CN1387037A - Chromatographic cake for separating or renaturating and purifying biolgical macromoleculae - Google Patents

Abstract

A chromatographic cake for separating or renaturating/purifying biologic macromoleculae is characterized by that the both ends of chromatographic medium is fixed by press plates, the annular cylindrical body has side hole, the press plate and cylindrical body constitute the cavity of chromatographic cake, and the distributor and sieve plate are arranged at both sides of chromatographic medium in said cavity. Its advantages are filling the filler from said side hole, and high column effect and smooth surface of filled chromatographic cake.

Description

Biomacromolecule separates or while renaturation and purifying chromatographic cake

The present invention relates to a kind of biochemical industry or design library part editor and reuse, the chromatographic cake of particularly a kind of biomacromolecule separation or while renaturation and purifying.

Chromatography and capillary electrophoresis are the most effective two kinds of biomacromolecule separation means known today, and the latter only can be used for analytical scale, and the former can be used for analysis, preparation and production scale, is separation means most important, best in the present bioengineering.Traditional idea is that the separating effect of chromatogram promptly is directly proportional with column length with the post number of plates.Yet, the oversize not only chromatographic column of pillar costliness, and, post voltage rise height, the high performance liquid chromatograph of necessary use value costliness.Though people know that from experience the separation of biomacromolecule is irrelevant with column length basically, but still does not know what degree pillar can be short to.Bibliographical information is arranged, can draw use short column by the metering of the solute in liquid chromatography replacement model and carry out effective separation of biomacromolecule, and adopt column length only to carry out biomacromolecule and separate for the membrane chromatography of the thick thin slice formation of 2mm filling post and the 2mm that cuts from continuous rod, effect is fine, but can only be used for analytical scale.Be used for preparative-scale or plant-scale performance liquid chromatographic column and generally load the particulate filler that diameter is 20-30 μ m, too high to avoid pressing at the conditions of high flow rate lower prop, thus reduce separating effect.And the length of its post is good with 10.Phamacia company is famous in the world boundary's (existing also production pressed chromatographic media in some) to produce the normal pressure chromatographic media.In order not make soft matrix fill itself become more and more little because of pressurized makes bed volume because of column length increases, thereby it is more and more high that post is pressed, also once provided thickness less, the cake type chromatographic column that diameter is bigger, yet be several such cake type chromatographic columns to be cascaded use in use, make actual these column length sums that are cascaded still than the big manyfold in post footpath, therefore, as previously mentioned, flow velocity can not be high during use.Many albumen of expressing with E.coli in bioengineering are too strong because of its hydrophobicity, and often the form with occlusion body exists in E.coli.Though the primary structure of protein is correct, yet its three grades or quaternary structure are wrong substantially.The hydrophobicity of these occlusion bodies is generally all very strong, and this just requires with high concentration denaturant such as 7.0mol/L guanidine hydrochloride (GuHCl) or 8.0mol/L urea dissolving occlusion body, and then carries out protein renaturation.Commonly used dialysis and dilution method are carried out protein renaturation, annealing efficiency low (being generally 5%-20%) not only, and also consuming time and can not reach the purpose of separating foreign protein.Someone once proposed the technology with high performance liquid chromatogram hydrophobic chromatography (HPHIC) realization metaprotein while renaturation and purifying, still, met water formation precipitation if run into albumen when sample introduction, and then chromatographic column will blocked or breaking-up.Someone also design and use once cross metaprotein renaturation and simultaneously purification devices or multifunctional protein purifier.But just show its result of use not to the major parameter of this device or chromatographic cake itself in advance with explanation, more it is not optimized.Therefore, the main deficiency that exists in the separation and purification in bioengineering downstream at present can be summarized as follows: the separation and purification of biomacromolecule is normally carried out in the glass column that is filled with the soft matrix chromatogram medium of bulky grain, plastic column or stainless steel column.Its shortcoming is that the post effect is low, needs filler many, and the amount that consumes moving phase is big, and the production cycle is long; When separating small-molecule substance with chromatographic column, its degree of separation is directly proportional with column length, and its post footpath was generally 1: 10 with the column length ratio.And the degree of separation of biomacromolecule is subjected to the influence of column length less, usually selecting column length for use is the pillar of 5cm, behind its filling granule filler, chromatographic system pressure enlarges markedly, need high pressure liquid chromatograph supporting with it, production cost is increased, it is high that granule filled column effect is given full play in influence, capacity is big, the advantage of favorable reproducibility; Common chromatographic column does not allow full-bodied sample to enter pillar, mustn't be deposited in column cap by small amount of sample yet, and the fixedly import and export of moving phase in use, makes troubles to operation; Common separation purifying technique will pass through renaturation, removes denaturant, thick purifying and consummateization of multistep could obtain purer product.Process route is long, and quality and loss of activity are big, the recovery low (being generally 5～20%); Common refolding method adopts dilution method or dialysis.And dilution method adopts the stepwise dilution step that reduces concentration gradually again, brings many difficulties can for separation and purification subsequently to the dilution of tens times in sample even hundreds of times; Dialysis long (step dialysis needs 24 hours usually) consuming time, and need repeatedly change dislysate.Other two kinds of methods all make target product that the polymerization precipitation takes place in renaturation process easily and influence annealing efficiency; Because purification procedures is many, containing the target product liquor capacity constantly increases.And each step all needs supporting with it equipment, and therefore, equipment investment is big, the production cost height.

The chromatographic cake that the purpose of this invention is to provide a kind of biomacromolecule separation or while renaturation and purifying, in the separation and purification in bioengineering downstream, the granule filled column be can under middle normal pressure power, make full use of and bring into play and height, big, the high repeatability and other advantages of capacity imitated, fully overcome the deficiencies in the prior art, reduce cost, improve output, and merge into a step and finish removing denaturant, thick purifying and consummateization of multistep in the separation and purification of biomacromolecule and the renaturation.

Realization of the present invention: be described further below in conjunction with accompanying drawing.

Accompanying drawing 1, chromatographic cake structure construction synoptic diagram of the present invention.

Accompanying drawing 2, cylinder AA cut-open view of the present invention.

Accompanying drawing 3, structure of distributor organigram

Accompanying drawing 4, distributor BB cut-open view.

Shown in accompanying drawing 1,2, chromatographic media (10) two ends are polygon or circle and are provided with turnover The pressing plate (3,4) of mouth (1,2), sidepiece are annular and the cylinder (5) that side opening (9) are arranged, pressing plate (3,4) and cylinder (5) form the cavity of chromatographic cake; Pressing plate (3,4) and cylinder (5) form Cavity thickness be 0.2～50mm, diameter is 5.0～1,000mm, the ratio of thickness and radical length Be less than or equal to 1; Chromatographic media (13) two in the cavity that pressing plate (3,4) and cylinder (5) form End is provided with distributor (7), sieve plate (6); The cavity that pressing plate (3,4) and cylinder (5) form Can more than the withstand voltage 20Mpa, mainly select stainless steel, titanium alloy or the various worker of acid and alkali-resistance, compression resistance Engineering plastics; Shown in accompanying drawing 3,4, distributor (7) is mainly selected the various engineering plastics of acid and alkali-resistance, Its upper surface or two-sided radial and guiding gutter (11) ring-type, the horizontal stroke of guiding gutter (11) of being provided with The cross section is triangle or semicircle, and radial and intersection ring-type guiding gutter (11) is provided with circle Distribution hole (12), the aperture is by increasing gradually with the increase of distributor (7) centre distance. Sieve plate (6) be micropore corrosion resistant plate or plastic plate, its aperture is between the diameter of chromatographic media (10) particle And between the diameter of large biological molecule, sieve plate (6) diameter is greater than the diameter of cavity. Sealing ring (8) Be various corrosion resistant engineering plastics. The roughness of chromatographic cake cavity inner surface is less than 1.6 μ m, so that Sealing and minimizing are to the Irreversible Adsorption of large biological molecule.

The present invention has the following advantages and good effect compared with the prior art: chromatographic cake can withstand voltage 20Mpa More than, the thickness L of cylinder is L/ φ≤1 with the ratio of diameter phi, L≤50mm, distributor and sieve plate Stressed littler, in use indeformable, can guarantee the planarization on sample introduction end chromatographic media surface, Be conducive to improve separative efficiency; The one or both sides of distributor all can have guiding gutter, and from distributor The center begin to be radial, and intersect with annular diversion trench, an aperture is arranged, from dividing in the intersection Cloth device center is to its periphery, and hole diameter increases gradually, and such distributor is conducive to the equal of mobile phase The even distribution; Separating rate is fast, system pressure low (being generally less than 5.0Mpa), its performance and perfusion look Compose similarly, be conducive to suitability for industrialized production; There is an aperture to can be used as that mobile phase is for subsequent use to be advanced on the column side face Outlet. But when cylinder length timing, adopt the side direction pressurization; Allow into full-bodied sample and product Give birth to a little precipitation. When carrying out chromatographic isolation, do not need fixedly import and export; Can make common renaturation Reach purifying process and simplified at least three times, make the associated production cycle shorten several times, equipment investment Also save a lot; Can reduce the Irreversible Adsorption to target product, make the rate of recovery of target product remarkable Improve; Can carry out simultaneously renaturation, purifying, separation foreign protein and the recyclable denaturant of albuminate. Reusable denaturant can reduce denaturant on the other hand to the pollution of environment on the one hand; Identical During column volume, with general pillar relatively, the available high-pressure homogenization method more filler of packing into has bigger Mass loading and volume load.

Claims (4)

1, biomacromolecule separates or while renaturation and purifying chromatographic cake, it is characterized in that:

A, chromatographic media (10) two ends are polygon or circle and the pressing plate (3,4) that is provided with import and export (1,2), and sidepiece is annular and the cylinder (5) that side opening (9) are arranged, and pressing plate (3,4) and cylinder (5) are formed the cavity of chromatographic cake;

The cavity thickness that B, pressing plate (3,4) and cylinder (5) are formed is 0.2～50mm, and diameter is 5.0～1,000mm, and thickness is less than or equal to 1 with the ratio of radical length;

Chromatographic media (10) two ends are provided with distributor (7), sieve plate (6) in the cavity that C, pressing plate (3,4) and cylinder (5) are formed;

The cavity that D, pressing plate (3,4) and cylinder (5) are formed can mainly be selected stainless steel, titanium alloy or the various engineering plastics of acid and alkali-resistance, compression resistance for use more than the withstand voltage 20Mpa.

2, separate or while renaturation and purifying chromatographic cake according to the described biomacromolecule of claim 1, it is characterized in that distributor (7) mainly selects the various engineering plastics of acid and alkali-resistance for use, its upper surface or two-sidedly be provided with radial and diversion trench (11) ring-type, the xsect of diversion trench (11) is triangle or semicircle, radial and intersection ring-type diversion trench (11) is provided with circular distribution hole (12), and the aperture is by increasing gradually with the increase of distributor (7) centre distance.

3, separate or while renaturation and purifying chromatographic cake according to the described biomacromolecule of claim 1, it is characterized in that sieve plate (6) is micropore corrosion resistant plate or plastic plate, its aperture is between the diameter of the diameter of chromatographic media (10) particle and biomacromolecule, and sieve plate (6) diameter is greater than the diameter of cavity.

4, separate or while renaturation and purifying chromatographic cake according to the described biomacromolecule of claim 1, it is characterized in that O-ring seal (8) is various corrosion resistant engineering plastics.

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