|Title of Invention||
"A DEVICE FOR DIALYSIS/PURIFICATION OF PROTEINS AND METHOD THEREOF"
|Abstract||In existing traditional methods of dialysis/purification of protein samples, the sample loading and unloading process is cumbersome involving clamping or tying of the semipermeable membrane, costing time and effort, and also resulting in losses, especially when sample volume is small. Modern methods employing tubes fitted with semipermeable membranes, require expensive centrifuges for their operation. A recent (1996) patented invention of Pierce Co. USA (US Patent number 5,503,741), involves uses of 'Dialysis Casette". The device, though an improvement over traditional methods, has several limitations viz. inflexibility regarding sample volume, cumbersome and expensive sample loading/collection process involving use of syringes and needles, non-reusability of casettes and high cost. In contrast, our invention offers several advantages. Low-cost, simple device, not requiring any sealing of membrane with thread or clamp. Offers considerable flexibility regarding volume of sample to be processed and does not require any external equipment e.g. centrifuge,for its operation. Sample loading is extremely easy and safe, as per design of device. No syringe, needle or any other external device is required. The sample recovery process is quick, efficient and simple. The unit is cost-effective as it can be used any number of times and need not be discarded after single-use. Only the membrane requires replacement. Mass production as well as scale-up of the device is also easy owing to simple design.|
|Full Text||This invention relates to a device for dialysis/purification of proteins and method thereof. The invention proposes a device which is simple to operate and low in cost and offers an easy loading and recovery of protein sample with a minimal sample loss.,
Traditional tubing dialysis/protein purification methods are cumbersome, involving boiling, soaking, tying or clamping of membranes and costing several hours of time and effort. They also result in loss of precious protein sample loss during processing. Improved modem methods employ commerciallt available tubes, having membranes fitted inside. When these tubes loaded with sample are centrifuged, smaller particles e.g. salts, low molecular weight contaminant etc. pass through, while the larger particles i.e. the proteins are retained. By employing membranes of different cut-off values, proteins of different molecular weights can be separated. The process though quick and efficient, has limitations.
Firstry, these tubes cannot be operated without a centrifuge, and as separation/purification of proteins is usually carried out at 4°C, a refrigerated centrifuge might become essential. Since a refrigerated centrifuge is expensive and not all lavs may possess it, especially uynder Indian conditions, the utility of such methods beomes limited. Secondly, the semipermeable membranes are fitted inside tubes of a fixed volume, which restiricts flexibility regarding sample volume which can be processed in a single unit. Thirdly, the units are very expensive, being imported and not manufactured in India.
A recent patented invention of pierce Co. USA (US Patent number 5,503,741), introduced in 1996 involves uses of Dialysis Casette' (detailed literature enclosed). The product consists of a semipermeable memberane sealed in a plastic frame. Sample is loaded and recovered with help of syringes. The device though an improovent over traditional methods of dialysis, has limitations.
Firstly, the semipermeable membrance in fixed in a frame, which restrictrs flexibility regarding volume of sample to be processed i.e. cassettes are of fixed size. For large volumes, multiple cassettes have to be used for same sample or larger sized cassettes have to be use.
Secondly, sample loading as well as recovery, involves use of syringe and needle which causes sample loss due to sticking. The losses can be significant when
handling small volumes. In case of multiple samples, a number of syringes equal to number of samples, are required which makes the loading and recovery of sample, time consuming, tedious and expensive besides posing the risk of accidentally puncturing the membrane or causing injury to user. Thirdly, cassettes are not reusable, and entire unit has to be discarded after single use. Fourthly, removal of membrane protecting agent viz. glycerol, from the membrane is quite cumbersome, requiring injection of distilled water into the cassette and running it in distilled water for a minimum of four hours. Lastly, the cassettes being imported from abroad are quite expensive. The fact that they cannot be reused, is also a limitation.
STATEMENT OF INVENTION
According to this invention there is provided a device for dialysis/purification of proteins comprising an upper hollow float chamber (a) which is closed at one end and open at the other end; a middle air tight seal(c) which is detachable and can be pressfitted over the open end of the said upper hollow float chamber; and at least one lower semi-permeable membrane sac disposed in the said airtight seal over the said aperture, wherein the said airtight seal has at least one aperture which makes the said lower membrane sac in flow-communication with the said upper hollow float membrane.
Further, according to this invention there is provided a method for dialysis/purification of proteins through the device comprising of putting the protein sample to be purified into the interior of one of semipermeable sac via the corresponding aperture in the said air tight seal, press-fitting the seal to the open end of the said hollow float chamber,
floating the device in a tank of buffer/distilled water for subjecting the sample to dialysis/purification as herein described.
The present invention is considerably more effective than previously known devices used for the same purpose and does not suffer from the aforementioned disadvantages. It offers several advantages, as detailed below:
i) Low-cost, simple device, which does not require any sealing of
membrane with thread, clamp etc., making the assembly process
quick and easy, ii) Offers considerable flexibility regarding volume of sample to be
processed. The same device can be used to process a wide range of
sample volumes, iii) Does not require any external equipment e.g. centrifuge for its
operation, iv) Sample loading is extremely easy, as sample can simply be poured
into the semipermeable membrane sac, as per design of device, v) The sample recovery process is quick, efficient and simple,
involving only inversion of device. Processed sample remains
untouched by hand, vi) Safe and easy sample loading and recovery, as no syringe, needle
or any other external device is required, vii) Since membrane is not fixed in any frame, but is provided
separately, its washing to remove any protecting agent is fast, easy
viii) The unit is cost-effective as it can be used any number of times and need not be discarded after single-use. Only the membrane requires replacement,
x) Low-cost, mass production of the device can easily be carried out, owing to its simple design,
The invention will now be described with reference to the accompanying drawings:
Sheet 1: Ilustrates the various components of the device. Sheet 2: Illustrates the process of assembling the device. Sheet 3: Illustrates operation of the device and the sample recovery process.
The device has the following components as indicated in Figure 1:-
a. A hollow cylindrical chamber, open at one end and closed at the other, made
of flexible plastic material, preferably transparent and chemically inert.
b. The hollow chamber top is flat and ensures that chamber can be conveniently
placed on any flat surface for sample recovery, without any need for external
support, rack etc.
c. A circular, detachable, air-tight seal with central aperture and made of flexible
plastic material. The seal fits tightly over the open end of the float chamber,
closing it effectively. The aperture provides passage for semi-permeable
membrane sac, sample loading as well as collection and also helps connect hollow
float chamber with semi-permeable membrane sac.
d. A semi-permeable membrane. This is in form of cut-sheets, which may be of
any geometrical shape. For convenience, circular cut-sheets are preferred.
Membranes with different cut-off values can be used, depending upon molecular
weights of proteins to be separated.
e. A float sheet with central aperture corresponding to diameter of float-chamber,
for keeping the device floating vertically. A larger sheet with several apertures can
be used for running muliple units simultaneously. The float sheet however is not
an essential part of the device, for if the hollow chamber is of a shape that it won't
topple over on being floated, use of float sheet can be avoided.
The device is assembled as illustrated in Sheet 2, as follows:-
Fig. 2 Semipermeable membrane sac formation- The sheet of semipermeable membrane is placed over the central aperture of the air-tight seal, on side which faces the float chamber on sealing , and with help of a glass rod, gently pushed
through the aperture. A sac is formed on removing the glass rod. The depth of
the sac formed can be easily adjusted depending upon the volume of sample to be
processed. In case of larger volume, more of the membrane can be pushed
through the aperture, forming a deeper, longer sac.
Fig.3 Sample loading - The sample to be processed, is poured directly into the
sac formed, via the opening at top of sac. This is quite easy, as opening is wide
enough. Alternatively, sample can also be poured into float chamber, as on
inversion, sample will automatically drain and fill the semi-permeable membrane
Fig.4 Sealing of semipermeable membrane sac, loaded with sample- This is done
by pushing the air-tight seal against edges of hollow float-chamber, so that it fits
tightly and forms an air-tight seal. The sac of semipermeable membrane, loaded
with sample is now sealed.
Fig.5 Attachment of float-sheet- The float-sheet with central aperture is passed
over the closed end of float chamber, till it comes to rest against rim of seal.
Device is now fully assembled and ready for use.
Referring to Sheet 3 for operation of device, it is carried out as follows:-
Fig.6 The fully assembled device is floated in a beaker/chamber filled with distilled water/buffer, as per methodology requirement. The compact size of the device enables it to be floated in a small beaker/container which can be conveniently placed in a refrigerator, if the requirement is for cold conditions during the dialysis/protein purification process.
Fig.7 After end of dialysis/ protein purification process, the sample is ready for recovery. The device is removed from the beaker/chamber containing buffer, inverted i.e. the float chamber is lower to dialysis sac, and placed on a flat surface. The flat end of the float chamber enables it to be conveniently placed on bench-top or in refrigerator.
Fig.8 Within a few minutes, the processed sample under the influence of gravity, drains from the semipermeable membrane sac, down into the float-chamber. Fig.9 The air-tight seal is removed and the sample can be poured out or processed ( e.g. lyophilized) in the container itself and thus remains untouched by hand.
Since the process does not involve any tying, clamping, use of syringe and needle etc. for loading or recovery of sample, the device makes dialysis/protein
purification simple, quick, safe and efficient. The ease of assembly and operation, flexibility in terms of sample volume processing and rapid, simple method of recovering the processed sample makes it extremely useful for laboratory procedures involving dialysis/protein purification.
The preparation of membrane sac, the assembly, the utilization, the dismantling, the sterilisation and the cleaning of the whole of the device according to the invention, are simple. The apparatus has overall small dimensions and is readily transportable.
Naturally, embodiments of the principle of the invention other than described by way of example may be carried out, without departing from the scope of this invention. For example, hollow chamber may be of a shape other than cylindrical, and may have conical top than flat, for convenient collection of small sample volumes. The conical top can be encased in a collar, to enable the device to stand on a flat surface on inversion. Advantageouly, the hollow chamber may have open top or a top which can be opened, thus eleminating the need for inversion. Moreover, material of construction of device can be varied and need not be plastic alone. Glass or any other suitable material can be used. Similarly, air-tight seal may have a shape other than circular and aperture in the seal may not necessarily be central or of any particular shape. The thickness of seal may also be varied depending upon convenience of use and constructing material, which can be any suitable material apart from plastic. The seal can be of "push-type" without any threads, or internally screw-threaded for engagement with externally threaded open mouth of hollow chamber, thus acting like " screw-type" lid, the basic function remaining unchanged i.e. to connect hollow chamber with membrane sac. „ Membranes formed by supports of various materials and covered with coatings of various nature may be employed for semi-permeable membrane sac formation. Alternatively, prefabricated sacs/tubes, available separately or attached to seal, can be used. The semi-permeable membrane sheets may be of natural or synthetic material and may or may not be provided with additional support e.g. of textile or other material. The sac may be elongated sufficiently to form a long tube, to carry greater volume of sample, may take various forms and be movable or stationary, the basic function being to act as semi-permeable barrier between sample and the surrounding medium i.e. buffer or distilled water. For special requirements involving processing of sample under sterile conditions, autoclavable material or any other material which can suitably be sterilized, may be used in construction of device.
1. A device for dialysis/purification of proteins comprising an upper hollow float chamber (a) which is closed at one end and open at the other end; a middle air tight seal(c) which is detachable and can be pressfitted over the open end of the said upper hollow float chamber; and at least one lower semi-permeable membrane sac disposed in the said airtight seal over the said aperture, wherein the said airtight seal has at least one aperture which makes the said lower membrane sac in flow-communication with the said upper hollow float membrane.
2. A device as claimed in claim 1 wherein said hollow float chamber is cylindrical with a flat top(b).
3. A device as claimed in claim 1 wherein the said hollow float chamber is made of flexible plastic.
4. A device as claimed in claim 1 wherein there are multiple membrane sacs of different pore size to separate molecules of different molecular sizes.
5. A method for dialysis/purification of proteins through the device as claimed in claim 1 comprising of putting the protein sample to be purified into the interior of one of semi-permeable sac via the corresponding aperture in the said air tight seal, press-fitting the seal to the open end of the said hollow float chamber, floating the device in a tank of buffer/distilled water for subjecting the sample to dialysis/purification as herein described.
6. A device for dialysis/purification of proteins and method thereof as herein described and illustrated with reference to the accompanying drawings.
|Indian Patent Application Number||2088/DEL/1997|
|PG Journal Number||29/2008|
|Date of Filing||25-Jul-1997|
|Name of Patentee||Dr. Parikshit Bansal|
|Applicant Address||Animal Biochemistry Division National Dairy Research Institute, Karnal-132001, India.|
|PCT International Classification Number||B01D 61/24|
|PCT International Application Number||N/A|
|PCT International Filing date|