cell lysate is carried out at 7000 to 9000 rpm for 15 to 25 minutes with pulsation on and off at regular intervals of 3 to 7 minutes.

(vii) Supernatant sample can be taken for C-DEAE after equilibration of C-DEAE matrix with 10 mM Tris buffer, pH 8.5, washed the column after loading with 10 mM Tris buffer, pH 8.5 solution and eluted the captured protein using linear gradient(40-50%) of lOmM Tris buffer pH 8.5 and l0mM Tris buffer, pH 8.5 containing 10 mM Ammonium sulphate pH 7.0. Linear flow rate of 56.55 cm/hr, needs to be the operational flow rate.

(viii) buffer exchanging eluate sample of C-DEAE by using 10mM buffer such as sodium citrate, at a pH of 7.0 to 9.0 and concentrating by using 10 10KDa cut off cassette;

(ix) The above concentrated sample can be taken for loading on to IEX after equilibration of SP-Sepharose matrix using 10 mM Sodium citrate buffer, pH 6.0 and subsequent washing with 10 mM Sodium citrate buffer, pH 6.0, eluted the captured protein using linear gradient (30-70%) of lOmM Sodium citrate buffer, pH6.0 and lOmM Sodium citrate buffer, pH8.0 along with 800 mM Sodium citrate buffer, pH 6.0. Linear flow rate to be maintained during IEX is 15.28 cm/hr.

(x) desalting and concentrating IEX eluate using 10 KD bio max cassettes with 40 mM Phosphate buffer solution, at a pHof 7.0to8.0;

(xi) concentrating by using stabilizers such as L-Alanine, Mannitol and 40 mM said buffer, at a pH of 7.0 to 8.0;

(xii) freezing formulated bulk for filling and lyophilization and subjecting the sample for stage wise drying at 15 to 20°C.

We shall now describe the present invention by way of illustrations which only describe the invention but does not restrict the scope of present invention.

Urate oxidase is a recombinant urate-oxidase enzyme produced by genetically modified Saccharomyces cerevisiae strain. The cDNA coding for rasburicase was cloned from a strain of Aspergillus flavus. Rasburicase is a tetrameric protein with identical subunits of a molecular mass of about 34 kDa. The monomer, made up of a single 301 amino acid polypeptide chain, has no intra- or inter-disulfide bridges and is N-terminal acetylated.
The drug product is a sterile, white to off-white, lyophilized powder intended for intravenous administration following reconstitution it is used in the treatment and prophylaxis of acute hyperuricaemia, in order to prevent acute renal failure, in patients with haematological malignancy with a high tumour burden and at risk of a rapid tumour lysis or shrinkage at initiation of chemotherapy. Urate oxidase is indicated for the initial management of plasma uric acid levels in pediatric patients with leukemia, lymphoma, and solid tumor malignancies who are receiving anti-cancer therapy expected to result in tumor lysis and subsequent elevation of plasma uric acid.

Urate oxidase has the following sequence:

Ser Ala Val Lys Ala Ala Arg Tyr Gly Lys Asp Asn Val Arg Val Tyr Lys Val His Lys Asp Glu Lys Thr Gly Val Gln Thr Val Tyr Glu Met Thr Val Cys Val Leu Leu Glu Gly Glu Ile Glu Thr Ser Tyr Thr Lys Ala Asp Asn Ser Val Ile Val Ala Thr Asp Ser Ile Lys Asn Thr Ile Tyr Ile Thr Ala Lys Gln Asn Pro Val Thr Pro Pro Glu Leu Phe Gly Ser Ile Leu Gly Thr His Phe Ile Glu Lys Tyr Asn His ILe His Ala Ala His Val Asn Ile Val Cys His Arg Trp Thr Arg Met Asp Ile Asp Gly Lys Pro His Pro His Ser Phe Ile Arg Asp Ser Glu Glu Lys Arg Asn Val Gln Val Asp Val Val Glu Gly Lys Gly Ile Asp Ile Lys Ser Ser Leu Ser Gly Leu Thr Val Leu Lys Ser Thr Asn Ser Gln Phe Trp Gly Phe Leu Arg Asp Glu Tyr Thr Thr Leu Lys Glu Thr Trp Asp Arg Ile Leu Ser Thr Asp Val Asp Ala Thr Trp Gln Trp Lys Asn Phe Ser Gly Leu Gln Glu Val Arg Ser His Val Pro Lys Phe Asp Ala Thr Trp Ala Thr Ala Arg Glu Val Thr Leu Lys Thr Phe Ala Glu Asp Asn Ser Ala Ser Val Gln Ala Thr Met Tyr Lys Met Ala Glu Gln Ile Leu Ala Arg Gin Gin Leu He Glu Thr Val Glu Tyr Ser Leu Pro Asn Lys His Tyr Phe Glu He Asp Leu Ser Trp His Lys Gly Leu Gin Asn Thr Gly Lys Asn Ala Glu Val Phe Ala Pro Gin Ser Asp Pro Asn Gly Leu He Lys Cys Thr Val Gly Arg Ser Ser Leu. Lys Ser Lys Leu.

Major equipment and Measuring / Monitoring / Recording devices used in the Urate oxidase process are listed below.


All the equipment used in the process is Qualified or Re qualified in terms of Design Qualification (DQ), Installation Qualification (IQ), Operational Qualification (OQ) and Performance Qualification (PQ).

The Process Flow chart employed for the production of Urate oxidase is is given below :-

Urate oxidase, a recombinant bio therapeutic protein is produced by E.coli (BL21) through an inducible vector system that carries the gene sequence for the above protein. The best expressing clone was selected from the transformed colonies and used for making the Master Cell Bank (MCB) followed by Working Cell Bank (WCB). MCBs and WCBs are stored at -80°C. WCB is taken for regular process activities where as MCB is kept as a backup for further making of WCBs when ever required.

The Lab Process Records are maintained for the process employed for the production of Urate oxidase.

The following were the Critical Process parameters, which were fully monitored and controlled during the execution of process validation batches.

Critical Process parameters:

1. Inoculum OD - Age during seeding

2. Loading protein concentration - CAPTO-DEAE chromatography

3. Loading protein concentration - SP-Sepharose chromatography

The following process parameters need to be monitored:

Process parameters:

1. Control range of temperature for fermentation , maintained between 3 7°C to 42°C

2. Growth profile of fermentation will be monitored and captured in LPRs.

3. Total protein yields in fermentation

4. Protein yields in Cell lysis

5. Total protein yields obtained at CAPTO-DEAE

6. Total protein yields obtained at SP-Sepharose

7. Total protein yields obtained at final bulk concentration

Yield data can only be for monitoring only.

In-process sampling during Urate oxidase manufacturing can be done as per in-house guidelines. Frequency of in-process sampling was described in the LPR.

The in-process test procedures employed are mentioned in the procedures and available as controlled documents.

The specifications including additional tests if any are defined in in-house specifications.
The above said specifications are controlled documents.

The analytical methods used for each test is validated. The Specifications and the methods are available as a controlled document.

Product for Elitek has inherent problems, which are based mainly on host based which would have implications trickled down all the process steps till to the end product formulation. Therefore, it is essentially important to work on a host such as E.coli for w0hich expertise is well established across the world.

WE CLAIM:

1. An improved process for preparing urate oxidase comprising:

(i) passing working cell bank (WCB) to thaw and inoculating the thawed WCB into incoulum medium;

(ii) Fermentation at temperature of 37 to 42°C for 16 to 24 hours;

(iii) cell harvesting the product of step (ii) using a batch centrifuge and taking harvested cell pellet for cell lysis with 35 to 45 fold dilution by tris buffer, at a pH of 8.5 by using sonicator for 35 to 60 minutes and collecting the supernatant product;

(iv) taking supernatant product for C-DEAE after equilibration of C-DEAE matrix with 10 mM tris buffer, at a pH of 7.5 to 8.5, washing the column after loading with 10 mM tris buffer, at a pH of 7.5 to 8.5 and eluting the captured protein;

(v) buffer exchanging eluate sample of C-DEAE by using 10mM buffer such as sodium citrate, at a pH of 7.0 to 8.0 and concentrating by using 10 10KDa cut off cassette;

(vi) loading the above concentrated product of step (v) to IEX after equilibration of SP-sepharose matrix using 10 mM buffer such as sodium citrate, at a pH of 6 to 7.0 followed by washing with 10 mM said buffer and 800 mM Sodium citrate, at a pH of 6.0 to 7.0 for eluting, the captured protein;

(vii) desalting and concentrating IEX elute using 10 KD bio max cassettes with 40 mM said buffer solution, at a pH of 7.0 to 8.0;

(viii) concentrating by using stabilizers such as L-Alanine, Mannitol and 40 mM said buffer, at a pH of 7.0 to 9.0;

(ix) freezing formulated bulk for filling and lyophilization and subjecting the sample for stage wise drying at 15 to 20°C.

2. An improved process as claimed in claim 1, wherein inoculated medium is kept for incubation in a rotary shaker till the time optical density measurement at 600 nm reaches between 1.6 to 2.2.

3. An improved process as claimed in claim 1 or 2, wherein fermentation, and induction is carried out for 10 to 12 hours at optical measurement of 35 to 40, using IPTG for the selective expression of Urate oxidase.

4. An improved process as claimed in claims 1 to 3, wherein centrifiigation of Cell lysate is carried out at 7000 to 9000 rpm for 15 to 25 minutes with pulsation on and off at regular intervals of 3 to 7 minutes.

5. An improved process as claimed in any one of claims 1 to 4, wherein linear gradient (40 to 50%) of 10mM tris buffer is carried out at a pH of 7.5 to 8.5 and 8 to 10mM tris buffer.

6. An improved process for as claimed in any one of claims 1 to 5, wherein using linear gradient (30 to 70%) of 10mM sodium citrate buffer, at a pH of 6.0 and 1mM said buffer, at a pH of 6.0 along with 800 mM said buffer, at a pH of 6.0. Linear flow rate is maintained during IEX is 15 28 cm/hr