This invention relates to Versatile Spin Filter Module
FIELD OF INVENTION:
The present invention relates generally to bioreactor for growing cultures. More particularly, it relates to spin filter perfusion technology preferably for effectively growing both adherent as well as non-adherent mammalian cells.
BACKGROUND OF THE INVENTION:
Increased demand for the biological products such as factor VIII, monoclonal antibodies, Urokinase, tissue-plasminogen activators and recombinant proteins has necessitated increase in production of such products. Due to complex structure of these products, mammalian cells are commonly employed as host cell for production of these biological products. Since the mammalian cells have low growth rate and productivity (e.g. cell density: usually 1.5 to 2xl06 cells /ml and doubling time usually 15 to 30 hrs), need for developing efficient technology to improve proliferation of mammalian cells, volumetric productivity, and production of protein has become imperative. Additionally growing demand of these products motivated the development of more efficient and reliable cell culture production technologies. The perfusion technology is known to increase cell concentration. Further spin filter perfusion technology being the best cell separating device available, are commonly employed for enhanced volumetric productivity, and production of protein. Despite the advantages of the increased animal cell concentration to 1-2 x 107 compared to 1.5 to 2x106 and productivity of protein averaged to 660mg/l/d with spin filter perfusion system, it has some inherent disadvantages with regard to limited scale up potential, unreliability, contamination risk, equipment failure, increased analytical costs long validation time.
Typical Spin-filters are cylindrical wire cage with a porous wall, which are placed internally or externally to the bioreactor. The cells are cultured by continuous
addition of fresh nutrient medium to the outside of the spin-filter and continuous removal of the spent medium from within the spin-filter. The maximum cell density that can be reached in such a process (without considering physiological limitations of the cell itself) depends upon the capacity of the spin-filter device to minimize cell loss at a specific perfusion rate and on the longevity of the culture.
In perfusion culture, cells are selectively retained in the bioreactor with continuous feeding of nutrients and spent medium withdrawn so that dead cells, cell debris, inhibitory by products and enzymes released by dead cells are selectively removed. To meet the increasing market demand, research has been conducted to increase the bioreactor volumetric productivity by various methods, such as novel bioreactor design and feeding strategies for perfusion culture. Presently available bioreactors largely have filtration based cell retention system and possess Spin filter modules made up of stainless steel, synthetic polymers, ceramic and polyester membrane modules. However, these units have different limitations. For example, stainless steel modules have limited longevity because the negatively charged cells prefer to attach on to the positively charged stainless steel scaffold which therefore get rapidly clogged. Consequently, toxic metabolites such as lactic acid and ammonia accumulate and results in low cell density and viability. Synthetic screens are constructed from polymers such as ethylene-tetrafluoroethylen (ETFE), polyamide or poly tetrafluoroethylen (PTFE). Since the cells, proteins and DNA are negatively charged show advantage over metallic screen. However, hydrophobic nature of protein play important role in adhesion to filter surface thereby resulting in filter fouling. This necessitates coating of hydrophobic surface by hydrophilic substance. The ceramic membrane also proved to be unsuccessful as the material appears to be not suitable for prolonged application. Similarly, polyester membrane based retention modules are manufactured as disposable units and are available in only certain standard sizes. Hence, the operating costs are higher and the results are not scalable. Apart from filter material, weaving pattern and orientations do help in providing solutions to fouling of filters. The other simple approach to attempt to reduce
fouling is providing rotating filters. These systems use fast rotating cylinder filters mounted either on the stirrer shaft or driven independently by a rotor. As can be seen from the above description, the draw backs associated with existing filter modules are:
 limited longevity due to clogging of screens,
 low cell density and viability.
 filter fouling and requiring coating of hydrophobic inability
 limited scale up potential, unreliability,
 contamination risk,
 equipment failure,
 increased analytical costs long validation time.
The inventors after prolonged R&D have been successful to develop a VSFM that obviate the drawbacks of the existing spin filters as herein before described. The VSFM of the present invention eliminates using modules made of steel, synthetic polymers, ceramic and polyester membrane modules. The spin modules of the present invention comprises of neutral, hydrophobic, biocompatible membrane, being mounted on chemical, temperature and corrosion resistant polymeric support
OBJECTIVES OF THE INVENTION
The main objective of the present invention is to provide a Versatile Spin Filter Module obviating the drawbacks associated with the existing spin filters for bioreactor.
The other objective is to provide a filter module that can be used for both adherent and non adherent cells
Another objective is to provide a filter module that can be used for mammalian cell culture.
Yet other objective is to provide a filter module that can withstand high temperature (autoclavable) is reusable and economical.
Yet other objective is to provide a filter module that is industrially feasible, economical, and can withstand clog-free operation and longer cycle time.
Still other objective is to provide a filter module that can promote high cell density and high volumetric productivity.
DESCRIPTION OF THE FIGURES:
Figure 1 (a - i) illustrate the different views of the features of the spin filter
module and methods of their arrangements.
Figure 2 depicts the properties of the membrane used in spin filter module.
Figure 3 illustrates Comparison of viable cell density and therapeutic protein productivity on different biocompatible membranes.
Figure 4 illustrates Comparison of viable cell density, cell retention and therapeutic protein productivity profile in stainless steel spin filter (ss) and VSFM.
Figure 5 shows Comparison of viable cell density, cell retention and therapeutic protein productivity profile in batch culture in T-flask (B) and perfusion culture with VSFM
Figure 6 illustrates Graphical results for stability and durability of VSFM at different rotation speed and dilution rate in terms of cell retention.
STATEMENT OF INVENTION:
Accordingly the present invention provides a versatile spin filter module comprising a cylindrical chemical and temperature resistant polymeric support designed for mounting membrane, a neutral, hydrophobic, biocompatible membrane, the said cylinder being provided with a bottom plate adapted to fixing on a shaft for inducing rotation, and a detachable clamping system for facilitating mounting of membrane.
According to one of the embodiment of this invention chemical and temperature resistant polymeric support may be constructed/fabricated/ manufactured from any of the following material: Poly (lactic acid) (PLA), polycarbonate, polyolefin (polyethylene, polypropylene), fluoropolymers (Teflon etc.), acrylic polymers, poly (vinyl chloride) PVC, polycaprolactone, poly (vinyl alcohol) (PVA) & silicone rubber (polydimethylsiloxanes). The support may be scalable between reactors of 0.5 L volume to 100 L volume. The diameter of the module may vary from 5 to 100 cm.
According to other embodiment of the present invention, a neutral, hydrophobic, biocompatible membrane may be fabricated from natural polymer such as rubber, silk, polyester and cellulose preferably from non-woven silk obtained from Sericare Bangalore.
According to another embodiment of the present invention, the pore size of the neutral, hydrophobic, biocompatible membrane may be varying from 5 to 30 microns.
According to yet other embodiment of the present invention, the detachable clamping system for facilitating mounting of membrane comprises simple wire tension attachment, clamping using a screw system to secure the mounted membrane, clamping using O-rings at multiple positions along the axial surface of the support structure, clamping using a wedge slide fabricated from the same material as the support.
According to yet other embodiment of the present invention, the bottom plate may be a conical piece and fixed to the shaft by inserting an Allen nut, threading and screwing, machining a quick coupler, fabricating a cross bar on the bottom conical piece which can be push fit into the impeller shaft.
According to yet another embodiment of the present invention, the bottom plate and screw used for fixing the said plate with shaft and the clamping system may be corrosion resistant material Co-Cr alloys or stainless steel.
According to still other embodiment of the present invention, the O-rings used for mounting/clamping membrane on polymeric material may be made of biocompatible material such as nylon, silica, rubber.
DETAILED DESCRIPTION:
The present invention is a bioreactor that uses spin filter technology. The bioreactor of the present invention solves the problems inherent in conventional spin filter based bioreactors. The bioreactor of the present invention further provides features heretofore unavailable in conventional spin filter bioreactors.
The versatile spin filter module (VSFM) of the present invention works for both adherent and non adherent cells and placed internally in the bioreactor. It is an internal membrane based rotating cell retention system that features scalability, biocompatibility, autoclavibility/sterizability by common techniques, selective cell retention, clog-free operation, non-fouling nature, no shear stress during rotation, and operated for longer duration at high perfusion rate. It was constructed using high quality steel and polymeric material. Its polymeric support is designed for quick mounting of the biocompatible membrane. The biocompatible membrane and its pore size can be chosen according to the size of cells cultured.
As shown in Figure 1(a), the versatile spin filter module comprises of a Polymeric support (2) for mounting a membrane (5) that can withstand high temperature, chemically inert and corrosion resistant, a clamping system comprising a clamp
lock (1) and O-Ring (4) for holding and securing the membrane, bottom conical
plate (3) adapted suitably for fixing the filter on shaft to induce rotation.
Fig 1(e) further illustrates the top and bottom view of the versatile spin filter
module (VSFM)
The polymeric support material can be used as the support structure for mounting
the membrane. Its function may be expanded to include but not be limited to the
following utility: (i) Structure support for membrane (ii) structure and separating
medium/membrane combined. The method of creation or fabrication of the
material is such that it satisfies the biocompatibility properties required of a
material for performing cell culture, the required pore size distribution for cell
retention and the required surface properties for either cell retention or cell
adhesion.
The support structure is made up of biocompatible autoclavable, corrosion
resistant, neutral, not conducive to attachment and chemical resistant any of the
following polymeric material: Poly (lactic acid) (PLA), polycarbonate, polyolefin
(polyethylene, polypropylene), fluoropolymers (Teflon etc.), acrylic polymers,
poly (vinyl chloride) PVC, polycaprolactone, poly (vinyl alcohol) (PVA) &
silicone rubber (polydimethylsiloxanes). It is generally contains cell culture and
cell culture media. It is further formed to have mesh structure and scalable
between reactors of 0.5 L volume to 100 L volume. The diameter varies between
5 and 100cm. Figure 1(f) shows the top and bottom view of the polymeric
support.
The O-Ring is made of biocompatible polymer such as nylon, silicon, rubber and
the detachable clamp lock is made up of corrosion free metals such as Co-Cr
alloys, stainless steels. Figure 1 (d) shows the enlarged view of the clamp lock and
Figure 1 (g) illustrates the details of mounting and securing the membrane on the
polymeric support.
The membrane is mounted, clamped and secured to the support structure by simple wire tension attachment. The membrane is simply rolled over the support structure and the ends brought together and secured by clamping the wire spring
attachment. The tension in the wire gives strength to the securing mechanism. Alternative devices can be visualized as the use of multiple positions clamping using a device which is a modified version of the one as shown Figure 1 (g): i. Clamping using a screw system to secure the mounted membrane.
ii. Clamping using O-rings at multiple positions along the axial surface of the support structure.
iii. Clamping using a wedge slide fabricated from the same material as the support.
The membrane used is fabricated from non woven silk. It is obtained from Sericare, Bangalore. The process of preparing this membrane has already been patented under name of Mr. B.Tandon Indian patent number 245840. However, the properties of membrane such as neutral surface, pore size for cell retention, non fouling property, non supportive for mammalian cell attachment and hydrophobicity are not the part of that patent. These belong entirely to the new work that was carried out and reported validating the application of membrane as screen material for spin filter for mammalian cells. This work is new and not reported/published elsewhere.
Silkworm is placed on a substantially flat surface which is free of anchor points (so that the silkworm cannot spin a cocoon across anchor point). As silkworm cannot find a suitable place to begin spinning its cocoon, it moves on the flat surface provided randomly and releases silk liquid through its spinneret. Several silkworms when are placed on the same flat surface the process of random movement of silkworms and liquid silk coming out from them leads to the formation of crisscross silk threads. Combination of several such threads leads to planar silk sheet. The silk sheet so obtained is cut into suitable sizes for further application and sterilized by gamma irradiation. Silkworm used for making flat silk sheet is Bombix Mori. The flat silk sheet is catalogued as Seri-FSS by Seri care, Bangalore. The pore size of the membrane varies from 5 to 30 microns.
Three different biocompatible membranes BM1, BM2 and BM3 were selected and their properties were analyzed as tabulated in the figure 2.
The bottom conical plate (3) adapted suitably for fixing the filter on shaft to induce rotation has been shown in further details in figure 1(b) (top view) and figure 1 (c) (bottom view). The plate is made of corrosion resistant Co-Cr alloys, stainless steels and is used to fix the filter on the shaft for the purpose of rotation.
The versatile spin filter module is attached or fixed to the impeller shaft of the reactor in a number of ways Fig.l (h) including but not limited to the following: i. Inserting an Allen nut through a hole drilled in the bottom conical piece.
ii. Threading the impeller shaft on the outside and screwing in the bottom corneal piece.
iii. Machining a quick coupler at the bottom of the shaft onto which the conical bottom piece can be mounted and fixed by turning.
iv. Fabricating a cross bar on the bottom corneal piece which can be push fit into the impeller shaft.
The VSFM is provided with the harvesting device (not shown in the drawing) consists of a tube (metallic or polymeric) reaching to the bottom of the versatile spin filter unit. It also has the option of including tube attachments to reach different depths of the module based on the process requirement. It consists of a quick coupler, tube (whose length and diameter can be varied) and connected to the main harvesting pump of the reactor.
The application of the versatile spin filter module covers all types of cell culture including but not limited to microbial, to plant, to insect and mammalian cell culture. The cell culture may be adherent or non-adherent depending on the process. The design of the versatile spin filter module is such that it can be easily
used with any membrane materials fabricated to meet the size specifications and cell culture requirements.
The versatile spin filter module and its accessories are made up of chemical and temperature resistant polymers and metals, since it is not affected by media constituents, it can be easily cleaned, and sterilized by chemicals and autoclaving.
Adherent cells can be cultured with versatile spin filter module either in single module with microcarrier beads or with dual module without microcarrier beads. In the dual module two types of membrane used with VSFM. In this design variation, there are two concentric cylindrical units for mounting the membranes. The inner cylinder has a membrane not conducive to cell attachments while the outer cylinder has a membrane that promotes cell attachment. Thus, while the outer cylinder membrane yields a monolayer of desired cells, the inner layer prevents clogging and easy removal of toxic metabolites and waste products from the reactor. This mode can be used for culturing corneal cells, skin cells etc. Fig l(i).
Figures 3 to 6 exhibit the efficacy of the VSFM of the present invention. Figure 3 illustrates Graphical Results of Performance of Different Biocompatible Membranes Used as VSFM Screen Material.
Suitable screen material, BM1, BM2 and BM3, was selected based on improvement of viable cell density and therapeutic protein productivity analyzed at different time intervals.
Figure 4 shows graphical results of performance of stainless steel spin filter and vsfm for non adherent hybridoma cells.
Performance of the two units was compared using three different parameters viable cell density, cell retention and therapeutic protein productivity estimated at different time intervals.
Figure 5 shows graphical results of performance of batch culture and perfusion culture with vsfm for adherent mammalian cells.
The performance of VSFM as a retention unit for adherent cells in batch culture (T-Flask) and perfusion culture (VSFM) was compared using three different parameters viable cell density, cell retention and therapeutic protein productivity, estimated at different time intervals.
Figure 6 shows graphical results for stability and durability of vsfm at different rotation speed and dilution rate.
The stability and durability of VSFM as a retention unit for adherent and non adherent cells in perfusion culture (VSFM) was estimated by running the VSFM at three different rotation speed 30rpm, 45rpm and 60rpm and at four different dilution rates (.05/hr, .06/hr,.09/hr and 0.12/hr) and cell retention was estimated at different time intervals.
The foregoing description is primarily intended for the purpose of illustration. The VSFM of the present invention may be embodied in other forms or carried out in other ways without departing from the scope of the invention. Modifications and variations such as altering size, shape, pore size, perfusion rate, readily apparent to the person skilled in the art still fall in the scope of this invention though not explicitly disclosed.

WE CLAIM:
1. A versatile spin filter module (VSFM) comprising at least one cylindrical chemical and temperature resistant polymeric support (2) designed for mounting membrane, a neutral, hydrophobic, biocompatible membrane (5), the said cylinder being provided with a bottom plate (3) adapted to fixing on a shaft for inducing rotation, and a detachable clamping system for facilitating mounting of membrane.
2. The VSFM as claimed in claim 1 where in the polymeric supports with membrane mounted on it are placed concentrically when more than one support is employed.
3. The VSFM as claimed in claim 1 where in the chemical and temperature resistant polymeric support is corrosion resistant and made of from any of the following material: Poly (lactic acid) (PLA), polycarbonate, polyolefin (polyethylene, polypropylene), fluoropolymers (Teflon etc.), acrylic polymers, poly (vinyl chloride) PVC, polycaprolactone, poly (vinyl alcohol) (PVA) & silicone rubber (polydimethylsiloxanes).
4. The VSFM as claimed in claim 1 where in the polymeric support is a mesh structure and scalable between reactors of 0.5 L volume to 100 L volume.
5. The VSFM as claimed in claim 1 where in the diameter of the module varies from 5 to 100 cm.
6. The VSFM as claimed in claim 1 where in a neutral, hydrophobic, biocompatible membrane is fabricated from natural polymer such as rubber, silk, polyester and cellulose preferably from non-woven silk by patented process (245840) and obtained from Sericare Bangalore.
7. The VSFM as claimed in claim 1 where in the pore size of the membrane ranges from 5 to 30 microns.
8. The VSFM as claimed in claim 1 where in the detachable clamping system comprises of a detachable clamp clock (1) and an O ring (4).
9. The VSFM as claimed in claim 1 where in detachable clamping system comprises simple wire tension attachment.
10. The VSFM as claimed in claim 7 where in the O-Ring is made of biocompatible polymer such as nylon, silicon, rubber and the detachable clamp lock is made up of corrosion free metals such as Co-Cr alloys, stainless steels.
11. The VSFM as claimed in claim 1 where in the bottom plate is a conical piece and fixed to the shaft by inserting an Allen nut, threading and screwing, machining a quick coupler, fabricating a cross bar on the bottom conical piece which can be push fit into the impeller shaft.
12. The VSFM as claimed in claims 1 and 10 where in the bottom plate and screw used for fixing the said plate with shaft and the clamping system is made of corrosion resistant material such as Co-Cr alloys or stainless steel.