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WO2014186819A1 - Procédé de formation de membranes de polyacrylamide - Google Patents

Procédé de formation de membranes de polyacrylamide Download PDF

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Publication number
WO2014186819A1
WO2014186819A1 PCT/AU2014/000376 AU2014000376W WO2014186819A1 WO 2014186819 A1 WO2014186819 A1 WO 2014186819A1 AU 2014000376 W AU2014000376 W AU 2014000376W WO 2014186819 A1 WO2014186819 A1 WO 2014186819A1
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WO
WIPO (PCT)
Prior art keywords
casting
membrane
process according
membranes
plates
Prior art date
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Ceased
Application number
PCT/AU2014/000376
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English (en)
Inventor
Hung Yoon Choi
Hani NUR
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NuSep Holdings Ltd
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NuSep Holdings Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2013901775A external-priority patent/AU2013901775A0/en
Application filed by NuSep Holdings Ltd filed Critical NuSep Holdings Ltd
Publication of WO2014186819A1 publication Critical patent/WO2014186819A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/40—Polymers of unsaturated acids or derivatives thereof, e.g. salts, amides, imides, nitriles, anhydrides, esters
    • B01D71/401—Polymers based on the polymerisation of acrylic acid, e.g. polyacrylate
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
    • B01D67/0002—Organic membrane manufacture
    • B01D67/0006—Organic membrane manufacture by chemical reactions

Definitions

  • the present invention relates to an improved process to form polyacrylamide membranes for biological separations using electrophoresis.
  • membranes are manufactured and used for separation of biological samples.
  • a specific eiectrophoretic separation teehnoiogy, PriME ⁇ Preparative Isolation by Membrane Electrophoresis) has generally used membranes that are thin polyacrylamide membranes which are produced in between two glass plates to achieve a glossy surface with desired thickness.
  • This production process has inherent safety concerns associated with multiple handling of glass plates and the weight of glass can limit the number of membranes produced per batch.
  • the use of an alternative to glass plates such as plastic films has reduce the risk associated with manual handling of glass and increased the batch production scale.
  • the solution phase polymerisation process to manufacture membranes involves the free radical co- polymerisation of acrylamide monomer and polyfunctional crosslinking agent N, '- methylene-bis-acrylamide (Bis).
  • a redox initiator system ammonium persulphate (APS) with W,W,W'./V ' -tetramethyletfiylenediamine (TEMED) is used to initiate the free radical polymerisation.
  • the membranes may be formed on a substrate of polyethyleneterapthalate (PET) by casting polyacrylamide polymer gel between two glass plates. Manual production is limited to small scale batches when glass plates are used due its heavy weight and thickness, also handling the fragile glass plates is a safety concern.
  • the present inventors have developed a process resulting in improved polyacrylamide membranes suitable for biological separations using electrophoresis.
  • the present invention provides a process for producing a plurality of polyacrylamide membranes, the process comprising:
  • At least one casting plate is a heat-resistant plastic film.
  • the process may further include:
  • the present invention provides a process for producing a plurality of polyacrylamide membranes, the process comprising:
  • step (c) providing a plurality of alternating layers of casting plates and membrane substrates on top of the casting plate in step (b);
  • the process may further include;
  • the membrane casting vessel is a tank having a removable front wall adapted to receive the solution, the casting pates, the membrane substrates and the weighting material.
  • the acrylamide monomer solution is N, '- methylene-bis- acrylamide (Bis) and the crosslinking agent is ammonium persulphate (APS) and ⁇ , ⁇ , ⁇ ', ⁇ ' -tetramethylethylenediamine (TE ED).
  • Further additives may be included with the solution such as Teric B18 (Surfactant) and buffer components such as MES (2-(N- morpholino)ethanesulfonic acid) and Bis-Tris (Bis(2-hydroxyethyl)amino- tris(hydroxymethyl)methane.
  • the casting plates can be glass plates and heat-resistant plastic films.
  • the glass pales can have a thickness of about 0.1 to 5 mm or 0,5 to 2 mm. In one embodiment, the glass plates are about 2 mm thick.
  • the heat-resistant plastic films can have a thickness of about 0.1 to 2 mm or 0.1 to 1 mm or 0.1 to 0.5 mm. In one embodiment the heat-resistant plastic films are up to about 1 mm thick. In another embodiment the heat- resistant plastic films are about 0.5 mm thick. It will be appreciated that the glass plates and the plastic films can be thicker or thinner, depending on the supplier specifications.
  • the heat-resistant plastic films can be made of polyethylene terephthalate (PET), polypropylene or polyethylene terephthalate glycol-modified (PETG), PVC (polyvinyl chloride) film; Polymethylmethacrylate (plexiglass); Teflon PTFE film
  • the heat-resistant plastic films are PET.
  • An exemplary heat resistant plastic film was the 3 "Black & White Laser transparency film (product no CG3300). The film is a PET coated film suitable for use with laser printers.
  • the membrane substrate is a PET fibre sheet.
  • two PET fibre sheets are placed between the casting plates to form th membrane substrate.
  • the PET fibre sheets are placed between heat-resistant plastic films to allow smooth glossy membrane surfaces to form.
  • the weighting material may be one or more glass plates. In an embodiment the weighting material is two or more glass plates. In another embodiment, a plurality of glass plates form the weighting material.
  • the process can be used to produce 0 or more membranes, 5 or more membranes, 20 or more membranes, 25 or more membranes, 30 or more membranes, 35 or more membranes, 40 or more membranes, 45 or more membranes, 50 or more membranes, 55 or more membranes, 60 or more membranes.
  • the process is carried out in a controlled atmosphere environment to maintain constant conditions during the casting process.
  • the membrane casting vessel is placed in a glove box and the process is carried out in the glove box.
  • the membranes are A4 sheet size or formed in sheets of about 315 mm X 315 mm in size and have a thickness of between about 0.1 to 0.3 mm.
  • the membranes are adapted for use in Preparative Isolation by Membrane Electrophoresis (PriME) that was originally developed by Gradipore Limited and described in US 6,328,869, US 6,402,913, US 6,919,006, and US 6,800,184.
  • PrimaryME Membrane Electrophoresis
  • Examples of uses of the membrane separation technology are described in Li G, Stewart R, Conlan B, Gilbert A, Roeth P, Nair H. Purification of human immunoglobulin G: a new approach to plasma fractionation. Vox sanguinis. 2002;83:332-8; Thomas TM, Quindere J, Thomas DE, Gee SC, Bate IM, Rylatt DB. Preparation of monoclonal antibodies using electrophoresis separation instrument, GradiflowTM.
  • the membranes formed between the heat-resistant plastic films in the casting process were found to have a smoother and more even glossy surface. This resulted in improved batch quality compared with the process only using glass as the casting plates.
  • a further advantage of the use of heat-resistant plastic films in the process is that glass plates are heavy and cumbersome to manipulate in the membrane casting process compared with handling of the heat-resistant plastic films. The heat-resistant plastic films can be discarded after use whereas the glass plates require a rigorous cleaning an process and need to be stored carefully to minimize breakage and scratching.
  • PET polyethyleneterapthalate
  • the present invention provides polyacrylamide membranes produced by the process according to the first aspect of the present invention.
  • Figure 1 shows an embodiment of material assembly fo casting membranes.
  • Figure 2 shows a schematic of macromolecule size-based separation using preparative electrophoresis membrane based technology.
  • Figure 3 shows a schematic macromolecule charge-based separation using preparative electrophoresis membrane based technology.
  • the casting plates can be glass plates and heat-resistant plastic films.
  • the glass plates (2 mm) were used.
  • the better performing heat resistant plastic film was the 3M "Black & White Laser transparency film (product no CG3300).
  • the film is a PET coated film but the percentage of coating is 3 proprietary information. The coating percentage may determine the transparency level and may be partly thermal properties of the film.
  • Other films that have similar properties could be used for membrane manufacturing process. However, the surface of the membranes could be slightly less or more glossy and intact depending on the type of films used without affecting intrinsic properties of membrane.
  • the films were developed for use in other areas and include useful features such as being waterproof, transparent, rigid, excellent mechanical strength, environment friendly and high temperature durable.
  • Application areas include laser printing, offset printing, plate- making, overhead projecting and advertising printing.
  • PET fibre sheets are used as a substrate for casting the membranes. These fibers are utilized to produce wicking media with open-cell pore structures that control liquid volume capacity and fluid transfer rates. The fibers offer a wide assortment of extruded profile geometries and can be engineered to meet various density, permeability, and wicking performance requirements. PET fibre sheets are FDA compliant for use in various biological assays and separation processes.
  • the substrates are engineered for excellent fluid transfer properties, have high thermal bonding properties for use in higher temperature applications. Good thermal bonding properties maintain part integrity during normal transportation, storage, and use.
  • the substrates are highly inert and resistant to chemicals and may be utilized with many acidic, basic, and organic solvents. Mechanical properties provide excellent part rigidity.
  • PET porous media may also be engineered for part softness or rigidity as required for the end-use application. It offers excellent tensile strength, which is beneficial for product that is supplied on reels and intended for high volume assembly automation.
  • the substrates can have one or more of the following characteristics and structure:
  • PET porous substrates may also be engineered for part softness or rigidity as required for the end-use application. PET porous substrates offer excellent tensile strength, which is beneficial for product that is supplied on reels and intended for high volume assembly automation.
  • the substrates come in sheets or rolls and were purchased from Shangshai Bolting cloth manufacturing, China.
  • the properties of the polyaerylamide gels are depended on the composition of reacting monomer, cross-linker, initiator as well as polymerisation environment such as oxygen level and temperature.
  • the experiments were carried out at different initiator concentrations to produce membranes with desired properties using different substrates.
  • the experiments were also carried out under controlled and uncontrolled environment with optimising the initiator concentration.
  • the acrylamide (monomer) and the cross-liker (N, '- methylene-bis-acrylamide (Bis) concentrations were kept constant to retain the desired pore size of particular membranes.
  • Table 2 shows the composition of initiator for 250 kDa membrane formulation using different plastic films.
  • the initiator concentration for the glass plate formed membranes was used as reference standard.
  • Table 3 shows the composition of initiator for 75 kDa membrane formulation using different plastic films in uncontrolled environment.
  • Polypropylene 0.366 0.366 Table 3 The composition of initiator using different plastic films for 75 kDa membrane in uncontrolled environment.
  • the membrane casting vessel was a tank having a removable front wall adapted to receive the solution, the casting pates, the membrane substrates and the weighting material.
  • a small scale casting vessel that makes A4 size membrane sheets and a large scale casting vessel that makes 315 mm x315 mm membrane were used. Both vessels were made of acrylic plate having a bottom and three fixed side walls. The fourth side wall was removable to assist with removing the cast membranes.
  • Membranes were prepared in a membrane casting vessel in the form of a tank by assembling the glass plates or plastic films as appropriate in the acrylamide reaction mixture solution.
  • Figure 1 shows an example of the assembly of materials for casting membranes. This procedure was carried out either in a controlled/ uncontrolled environment in a membrane casting tank containing the reaction mixture. The polymerisation starts within 25-30 minutes after adding the initiator. Therefore, the assembly should be completed within about 20 minutes of adding initiator to the monomer solution. The reaction is completed within about 2 hours. The membranes can be left curing overnight for up to about 18 hours. The cast membrane and casting plate block is removed from the vessel and the membranes were separated after curing and stored in appropriate storage buffer.
  • Membranes were cut to size and assembled into a membrane cassette for use in the electrophoresis apparatus.
  • the cassette has an outer housing unit component containing gasket.
  • a restriction membrane was placed within the housing unit followed by a support grid having channels.
  • a separation membrane was then placed on the grid.
  • a collar of the grid faced the restriction membrane and the grid channels face upwards towards the separation membrane.
  • An another grid was placed on the separation membrane so that the channels face towards the separation membrane.
  • Another restriction was placed On top of the grid followed by inner housing component ⁇ containing a gasket) on top of the cartridge stack. Clips (6 in total) on the inner and outer housing are aligned and gently pressed until they are fully clipped. Buffer stream inlet tubes were then provided to the assembled cassette.
  • the PrlME technology is a preparative electrophoresis membrane based technology based on two major techniques. One is polyacrylamide membrane and the another is protein electrophoreses. By choosing a selected pore size of the separation membrane and a suitable buffer (or buffers), it separates protein molecules based on their molecule weights and isoelectric point within a particular electrical field and buffer environment. The separation process is simple and efficient and the working principle is demonstrated by Figure 2 showing macromolecule size-based separation and Figure 3 showing macromolecule charge-based separation.
  • the BF400 (NuSep Ltd) is a laboratory scale biological separations apparatus incorporating the PrlME technology. With a processing volume of between 5 and 50 ml, it is a versatile system capable of processing samples from a diverse range of biological complexes.
  • the control panel has time setting key, voltage key, buffer pump start key, stream pump start key, cover indicator, electrical reverse key and start and stop key.
  • the apparatus has a fixed stream pump and buffer pump, stream pump gives a fixed stream flow rate to both stream 1 and 2 at.20 ml/min, the buffer pump circle the buffer at 2 l/min. Electrophoresis conditions
  • Frozen pooled plasma (Serologicals, Atlanta, GA) from healthy donors was thawed at 37 ' C.
  • One millilitre of plasma was diluted in 1 :10 with Tris-Borate buffer pH 8 9 [32 mM (3-88 g/l) Trizma®Base, 96 rnM(5-93 g/l) and placed into stream 1 (S1 ) of the BF400.
  • a membrane cartridge comprising a 150 kDa pore size separation membrane sandwiched between two 5-k Da pore size restriction membranes, was used in the separation unit.
  • Tris- Borate buffer ⁇ 1 -8 I was circulated in the buffer tank and kept cold with ice.
  • S2 An electric potential of 250 V with the positive electrode configured at stream 2 (S2), was placed across the membrane sandwich to perform the electrophoresis.
  • the product in S2 was harvested every 60 rniri for a total of 360 min. After each harvest, 0-ml of fresh buffer was used to replenish S2.
  • SDS-PAGE sodium dodecyl sulphate- poiyacrylamide gel electrophoresis
  • the membranes were visually inspected for bubbles, white patches, thickness and appearance.
  • the membranes are then characterised with respect to its pore size using specific proteins following PRIME instrument and technology.
  • the pore size is determined from the transfer rate of the specific protein through the membranes.
  • Production of polyacrylamide membranes includes the following steps:
  • the casting plates When not in use, the casting plates should be separated with clean A4 paper and stored in a suitable container.
  • Steps iii and iv Repeat Steps iii and iv until all sheets of substrate are loaded. Place another glass plate on top of the last casting plate/film.
  • the components and weights used for manufacturing a batch of membrane is shown in Table 4.
  • the protein transfer test results are also comparable, i.e. the desired pore size of the membranes have not been affected.
  • the protein transfer results are provided in Table 5.
  • the total weight of the reaction chamber was significantly reduced as well as significantly reduced concern regarding the presence of broken glass during manufacturing and cleaning process.
  • the thickness of each plastic film (0.1 mm) is 20 times thinner than the glass plate, i.e. batch size can be increased significantly, 40 membranes per batch have been produced.
  • the initiator concentration was successfully optimised to produce membrane using suitable plastic films.
  • the validation batches reproduced the membranes with desired properties.
  • Table 6 show the results for 250 kDa in controlled environment.
  • the standard initiator concentration is used for polymerisation for batch HN250K061113.
  • the polymerisation time was longer and the gel consistency was more elastic. This could be due the dissolved oxygen in the plastic film which inhibits polymerisation.
  • the gel was sticking to 3M films ⁇ could not be separated) and also on the glass plates.
  • Polypropylene shows best results: produced membrane with no dry patches and easy to separate.
  • the initiator concentration was optimised to 2 times (Batch: HN250K141 1 13_1 ) and 3 times (Batch:
  • HN250K1411 13_2 higher than the standard.
  • the batch HN250K141113_1 produced membranes with desired properties.
  • Batch: HN250K141113_2 failed due to early polymerisation.
  • the validation batches produced reproducible results.
  • Table 7 show the results for 75 kDa membrane production in uncontrolled environment using different plastic films kDa.
  • the initiator concentration was optimised to 9 times than the standard to produce membrane with desired properties.
  • Membranes can be manufactured successfully using plastic films replacing glass plates.
  • the product performance vvas confirmed by physical inspection and protein transfer QC test and the results were comparable.
  • the smooth surface of plastic films has produced membranes with glossy surface comparable with glass plates.
  • the plastic films are disposable, therefore reducing the processing time significantly.
  • the cleaning of glass plates involves hand washing, washing in the dishwasher 3 times and drying, also further cleaning with ethanol.
  • the membranes formed between the heat-resistant plastic films in the casting process were found to have a smoother and more even glossy surface. This resulted in improved batch quality compared with the process only using glass as the casting plates.
  • a further advantage of the use of heat-resistant plastic films in the process is that glass plates are heavy and cumbersome to manipulate in the membrane casting process compared with handling of the heat-resistant plastic films. The heat-resistant plastic films can be discarded after use whereas the glass plates require a rigorous cleaning an process and need to be stored carefully to minimize breakage and scratching.

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  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Manufacturing & Machinery (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)

Abstract

L'invention concerne un procédé de production d'une pluralité de membranes de polyacrylamide consistant : (a) à mettre une solution de monomère d'acrylamide et un agent de réticulation dans un récipient de coulée de membrane; (b) à placer une plaque de coulée sur le fond du récipient; (c) à placer un substrat formant membrane sur la plaque de coulée; (d) à placer une plaque de coulée sur le substrat formant membrane; (e) à répéter les étapes (c) à (d) pour former une pluralité de couches de plaques de coulée et de substrats formant membrane; (f) à placer une substance de charge sur la pluralité de couches; et (g) à permettre la polymérisation du monomère d'acrylamide au niveau des substrats formant membrane pour former des membranes de polyacrylamide, au moins une plaque de coulée étant un film en plastique résistant à la chaleur.
PCT/AU2014/000376 2013-05-20 2014-04-08 Procédé de formation de membranes de polyacrylamide Ceased WO2014186819A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2013901775 2013-05-20
AU2013901775A AU2013901775A0 (en) 2013-05-20 Plastic Film based Polyacrylamide Membranes

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WO2014186819A1 true WO2014186819A1 (fr) 2014-11-27

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018067736A1 (fr) * 2016-10-04 2018-04-12 Sage Science, Inc. Appareils, procédés et systèmes pour le traitement automatique d'acides nucléiques et la préparation électrophorétique d'échantillon
US10131901B2 (en) 2014-10-15 2018-11-20 Sage Science, Inc. Apparatuses, methods and systems for automated processing of nucleic acids and electrophoretic sample preparation
US10473619B2 (en) 2012-10-12 2019-11-12 Sage Science, Inc. Side-eluting molecular fractionator
CN111499899A (zh) * 2020-04-13 2020-08-07 辽宁省肿瘤医院 一种不同硬度的体外细胞培养基底材料聚丙烯酰胺凝胶薄膜的大批量制作方法与应用
US11542495B2 (en) 2015-11-20 2023-01-03 Sage Science, Inc. Preparative electrophoretic method for targeted purification of genomic DNA fragments
US11867661B2 (en) 2017-04-07 2024-01-09 Sage Science, Inc. Systems and methods for detection of genetic structural variation using integrated electrophoretic DNA purification

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4718998A (en) * 1986-05-20 1988-01-12 Fuji Photo Film Co., Ltd. Element for electrophoresis
US4818360A (en) * 1987-08-28 1989-04-04 Bios Corporation Method and apparatus for blotting from electrophoresis gels
US6607645B1 (en) * 2000-05-10 2003-08-19 Alberta Research Council Inc. Production of hollow ceramic membranes by electrophoretic deposition

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4718998A (en) * 1986-05-20 1988-01-12 Fuji Photo Film Co., Ltd. Element for electrophoresis
US4818360A (en) * 1987-08-28 1989-04-04 Bios Corporation Method and apparatus for blotting from electrophoresis gels
US6607645B1 (en) * 2000-05-10 2003-08-19 Alberta Research Council Inc. Production of hollow ceramic membranes by electrophoretic deposition

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10473619B2 (en) 2012-10-12 2019-11-12 Sage Science, Inc. Side-eluting molecular fractionator
US10131901B2 (en) 2014-10-15 2018-11-20 Sage Science, Inc. Apparatuses, methods and systems for automated processing of nucleic acids and electrophoretic sample preparation
US10738298B2 (en) 2014-10-15 2020-08-11 Sage Science, Inc. Apparatuses, methods and systems for automated processing of nucleic acids and electrophoretic sample preparation
US11542495B2 (en) 2015-11-20 2023-01-03 Sage Science, Inc. Preparative electrophoretic method for targeted purification of genomic DNA fragments
WO2018067736A1 (fr) * 2016-10-04 2018-04-12 Sage Science, Inc. Appareils, procédés et systèmes pour le traitement automatique d'acides nucléiques et la préparation électrophorétique d'échantillon
CN110088612A (zh) * 2016-10-04 2019-08-02 塞奇科学股份有限公司 用于核酸的自动化处理和电泳样品制备的装置、方法和系统
US11867661B2 (en) 2017-04-07 2024-01-09 Sage Science, Inc. Systems and methods for detection of genetic structural variation using integrated electrophoretic DNA purification
CN111499899A (zh) * 2020-04-13 2020-08-07 辽宁省肿瘤医院 一种不同硬度的体外细胞培养基底材料聚丙烯酰胺凝胶薄膜的大批量制作方法与应用
CN111499899B (zh) * 2020-04-13 2023-03-07 辽宁省肿瘤医院 一种不同硬度的体外细胞培养基底材料聚丙烯酰胺凝胶薄膜的大批量制作方法与应用

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