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Title:
HDMF AND RECOMBINANT PRODUCT FILTRATION SYSTEM
Document Type and Number:
WIPO Patent Application WO/2015/040501
Kind Code:
A1
Abstract:
The present invention discloses a filtration system characterized by a one step process for isolation and purification of biological products including recombinant products derived from High cell Density Microbial Fermentation (HDMF) but is not limited to HDMF and can be applied to other fermentation systems for separation of high value biological products.

Inventors:
PATIL BAPUSAHEB MALGONDA (IN)
PATIL VIKRANT BAPUSAHEB (IN)
Application Number:
PCT/IB2014/058630
Publication Date:
March 26, 2015
Filing Date:
January 29, 2014
Export Citation:
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Assignee:
PATIL BAPUSAHEB MALGONDA (IN)
PATIL VIKRANT BAPUSAHEB (IN)
International Classes:
B01D29/00; B01D36/00; C12M1/00
Domestic Patent References:
WO2006050625A22006-05-18
Foreign References:
US1082862A1913-12-30
US2885082A1959-05-05
Other References:
"FILTECH 2009 - Conference Proceedings", vol. 1, 2009, FILTECH, article HERMANN KATINGER, BAPU PATIL, VIKRANT PATIL: "Filtration of high density microbial fermentation biomass using BASP Biotech filter", XP007922728
"BASP Rotray Leaf Filter FE 05000", 15 November 2010 (2010-11-15), XP007922723, Retrieved from the Internet [retrieved on 20140610]
"BASP Rotray Leaf Filter FE 05000", 15 November 2010 (2010-11-15), XP007922727, Retrieved from the Internet [retrieved on 20140610]
Attorney, Agent or Firm:
PANDURANGI, Abhishek (A-403 Athene Building,,Lodha Paradise, Near Majiwada, Thane, 1,Maharashtra, India, Thane 400601, IN)
Download PDF:
Claims:
We claim,

1. A one-step process for Microbial HDMF broth and recombinant broth Filtrations to produce filtrates suitable for downstream purification with minimal discharge of effluentcomprising: a) preparing a pre-coat solution in a pre-coat tank (53) by mixing an inert precoat powder in a carrier liquid / water with the aid of an inert gas followed by circulating pre-coat solutionby pumping over rotary leaf filter (51) to achieve pre-coated filter-stack with desired thickness; b) feeding the Microbial HDMF and recombinant or otherwise broth containing biomass and liquid products, to the filter vessel through inlet nozzle under pressure through the precoat layer, simultaneous deposition of the moist biomass on the pre-coated filter screen to get filtered and passing out through filtrate outlet nozzle; c) displacing the filter vessel holdup /heel as well as the entrapped liquid in the biomass wet cake by slow introduction of clean dry compressed Air/ Nitrogen and transferring back the same to feed tank or filtered through the Guard Filter(55); d) deactivating the biomass in the filter vessel after dewatering; e) rotating the filter stack after deactivation to dislodge the wetcake followed by cleaning the filters using spray header and rotating filter stack for next batch and f) squeezing Wetcake for disposal.

2. The process according to claim 1, wherein the precoat powders sterile grade are selected from Diatomite, Perlite, Cellulosic origin.

3. The process according to claim 1, wherein the pre-coating is done

under the differential pressure of 2 to 3 psig.

4. The process according to claim 1, wherein the pre-coat layer thickness varies from 1.5 mm to 15 mm including body feed or otherwise.

5. The process according to claim 1, wherein the carrier liquid is water or suitable solvent.

6. The process according to claim 1, wherein the post pre-coating, the filter vessel is drained to remove entrapped carrier solvent / water / clear liquid product by purging of clean compressed air/ N2 to de water the pre-coat layer.

7. The process according to claim 1, wherein the Filter stack is stationary during filter filling, pre-coating, body-feeding, filtering, dewatering, hold up/heel filtration, filter draining andwetcake biomass deactivating.

8. The process according to claim 1 , wherein the Filter plate stack is spun/rotated after filtering the feed batch to dislodge the wetcake or slurring it for discharging.

9. The process according to claim 1, wherein the wetcake is slurried by spraying water before dislodging.

10. The process according to claim 1, wherein the Circulation of HDMF broth is continued till filtrate clarity is achieved but not more than 15Minutes.

11. The process according to claim 1 , wherein the inlet valve on the Guard Filter (55) is opened to allow the filtrate to pass through Guard Filters for trapping accidental release of leaked turbid filtrate to the filtrate receiving tank once the filtrate is clear.

12. Microbial HDMF and recombinant Filtration System comprising: a) A Rotary Leaf Filter (51) with filter plate stack for filtering broth after precoating; mounted on central shaft together with Geared motor, for dislodging spent wet-biomass slurry after dewatering under air pressure and deactivation; mounted on trolley with CIP and SIP facility b) Control Panel (52) with Variable Frequency Drive to facilitate

rotation,; c) A Precoat Tank (53) with compressed air connection for proper mixing of precoat in water; d) A Centrifugal pump(54) to circulate the chemically inert precoat slurry over the metallic Filter plates until all precoat powder is held on filter plates and clear water, free of precoat powder is seen during circulation; e) a Diaphragm Pump (AA) to feed HDMF broth or otherwise having dissolved liquid products to Rotary Leaf Filter through inlet nozzle to get filtered liquid through outlet nozzle;

A 'Guard Filter' (55) to prevent accidental leakage of unfiltered turbid liquid into filtrate tank.; g) a filtrate outlet of Rotary Leaf Filter (51) is connected to inlet on Guard Filter (55) to receive clear filtrate;the clear filtrate travels to filtrate storage tank (BB);and h) Interconnecting piping, valves, instruments, CIP/SIP provision, control panel etc. provided for the entire system with tri-clover fittings for quick assembly & dismantling; and i) optionally a Wet cake squeezer (56) to squeeze slurried biomass for disposal can be provided particularly for large biomass quantities.

The Microbial HDMF and recombinant Filtration System according to claim 12, wherein the CIP comprises Cleaning of filter by filter stack rotation; jet sprays of hot water/ steam using spray header and introducing clean air/ N2/ Dry steam from filtrate side to ensure perfect cleaning.

The Microbial HDMF and recombinant Filtration System

according to claim 12, wherein the spray header comprises jet sprays positioned above each filter plate. 15 The Microbial HDMF and recombinant Filtration System according to claim 12, wherein the spray header is in rotatable position to give more flexibility in cleaning.

16. The Rotary Leaf filtration System according to claim 14, wherein the backwashing of the filter is done by introducing water/ solvent or Air/ N2 /steam at 0.15 kg/cm2 pressure from filtrate side to assist opening of clogged filter media pores.

17. The Rotary Leaf filtration System according to claim 12, wherein the SIP- sterilizing in position comprises introducing steam at 0.15 kg/cm2 or more at 121°C Temperature followed by blowing dry steam Air/ N2 for final drying before next batch.

Description:
HDMF and recombinant product filtration system Technical field

The present invention addresses a High Density Microbial Fermentation (HDMF) and recombinant product filtration system. More particularly the invention relates to a HDMF and recombinant product Filtration System; useful for separation of high value liquid products in the manufacture of human biological medicinal products. The invention provides enormous cost reduction due to one-step filtration; eliminates the need for centrifuging huge amount of high purity water for dilution and reduces electricity consumption. This one-step process addresses unmet industrial needs of the downstream processing stage in High Density Microbial Fermentation broth filtration and further benefits in purification thus contributes in developing entire biotechnology platform.

Background and prior art

Microbial technology exploits microbial wealth for human requirement, like production of microbial metabolites and products such as enzymes, organic acids, antibiotics, drugs and pharmaceuticals, in processes like recombinant protein expressions, fermentations and downstream processing.

Over 151 recombinant pharmaceuticals approved for human use produced are by microbial cells either bacteria or yeast. The microorganisms are important in any microbial fermentation. The microorganisms in the fermentations process either produce valuable pharmaceuticals by converting the substrate to valuable fermentation products or they themselves are the products sought in the fermentation process. In both the circumstances, the goal of the fermentations process is to obtain the highest number of microbes per unit volume, which may be termed as achieving high cell density fermentation. For example, enzymes are used in high volume to produce bio- products using High cell Density Microbial Fermentations(HDMF). HDMF allows increased product volume for improved volumetric productivity. Factors that are most critical on biopharmaceutical manufacturing costs are the fermentation titer i.e. concentration of dissolved constituents in solution and the overall yield. The fermentation titer depends mainly on the host cell expression system, the genetic stability of the host cell or cell line and the cell density. The overall yield on the other hand is a result of the downstream processes. Purification costs are significant in biopharmaceutical production.

Fermentations produce wide variety of valuable recombinant proteins, Monoclonal antibodies, Vaccines, Nucleic-acid based products, Therapeutic enzymes etc. biotech-products using various microorganisms including animal cells and mammalian cells-CHO-Chinese Hamster Ovary cells for the production of complex protein such as antibodies.

During the past twenty-five years, regulatory requirements have directed industries to use modern technology to improve process purity and productivity control. The recombinant protein industry has become focused on process efficiency, speed, yield and cost. Despite that the centrifugation followed by final filtration method has remained, essentially unchanged since the midl950s. Microbial based processes present harvesting difficulties because of high cell densities, temperature control requirements and sensitivity of secreted product to shear and oxidation. Production scale centrifugation needing dilution with high purity water adversely affects product quality is of limited value because of low yields, high shear, high temperature and foaming. In addition, post-centrifugation filtration typically requires multiple membrane filters.

In High Density Microbial Fermentations using recombinant yeast such as Pichia pastories, but not limited to HDMF on yeast, biomass concentration of up to 350gmswet biomass i.e. up to 1 15gms dry matter per litre are accumulated. For the separation of biomass from the liquid phase conventional centrifuges are inefficient and require repeated centrifugation steps, needing huge amount of high purity water for dilution, in order to harvest products, which are dissolved in the aqueous liquid phase. This methodology leads to a very uneconomic process, including loss in yield and quality of products and increase in liquid volumes, which complicate the further steps in downstream purification (DSP). Although, there are alternatives such as the use of decanter centrifuges, which are very expensive, consume high energy and the quality of filtrate is not satisfactory, hence requiring further multiple filtrations.

In a continuous aerobic HDMF to produce high yields of yeast single cell protein product, by adding media containing high mineral salts concentrations to the ferment in the fermenter, cultures grow within the Fermenter and produce the required molecules that have to be extracted from the liquid solution. However, the separation - filtration processes contributes large cost component of pharmaceutical manufacture. The particle retention efficiency, nature of particle/biomass of cells or wet cake characteristics particle capacity of the filter, and the liquid flux all impact the product cost. Therefore, High cell microbial filtration separation of liquid from biomass in HDMF is challenging. Typically, the HDMF broth separation process requires 20 to 30 times dilution by pure water and repetitive centrifugation and filtration to produce a clear filtrate that can be used for further purification by Ion Exchange chromatography and Ultra Diafiltration for separating diluents and other constituents.

The conventional downstream process involving filtration and further purification of the HDMF broth is described as follows (Fig.2):

• In a 15KL fermenter, HDMF broth generation is around 12 KL and biomass produced is 4KL.

• Post the fermentation process, the broth is diluted 20 to 30 times by adding pure water facilitating centrifuging. The water volume used as a diluent depends on the characteristic consistency of the biomass.

• The output of the Fermenter- HDMF broth (Fig. IBis diluted and fed into Centrifuge (Fig.2-03)for biomass separation.

• The separated wet-biomass is discharged into Decontamination Tank(Fig.2, 04) and disposed after decontamination in slurry/ wetcake form. • The turbid filtrate from centrifuge is stored in 2KL tank (Fig.2, 05). After pH adjustment in Tank (05), the turbid filtrate is pumped through Heater (Fig.2, 06) into 15KL Intermittent Storage Tank (Fig.2, 07).

The main disadvantage associated with the Centrifuging (Fig.2, 03) is the compressible biomass wetcake. Due to g-force generated by 45kVh electric motor, the separated biomass forms compact wetcake along the periphery. The compacted-cake resists filtration. Hence to recover the extract i.e. the liquid from the Supernatant Biomass, the broth is diluted 20 to 30 times its volume, with pure water.

The decanters are expensive, consume high energy and hence they are sparingly used.

• Final filtration Unit (Fig.2, 08) -Under pump pressure the turbid filtrate is further filtered in series of micron filters (10μ, 5μ.ο.45μ and 0.22μ Micron) to obtain clear filtrate for further downstream processing i.e. to get purified product.

The use of various separators and filters of different depth and pore sizes in series involves huge investment in equipment, high purity H2O generator and auxiliary equipments and sterile space.

The process of HDMF Broth separation typically involves serial dilution by adding high purity water, repeated use of centrifugation in 20 to 30 batches, followed by multiple microfiltration steps.

• Decontaminated slurry (Fig.2, 04) is pumped out, for disposal through outlet (Fig.2, 03) (CC).

• The clear filtrate output from (Fig.2, 08) the filtrate storage tank is sent for downstream processing for further purification. Considering the characteristic consistency of the HDMF broth, in order to separate the biomass, 20 to 30 times dilution, centrifugation followed by pre- filtration and fine filtrations in 20 to 30 batches are needed to separate about 4kl biomass (Fig.203)with 220KL to 330KL diluents, i.e., High purity water.

In case of conventional process, dilution of broth adversely affects downstream processing. Diluted water weakens the gel in the chromatography column and hence should be avoided.

The use of various equipment's including centrifuges or expensive decanters, battery of filters of various sizes and huge quantity (20 to 30 times) of high purity water that is required for dilutions of the broth, deactivation and disposal of biomass makes the process both costly and time consuming. Also, the final product yield using conventional method and the quality is inferior too.

The centrifugation and auxiliary equipments consume enormous amount of capital investment and operating costs and electricity consumptions contribute to high investment, cumbersome inappropriate centrifuging, laborious operations in sterile environment is expensive adds to the cost of end product

In view of the foregoing, it is evident that the HDMF broth needs large diluent volume; further deactivation of spent Biomass makes the conventional elaborate method unsuitable due to loss of expensive product and inferior quality of the end product. Also, it is clear that the prior art methods escalates the operating costs, consumable and utility costs and needs large sterile space too.

Therefore, there is a need in the art to provide reliable and cost-effective one- step method of processing the HDMF broth without affecting the overall yield and quality of the fermentation product. Also, there is need in the art to device a simplified filtration and purification system, wherein the use of centrifuge, micron filters and use of huge amounts of high purity water involved in broth dilution can be avoided.

The downstream processing equipment and auxiliary equipment sizes, investment and operating costs would reduce substantially as the liquid feed would be only around 10,000 litres as against 210,000-310,000 litres. Therefore, development of appropriate HDMF or otherwise Broth processing system with maximum product recovery and reliable quality was the urgent unmet need in the technology of high density and other microbial fermentations filtering and downstream purification.

The present inventors felt; it would be an enormous improvement of the entire biotechnology platform, if the critical step of biomass separation and purification could be replaced by an efficient Process for HDMF and recombinant products Filtration and Purification technology, that meets the following criteria: a) Aseptic design and scalability

b) Separation without dilution HDMF harvests, c) Filtrates those are free of particles to enable filtration and purification d) Eventually enable washing and deactivation of the biomass while still in the filtration unit for disposal with Zero discharge of effluent.

e) The Chromatography Columns and Ultra/ Diafiltration units need to handle only about 10,0001itre liquid after separating biomass, as against 210,000 - 310,000Litresto save time, capital and operating and consumable cost.

Accordingly, considering the above unmet criteria, the present invention provides a highly scalable, an efficient and economical Filtration and purification system that facilitates economical quality processing of high value liquid HDMF and recombinant products in aseptic conditions.

Also, the present invention devises a cost-effective method to separate high value liquid products obtained from microorganisms and recombinant products wherein repeated water dilution of the HDMF biomass for centrifugation, and repeated filtration can be avoided to ensure better and faster purification in compact unit. The separation of liquid products from biomass can be carried without dilution of HDMF harvests or fermentation broths containing recombinant products. The spent Biomass can be deactivated while it is still in the filtration system.

Abbreviations:

HDMF: High Density Microbial Fermentation

cGMP: Current Good manufacturing processes

CIP: Cleaning in Position SIP: Sterilization in Position

NTU: Nephelometric Turbidity Unit

PTFE: Polytetrafluoroethylene

Brief Description of Photos and Drawings:

Fig. 1 depicts a typical HDMF process with fermentation medium capacity of 15KL wherein each component with capacity is denoted as follows: Tl- Antifoam tank: 250Z; GT1 - Tank: 2 KL; GT2 - Tank: 2 KL; Fl - Seed fermenter: 2001; F2 - Production fermenter: 15 KL; T2-Trace element solution: 100Z; T3- Feed medium tank: 7 KL and T4-Batch medium tank: 7KL.

Fig.2 depicts a typical HDMF broth filtration system wherein each component with holding capacity is designated as follows: AA -HDMF broth feed 1 1 KL; 01- Diluent Storage Tank 15KL (450 KL); 02- Dilution Tank with Pump: 2 KL; 03-Centrifuge with motor, control. (Westphalia-HFE-45 Flow Capacity 5- 7KL/Hr with 45kW Motor); 04-Biomass Decontamination System: 7 KL; 05 - pH adjustment tank: 2 KL; 06- Heat Exchanger; 07-Storage Tank: 15 KL and 08: Filtration Unit to filter 450 KL Total Dilute Filtrate.

Fig 3 depicts the one step HDMF system of the instant invention wherein each component comprises

(AA)- Diaphragm Pump with flameproof motor for broth feeding.

51- One step Filtration system with proprietary vertical filter stack, with pump for filtrate circulating, Skid Mounted Unit with CIP / SIP built in facility; 52- Control Panel for Filtration system; 53-Pre-coat Tank; 54- Pre-coat tank with pump; 55- Guard filter with 10 and 0.22 Micron filter; 56- Optional: Wetcake squeezer for biomass de watering and unloading and BB- Clear Filtrate outlet to downstream processing.

Fig.4depicts the downstream processingof the present invention wherein each component of the unit and its holding capacity is designated as follows: 11- Buffer storage tank (Glycine/ CaC ) : 2 KL; 12-Buffer storage tank (NaCl):2 KL; 13A/B-Ion exchange chromatography; 14 - Storage tank: 1 KL; 15A/B-Ultra and Diafiltration Unit with Pump; 16-Feed Tank: 200Z/17- Storage tank (HCl/CaCh): 200Z; 18: CIP Tank: 200Z; 19 -Final product tank: 501;20- Heat Exchanger ; 21: Conductivity adjustment unit;

22: SIP/CIP Steam Sterilization and cleaning in place: 200 1.

Summary of the invention

In accordance with the above objectives, the present invention provides novel filtration and purification system useful for separation of high value liquid products obtained from Microbial and recombinant products from HDMF broth or otherwise.

Accordingly, in a preferred embodiment, Fig.3 the invention provides a Fermentations including HDMF Broth Filtration System, wherein the system comprises the following components: a)Rotary Leaf Filter (51), having Precoat able filter-plate stack, Geared Motor, Control Panel with Variable Frequency Drive for filter stack rotation; mounted on trolley with CIP and SIP facility; b) Control Panel for filtration system (52); c) Precoat Tank (53) to mix precoat powder with clean water by

introducing air/N2- inert gas;

d) Precoatfeed pump(54) to circulate precoat powder mixed in clean water by introducing air / N2- inert gas.

The mixture is circulated to form required fine layer on the filter plates and allow water to pass through Rotary Leaf Filter (51)through bottom nozzle back to Precoat Tank (53)till adequate layer is formed and clear filtrate/ water is obtained;

Having created fine layer of chemically inert and pure appropriate specific layer on the filter plates with adequate quantity of filter aid, the clean dry pressurised air/N2 is introduced to drain off entrapped water in the precoat layer on filter plates. e) Guard Filter (55) protects filtered product stored in filtrate tank (BB) in case of accidental leakage through the filter plates. It has coarse filter AISI316, to trap leaked biomass and 0.2 micron fine filter to trap fine particles if any in filtrate as a guard filter. f) The Diaphragm pump (AA) connected to Fermenter pumps broth into the filter. The wet biomass gets deposited on filter plates and clean filtrate passes through outlet nozzle into Guard filter and then into Filtrate storage tank (BB).

Filtrate Product Tank (BB) equipped with centrifugal Pump feeds the particle free clear liquid product into purification system. g) The system has interconnecting piping, valves, instruments, view glasses, control panel to monitor the entire filtration and Biomass deactivation system within main filter. The system has tri- clover fittings on piping for quick assembly & dismantling,

h) Screw-press: biomass in the filter after deactivation can be slurried and discharged or can be squeezed in screw-press(56), dewatered and disposed off (CC) .

The present invention is designed for most critical filtration of HDMF broths in a sterile environment. The entire broth can be filtered with no waste in one- step without the use of centrifugation. The process of the present invention can be used for the production of recombinants, enzymes, vaccines, metabolites and DNA material. The real advantage of the present invention is that it is a one-step process, which does not require dilution of the HDMF or other fermentation broth containing recombinant products, centrifugation which leads to generating fine particles and series of cartridges filters and 0.22 membranes filters.

The instant technology of the present invention finds application in pharmaceutical and biotechnology fields to produce HDMF and recombinant products such as vaccines, enzymes, and metabolites, as well as transgenic recombinant products with great yield and high purity. The process lowers the production cost by about 60% compared to commercial process currently in the marketplace.

This novel filtration technique eliminates dilution(20 to 30 times)hence the quantity to be filtered is less than 10KL and hence efficient than the conventional filtration- separation techniques. The HDMF or otherwise broth can be fed directly into the system without dilution. The filtration system performs deactivation of the biomass in the filter itself. The produced filtrate is clear and suitable for chromatography that does not affect gel in column chromatography during purification. The size of chromatography purification system and Diafiltration system is also reduced to process less than 10 KL liquid as against 210KL or 310KL.

The invention provides a cleaning in position and sterilizing in position facility wherein the filtration system can be cleaned by front and back washing and further sterilized in place. Biomass wetcake deactivation, slurring and discharging the squeezed- dewatered wetcake in solid- wetcake form is performed in the filtration system itself. This is a batch process. The undiluted filtrate having liquid product can be purified at much lower costs due to enormous reduction in filtrate volume.

The present invention is an enormous development in industrial biotechnology to derive high value recombinant and medicinally important biological molecules.

Detailed description of the invention

The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

Accordingly, the present invention discloses novel filtration system useful for deriving HDMF and recombinant products from HDMF broth obtained from microorganisms or recombinant microorganisms The present invention is designed for filtration of HDMF broths in sterile environment according to cGMP. The entire HDMF broth can be filtered without 20 to 30 times dilution with high purity water and eliminating centrifugation with no waste in one- step without the use of centrifugation and serial micron filtrations. The process of the present invention can be used for the production of HDMF and recombinant products-enzymes, vaccines, metabolites and DNA particulates. The real advantage of the present invention is that it is a one-step process, which does not require huge quantity of high purity water for dilution affecting the downstream purification of the HDMF biomass, for centrifugation, consuming lot of electricity, intermittent storage tanks with pumps, heat exchangers, conductivity adjustments before microfiltration, Biomass deactivation system and use of series of Pre-filters and 0.22 membrane fine filters to filter enormous amount of diluted broth.

Accordingly, in a preferred embodiment, the invention provides HDMF filtration system as described in Fig. 3, wherein the system comprises: a) Rotary Leaf Filter (51) with filter plate stack for filtering broth after precoating; mounted on central shaft with Geared motor, Control Panel with Variable Frequency Drive to facilitate rotation, for spent wet-biomass slurry dislodging after dewatering under air pressure and deactivation; mounted on trolley with CIP and SIP facility;

b) Precoat Tank (53) with compressed air connection for proper mixing of precoat in water; The centrifugal pump to circulate the chemically inert precoat slurry over the metallic proprietary Filter plates until all precoat powder is held on filter plates and clear water, free of precoat powder is seen during circulation;

c) The 'Guard Filter' (55)to prevent accidental leakage of unfiltered turbid liquid into filtrate tank;

d) Interconnecting piping, valves, instruments, CIP/SIP provision, control panel etc. provided for the entire system with tri-clover fittings for quick assembly & dismantling; and

e) Optionally a Wet cake squeezer(56) to squeeze slurried biomass for disposal.

f) The liquid product in filtrate is purified in simplified Downstream Processing using Ion Exchange Chromatography and Ultra/ Dia- membrane filtration in extremely small setup and at tremendous savings.

In a typical embodiment, the commercial scale HDMF filtration system is mounted on trolley with CIP and SIP facility, designed according to the invention has a capacity of 501itre.

The HDMF filtration unit is also be scaled to a larger capacity as per fermenter size or customer's requirement.

The HDMF filtration system according to the invention consists of a rotary leaf filter (51) which is made up of Stainless Steel AISI 316, having a vessel diameter of 500mm with filtering area of 1M 2 and with wetcake holding capacity of about 15Z (considering maximum 350gms/litre moist wetcake). Using this specification, one can run a batch size having 501 HDMF Brothor a higher volume broth based on the characteristic of wetcake and its quantity employing the HDMF filtration system.

The rotary leaf filter (51) according to the invention consists of Precoatable filter plate stack with sterile filter aid powders to aid in filtration of HDMF Broth.

The HDMF filtration system has wet-cake washing, dewatering, deactivation, slurring and discharging; Cleaning in Place (CIP) by Front spray washing while rotating and Back washing and Sterilizing in Place (SIP) etc. facility. The Variable speed drive unit for filter stack rotation comprises of a Gear Box coupled with 1 HP (0.75 KW) Ex-proof Motor with VFD control system.

The Precoat Tank (53) according to the invention is a Vertical Vessel with air bubbling arrangement for proper mixing of Pre-coat powder in a clean carrier water/ solvent, made up of Stainless Steel AISI316 having a vessel diameter of 450mm with water holding capacity 50 Litres.

The Guard Filter (55) according to the invention is a Stainless Steel 316Filter vessel 035Ommwith welded dished bottom & top dish bolted with quick opening cover having a vessel diameter of with wetcake holding capacity of 30Litreshaving sintered SS316 filter plate to arrest accidental release of fine particles in filtrate storage tank (BB).

The Pump with motor (54)for precoating filter screen with precoat powder in water, according to the invention is a centrifugal type (Flow: 120 LPH) coupled with 1 HP (0.75 KW) Ex-proof Motor. The centrifugal pump circulates the chemically inert precoat slurry over the metallic Filter plates until all precoat powder is held on filter plates and clear water, free of precoat powder is seen during circulation. Then the circulation is stopped and air is introduced in the filter under gentle pressure to dewater precoat layer. Then undiluted HDMF broth or feed to be filtered is introduced with pump into Filtration system. The biomass gets deposited on pre-coated filter stack and sparkling clear filtrate passes into Guard Filter and then into filtrate receiver.

The interconnecting piping, valves according to the invention are provided on the entire system with tri-clover fittings for quick assembly & dismantling.

The filtration system according to the invention is manufactured as per cGMP norms and Quality Control/ Quality Assurance Plan. All necessary material, inspections & tests were carried out during the course of manufacturing with full documentation.

The Material of construction of the filtration system according to the invention is provided with the specification below.

Contact Parts : Stainless Steel AISI 316

Filter Media : Stainless Steel AISI 316L

Gaskets, '0' rings : PTFE/ Silicon.

According to another embodiment, the invention provides a one-step process for High Density Microbial Fermentation using the HDMF Filtration System. The process comprises: a) preparing a pre-coat solution in a pre-coat tank(53) with pure water by mixing an inert pre coat powder specially selected by introducing an inert gas N2 or Compressed dry air slowly, followed by circulating pre- coat solution by pumping over filter leaves in filter vessel to achieve proper pre-coat of desired thickness;

b) feeding the HDMF broth containing biomass with liquid product by pumping into the filter vessel through diaphragm pump (AA), to filter through the pre-coated filter leaves stack with simultaneous deposition of the biomass on pre-coated filter screens followed by allowing the clear filtrate passes out into Guard Filter (55) and then in to filtrate storage Tank (BB) for further downstream processing;

c) displacing the filter vessel holdup/heel volume as well as the entrapped liquid in the biomass/ wet-cake by slowly introducing clean dry compressed Air/ Nitrogen and then transferring back the same to feed tank if found turbid or pass through the Guard Filter (55) to Filtrate storage Tank (BB);

d) deactivating the biomass by introducing steam in the vessel at 121°C; e) slurring the deactivated biomass, by filter stack rotation by spraying hot water through spray nozzles for disposing;

f) draining entrapped water into Guard filter (55); and

g) cleaning and steam sterilizing the entire system as per SOP.

The novelty and inventiveness of the instant invention lies in the use of Pre coating the Filter stack in the rotary leaf filter with suitable Sterile Filter Aid (Chemically inert pre-determined pre-coat powder based on broth characteristic)to achieve particle free clear filtrate suitable for chromatography in one-step thereby eliminating, adversely affecting high purity water for dilution for HDMF broth for centrifuging and involving series of cartridge pre-filtrations and 0.45 and 0.22μηιίθΓοη fine filters. Thus the use of the instant HDMF filtration system with pre-coated rotary leaf filter (51) provides substantial cost reduction for broth filtration/separation.

The invention is described in stage wise manner herein below.

Pre-coating:

The pre-coat tank (53) is filled with water as required. The selected particular filter aid powder is slowly added while air is introduced to make homogenous mixture.

"Pre-coating' is a thin lto 1.5mm thick porous layer formed over metallic filter screen to filter HDMF broth to retain biomass and let out clear filtrate free of sub micronic suspended particles with least resistance.

Pre-coating is done by circulating mixture of pure water and inert Filter-Aid powder i.e. microcrystalline cellulose of highest purity (around 99% or better) of appropriate grade. The pressure during precoating is around 2kg/cm 2 and flow velocity 1.5 times or more that of the actual slurry to be filtered. Pre-coat powders as used in the invention confirm to pharmacopeia requirements or criteria from Pharm. Eur.II (inc. BP 93, BPC, DAB 10) and NF XVII. They are sterile and have different grades to suit the specific product requirements.

Pre-coating of the filter media according to the invention has the following advantages:

• Desired clarity is obtained quickly with minimal differential pressure. • The metallic filter media supports the precoat and the micro porous powder layer provides depth filtration assuring sparkling clear liquid by retaining sub micronic suspended solids.

• The metallic screen laden with non sticky powder does not clog and hence easy to clean and sterilize, assures longer life, consistency and integrity.

According to another embodiment, selection of pre-coat powder is important and depends on proper grade, particle shape, size distribution, surface area, surface charge, adsorption or absorption quality, process compatibility etc. The above criteria need to be critically considered while selecting precoat powder to ensure sparkling particle free clear filtrate. Similarly the characteristics of liquid i.e. specific gravity, surface and interfacial tensions, viscosity, nature, physical properties etc. are to be determined. Lab scale trials on actual broth are required for appropriate selection, determine quantity and precoating procedure. The selection of pre-coat may also depend on the microorganism involved in production of liquid products.

Based on preliminary selection of type, grade, quantity, number of layers, the actual trials were conducted for finalizing the pre-coat selection to set the operating procedure in place.

In a representative embodiment, the invention provides a one-step process for filtration having liquid product (Trypsin/Trypsinogen) that is secreted on the cells in the culture supernatant expressed by recombinant Yeast (Pichiapastories) in HDMF fed-batch fermentation. More than 18 different types of pre-coat Pharma grade Filter aid powders were tested to select most suitable pre-coat powder to filter the HDMF broth. Few such pre-coats are of Diatomite, Perlite and Cellulose origin

The process of Pre-coating should not take more than 15-20 minutes. The resultant differential pressure for pre-coating should be within 3 psig maximum and flow velocities 1.5 times the feed flow rate.

The quantity of Pre-coat powder required varies between 100 grams to 150 grams per square meter of filtering area. However it depends on nature of suspended solids and nature of liquid, broth etc.

The pre-coat layer thickness normally varies from 1mm to 1.5 mm. Further, it depends on nature of filtrate and wetcake, quantity and nature of suspended solids, size, shape etc. Pre-coating must be done using clean/ filtered fluid & on clean filter media. Under any circumstances the sticky particles in the feed should not reach filter screen/ cloth.

'Proper velocity & concentration' is the key for proper pre-coating, hence 0.2 to 0.3% concentrations of pre-coat powders are maintained in clean carrier solvent/liquid. Excess concentration results in settling of pre-coat at the bottom of the filter vessel. Clear filtrate is proof of filter integrity. Once pre- coating is satisfactory, circulation is stopped and filter vessel is drained to remove entrapped water as much as possible. Light purging of clean compressed air/N2 is good to dewater the pre-coat layer.

Filtration: The HDMF or otherwise broth is fed into the BASP Biotech rotary filter vessel keeping the vent valve crack open to allow only air to pass and avoid spillage / overflow. The vent/ overflow valve is closed as soon as filter vessel is full.

During filtration quick opening of valves will impinge the filter media and/or disturb the precoat layer. Filter stack is stationary during pre-coating, precoat dewatering, broth filtering, wetcaked watering under air pressure, vessel hold up/heel filtration and filter draining.

Feed circulation & filtration:

The HDMF broth is circulated until filtrate obtained is sparkling clear. The feed gets filtered through the pre-coat layer and screen filtrate passes through outlet. The biomass wet-cake gets deposited on the pre-coated filter screen. Circulation is continued till filtrate is clear. The clarity can be seen through the sight glass tube on the filtrate outlet line. Samples are collected for lab analysis. Once the filtrate is clear, the inlet valves on the Guard Filter and Filtrate receive rare opened to allow the filtrate to pass through into Filtrate Tank. The wetcake forms over filter plate stack. At the end of filtration the wetcake is dewatered for maximum product filtrate recovery.

Vessel hold-up filtration & Wetcake dewatering:

The filter vessel hold-up/heel volume can be transferred back to feed tank or filtered through the Guard Filter. Slow introduction of Air/ Nitrogen at 1 to 2 kg/cm 2 displaces vessel hold-up/heel volume as much as possible. Holdup filtration and dewatering may take half an hour or more, based on the wetcake characteristic. Wetcake Washing and Dewatering:

Wetcake washing is generally done as per process requirement by introducing water over wetcake broth under pressure. In filtering HDMF broth, cake washing is not done to avoid dilution of filtrate. However, dewatering is done by introducing clean dry compressed air/ N2 to collect entrapped liquid.

Deactivation of wetcake:

Once the wetcake is washed and biomass and precoat layer de watered, it can be deactivated at 0.15 kg/cm 2 or more 121°C by steam sterilizing, as per SOP.

Filter Cleaning In Position (CIP):

The filter is cleaned after deactivation of wetcake before and after every filtration batch as per SOP, to restore the filtration efficiency. Cleaning of filter while rotating filter stack and front-washing with jet sprays, is normal. It is possible to introduce air/ steam from filtrate side to loosen the entrapped particles if any for faster and reliable cleaning.

The filter stack is spun after filtration and dewatering to dislodge the wetcake. It is good practice to use spray wash to slurry the wetcake during filter stack rotation. The washed slurry can be drained and the filter screen can be cleaned by introducing compressed air from filtrate nozzle to open the blocked screen holes.

The present inventors have developed special spray header for effective cleaning of filter stack since HDMF broth is sticky and difficult to dislodge in position i.e. without opening the vessel. The spray header is provided with unique jet sprays positioned above each filter plate. The spray header position can be adjusted for cleaning.

The spray forces the wash liquid to penetrate into wetcake, loosen and slurries while the filter stack rotates at slow speed. The slurry is then drained.

After slurry discharge, once the filter is reasonably clean, the slurry outlet valve is closed and water level is maintained above filter plates while spinning/ rotating the stack at higher speed, to loosen the fine sticky particles if clogged in filter screen.

Backwashing:

In case, filter media gets choked it is possible to backwash the filter by gently introducing clean water while introducing Air/ N2 at 0.15 kg/cm 2 pressure to assist opening of clogged filter media pores.

Fill clean water keeping vent valve crack open, just above top plate and rotate the filter stack. Use water for cleaning generously. Repeat until the filter plates and filter vessel is clean. The number of cleaning cycles depends on the sticky/ slimy product reaching filter screen. Two or three washes ensure thorough cleaning. This can be seen through the view glass. Steam, Air, Detergent, Solvent etc. can be used as required. The complete system is Cleaned In Place (CIP) by circulating demineralised water throughout the system.

Sterilizing In Position (SIP): After CIP, the complete system is Sterilized In Position (SIP) by introducing steam at 0.15 kg/cm2 at 121°C Temperature. Dry Air/ N2 is blown for final drying before next batch.

The following examples, which include preferred embodiments, will serve to Illustrate the practice of this invention, it is understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

Examples: Example 1

Pre -coating:

Pre-coat Powder IKg was mixed in 30 Litres of water with the help of blowing inert air/nitrogen for homogenizing the slurry. Pre-coating was done by circulating the premix solution at flow rate around 25 LPM (litre per minute) for about 10 minutes. The filtrate clarity was found to be excellent.

Example 2

Filtration:

Typically, the technology was scaled to improve the yield of Trypsin/Trypsinogenpresent in supernatant- broth, which is produced from recombinant human Trypsin expressed on the yeast (Pichia pastories). Typically, a cultural supernatant containing 300/350 grams/litre of moist biomass produced in the bioreactor containing Trypsin /Trypsin ogen was fed to the filtration system of the present invention.

HDM Fermentation broth about (45 Litres), from the Ferm enter, having wet cell density of approx. 350gms/litre was fed to the instant Filtration system @ lKg/cm2 @ 21°C. Filtrate was not re-circulated. Once through filtration with Guard Filter in line was done.

Initially filtration was done under pump pressure. As filtrate flow reduced, clean compressed air was slowly introduced @ 1.5 kg/cm2 for filtering the biomass inside the filter vessel. 35 Litres of filtrate was collected in 15 minutes i.e. flowrate of 2.33 LPM. Filtrate was sent for lab analysis.

Filtrate was clear and acceptable as per lab analysis data shown below: Lab analysis:

Example 3:

Hold up transfer, wetcake deposition:

A thick and uniform wetcake bed was formed. It was observed that the top biomass layer was around 15 mm formed over a bottom pre-coat layer of around 3 mm. Since the biomass had filled 75% of the gap between leafs & hence no more filtration was done, the remaining 10 Litres of unfiltered biomass slurry i.e. filter vessel holdup was transferred back to feed vessel for filtering in next batch. Example 4

Filter cleaning & sterilizing (CIP/ SIP):

Filter stack was cleaned in position by spraying water through spray header while rotating the filter stack. Cleaning cycles took 30 minutes. After cleaning dry air was blown for 5 minutes. Finally the filtration system was sterilized in place for 30 minutes for reuse in next batch.

Example 5A

Comparison of the instant filtration system vis-a-vis existing separation systems / methods and parameters employed in operating is provided below.

Example 5 B Comparison of the downstream processing unit employed in the instant invention with the conventional method with reference to Fig 4 is provided below:

Components Equipment Conventional Present Invention in Fig 4

21 Conductivity Adjustment Unit Required Required

1 1 Buffer Storage Tank 2 KL 2 KL

(Glycin/CaC )

12 Buffer Storage Tank (NaCl) 2 KL 2 KL

Ion exchange chromatography (IEC) 2X 40L

Quantity of Feed to be 235KL 12KL Max

handled

14 Storage tank 1 KL 1 KL

15A/B Ultra & Diafiltration Unit with 4M 2 0.25M 2

Pump

Quantity to be processed 200,000 10,000

16 Feed Tank 200 1 200 1

17 Storage (HCl/CaCl 2 )Tank 200 1 200 1

18 CIP Tank 200 1 200 1

19 Final Product Tank 50 1 50 1

20 Heat exchanger - -

22 SIP/CIP Steam Sterilization and cleaning in place

(SIP-CIP) system for all tanks @121 °C; 20 min.

HDMF Broth Dilution with clean/high purity water adversely affecteddownstream purificationcausing the gel in the chromatography column to weaken and also causes tremendous load on Diaiiltration system.

Diaiiltration system with 4M 2 was not required for the instant HDMF Broth filtration purification system since the system does not require the dilution of the feed. However smaller size unit of Ultra / Diafiltration with 0.25M 2 membrane area sufficed. Industrial advantage:

The invention provides a filtration - purification system employing a one step process which is aseptic, easily scalable, uses lower pressure (e.g., less than 2K/cm 2 ), consumes minimal electrical power and does not require high purity water for dilutions that adversely affects downstream processing prior to centrifugation, intermittent storage tanks with pumps, heat exchangers, conductivity adjustments before microfiltration, biomass deactivation system and use of series of pre-filters and 0.22 μ membrane fine filters to filter enormous amount of diluted broth.

This novel one step filtration process eliminates need of high purity water required for multiple dilutions (20 to 30 times), besides that the dilution with high purity water adversely affects the downstream purification including the gel in ion chromatography column and the product, also dilution affects pH of the broth. Moreover, in the instant invention addition of buffers or other preservatives as additives is not required.

In conventional HDMF filtration systems, dilution increases the fermentation broth volume to 220,000 I to 330,000 I requiring a processing time of 120 hrs as against the present invention wherein volume of the undiluted HDMF broth in the range of 10KL or even lesser is produced in 15 KL fermenter requiring downstream processing time of 8 hrs. This further helps to resize the Ion Exchange Chromatography and ultra/ diafiltration purification system. The instant HDMF filtration system needs very low investment, negligible consumption cost, saves enormous energy cost, labour cost, ensures perfect product quality, drastically reduces processing time, improves and ensures product purity.The overall cost savings by using the invention is estimated to be around 60%. The cost savings is attributed to saving of high purity water due to the elimination of multiple dilutions and not using multiple micron filters at each filtration stage i.e. 10μ/5μ,0.45μ and 0.22μ. In prevalent commercial processes, multiple filters used after each centrifugation step, are not required by this invention.

The filtrate thus obtained is particle free and suitable for direct chromatography having 11.5NTU. The washing and deactivation of biomass can be done in the filtration unit itself after which the spent biomass can be sold for value.

The biomass can be squeezed after deactivation of spent biomass and can be sold for value.

Therefore, the one step Process for filtration of Microbial HDMF and recombinant using the Microbial HDMF and recombinant Filtration System of the instant invention is reliable, simple, user friendly and most economical for HDMF filtration. The invention is highly scalable with zero discharge of effluent.

The present invention is an enormous development in industrial biotechnology to derive high value recombinant and medicinally important biological molecules. 32