WO2002095390A1 - Colonne chromatographique remplie d'une substance tassee, son procede de production et ses applications - Google Patents

Colonne chromatographique remplie d'une substance tassee, son procede de production et ses applications Download PDF

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Publication number
WO2002095390A1
WO2002095390A1 PCT/CN2002/000333 CN0200333W WO02095390A1 WO 2002095390 A1 WO2002095390 A1 WO 2002095390A1 CN 0200333 W CN0200333 W CN 0200333W WO 02095390 A1 WO02095390 A1 WO 02095390A1
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Prior art keywords
chromatographic
column
cake
chromatographic medium
medium
Prior art date
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PCT/CN2002/000333
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English (en)
French (fr)
Inventor
Xindu Geng
Yangjun Zhang
Original Assignee
Northwest University
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Publication date
Priority claimed from CN 01115263 external-priority patent/CN1387037A/zh
Priority claimed from CN 01115264 external-priority patent/CN1387038A/zh
Application filed by Northwest University filed Critical Northwest University
Priority to EP02732319A priority Critical patent/EP1396721B1/en
Priority to US10/477,977 priority patent/US7208085B2/en
Priority to DE60228078T priority patent/DE60228078D1/de
Publication of WO2002095390A1 publication Critical patent/WO2002095390A1/zh

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/56—Packing methods or coating methods
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
    • B01D15/206—Packing or coating
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/22—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the construction of the column
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/56—Packing methods or coating methods
    • G01N2030/562—Packing methods or coating methods packing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G01N30/6052—Construction of the column body
    • G01N30/606—Construction of the column body with fluid access or exit ports

Definitions

  • the present invention relates to a chromatographic cake and a method and application for manufacturing the same, and in particular, to a chromatographic cake for biological macromolecule separation or simultaneous renaturation and purification, and a method and application for manufacturing the same.
  • Chromatography and capillary electrophoresis are two commonly used means of biological macromolecule separation. Capillary electrophoresis can only be used on an analytical scale due to the limited amount of mobile phase processed. Chromatography is currently the most important and effective separation method for biological macromolecule separation, renaturation, and purification. It can be used for both analytical and production scales.
  • high-performance liquid chromatography columns used for production scale are generally packed with granular fillers with a diameter of 20-30 ⁇ m, and the length / diameter ratio of the column is preferably 10 to avoid excessive column pressure and high pressure at high flow rates. seperate effect.
  • the soft matrix packing itself becomes smaller and smaller due to the pressure, resulting in higher and higher column pressure. It is provided by the world-renowned Phamacia company for the production of atmospheric and medium pressure chromatography media.
  • a kind of cake-type column with a small thickness but the column length is at least greater than lOCm
  • has a larger diameter but several such cake-type columns need to be connected in series during use, so these columns are actually connected in series. The sum of the lengths is still many times larger than the diameter of the column, and the flow rate cannot be high during use.
  • HPHIC high-performance liquid hydrophobic chromatography
  • the usual renaturation method uses the dilution method or dialysis method, and the dilution method must use a stepwise dilution step that gradually reduces the concentration.
  • the sample can be diluted dozens of times, or even hundreds of times, which will cause many difficulties in subsequent separation and purification.
  • the dialysis method takes a long time (one step of dialysis usually takes 24 hours), and the dialysate needs to be changed multiple times. In addition, both of these methods can easily cause the target product to polymerize and precipitate during the renaturation process and affect the renaturation efficiency. 6.
  • the current separation and purification method has many steps, the volume of the solution containing the target product is constantly increasing, and each step requires supporting equipment, so the equipment investment is large and the production cost is high.
  • the column packing can be obtained with axial compression and radial compression techniques, but the two methods are only applicable to column length / diameter ratio greater than 1.
  • the traditional process of filling the column is from the axial direction, and it is generally only applicable to columns with a column length / diameter ratio> 10. 2. Due to the high requirements for column packing technology, it is easy to cause uneven packing, such as tight exits and loose inlets, and poor reproducibility of column packing. 3.
  • the chromatographic column needs to be repacked only after it has been subjected to high pressure for a long time, or before repacking, repairing the recessed column inlet can extend its service life, and repairing the recessed column inlet must be disassembled.
  • the stigma can only be achieved by removing the sieve plate.
  • the area of the sieve plate is smaller and easier to remove.
  • the area of the "chromatographic cake" sieve plate is generally 1 to 100 times the size of a typical chromatographic column, and the distributor and the sieve plate are closely fitted, and It is difficult to remove it by pressing it into the groove of the cylinder. If it is forcibly removed, it will often damage the distributor. 5.
  • the object of the present invention is to provide a chromatographic cake with better effect, which is used for the separation or simultaneous renaturation and purification of biological macromolecules.
  • a chromatographic cake includes a chromatographic medium column with mobile phase inlet and outlet holes and a chromatographic medium filled in the inner cavity of the chromatographic medium column.
  • the ratio of the thickness of the inner cavity of the chromatographic medium column to the radial length is less than or equal to one.
  • a preferable size of the inner cavity of the chromatography medium column is: a thickness of 0.2 to 50 mm and a radial length of 5.0 to 1000 mm.
  • the chromatographic medium column includes a combination of upper and lower pressure plates with inlets and outlets for the mobile phase, and a column with at least one side hole.
  • the sides of the upper and lower pressure plates of the chromatographic medium column which are close to the inner cavity of the chromatographic medium column are respectively equipped with sieve plates.
  • the pore size of the sieve plate should be larger than the biomacromolecules in the mobile phase and smaller than the chromatographic medium.
  • distributors are respectively installed between the upper and lower pressure plates of the chromatography medium column and the sieve plates.
  • the distributor is a flat plate having the same cross-sectional shape as the internal cavity of the chromatographic medium column, and at least one side surface of the distributor is provided with radial and annular flow guide grooves, and the radial and annular flow guides meet Distribution holes are provided everywhere.
  • the cross section of the guide groove is triangular or chord-shaped.
  • the pore diameter of the distribution holes gradually increases as the distance from the center of the distributor increases.
  • a sealing ring is installed between the upper and lower pressure plates and the column body.
  • the advantages of small particle packing with high column efficiency, large capacity, and good reproducibility can be fully utilized and exerted under normal pressure to reduce costs and increase yield, and
  • the separation and purification of biological macromolecules and the removal of denaturants, crude purification and multi-step fine purification are combined into one step.
  • the chromatographic cake of the present invention can work under the conditions of 1-200 kg / cm 2 .
  • a second object of the present invention is to provide a method for preparing a chromatographic cake with high speed and low cost and high column efficiency.
  • a method for preparing a chromatographic cake includes the following steps:
  • the chromatographic medium column includes a combination of upper and lower pressure plates with mobile phase access holes and at least one side hole, and a chromatographic medium surrounded by the upper and lower pressure plates and the column body.
  • the ratio of the thickness of the column cavity to the radial length is less than or equal to 1;
  • the side hole of the chromatographic medium column can directly communicate with the homogenization tank on the high-pressure homogenous column packing machine and enter the packing column.
  • the chromatographic cake When preparing a chromatographic cake with a diameter of less than 50 mm, the chromatographic cake can be directly loaded on a high-pressure homogenizing column packing machine, and the chromatographic medium homogenate liquid is added to the inner cavity of the chromatographic medium column from the side hole of the chromatographic medium column according to a common column loading method .
  • the column When preparing a chromatographic cake with a diameter of more than 50 mm, in order to make the chromatographic medium packed more tightly, the column should be packed by suction filtration first, and then the chromatographic cake is loaded on a high-pressure homogenizer column loader to remove the chromatographic medium homogenate The side hole of the column is added to the inner cavity of the chromatography medium column.
  • Supplementing the chromatographic medium through the side holes of the chromatographic medium column is to eliminate the dead volume in the chromatographic cake, because after using the chromatographic cake for a period of time, the dead volume will increase due to the loss of the chromatographic medium, which affects the separation effect.
  • the method of adding chromatographic medium is the same as that of packing.
  • the chromatographic cake is rinsed through the side holes of the chromatographic medium column in order to connect the sparger and the sieve plate.
  • the accumulated impurities and bio-macromolecules that are not in the chromatographic cake are removed, because these accumulations will increase the pressure of the chromatography system on the one hand, and contaminate the subsequent samples to be separated on the other.
  • the method of rinsing the chromatographic cake is to use the side hole as an inlet, the inlet and outlet of the upper and lower plate of the chromatographic cake as an outlet, and rinse with buffer solution or water.
  • the replacement of the failed chromatographic medium through the side hole of the chromatographic medium column is to remove the failed chromatographic medium, which causes a significant increase in the system pressure, and the column efficiency becomes very low, and fill with a new medium.
  • the replacement method is to take the inlet and outlet of the upper and lower platens of the chromatographic cake as an inlet, use the side holes as an outlet, and flush out the failed chromatographic medium with water, and refill the chromatographic medium with the above-mentioned column packing method of the present invention.
  • the method of the present invention is very convenient and easy to install the column, saving time and effort. Because the column is directly installed without opening the pressure plates at both ends after leak detection, it can ensure that the chromatographic cake does not leak and the surface of the medium is flat, which is beneficial to improving the column efficiency; No larger homogenizers and additional auxiliary equipment are needed when filling larger chromatographic cakes, reducing production costs.
  • FIG. 1 is a schematic structural diagram of a chromatographic cake of the present invention.
  • FIG. 2 is a cross-sectional view taken along AA in FIG. 1.
  • FIG. 3 is a schematic structural diagram of a chromatographic cake distributor according to the present invention.
  • FIG. 4 is a BB sectional view of FIG. 3.
  • Figure 5 is a chromatogram of a radially packed chromatographic cake.
  • Figure 6 is a chromatogram of an axially packed chromatographic cake.
  • Figure 7 Chromatogram of a radial packed chromatographic cake.
  • Figure 8 shows a chromatogram of a reloaded column chromatography cake.
  • Figure 9 shows the separation chromatogram of the chromatographic cake.
  • Figure 10 shows the chromatographic separation of the target.
  • Figure 11 shows the chromatographic separation of seven standard proteins by a 10x50 R.D. chromatographic cake.
  • Figure 12 shows the separation of five standard proteins by a 10 X 200mml. D. chromatographic cake.
  • Figure 13 shows the separation of five standard proteins by a 10 X 300mml. D. chromatographic cake.
  • Figure 14 shows the renaturation and purification of 10X 50ramI.
  • D chromatographic cake for recombinant human interferon-Y.
  • FIG. 15 is a schematic diagram of various functions of the chromatographic cake of the present invention. The best way to implement the invention
  • the chromatographic cake of the present invention includes a stainless steel chromatographic medium column and a chromatographic medium 10 filled in the inner cavity of the chromatographic medium column.
  • the cross section of the inner cavity of the chromatographic medium column is circular, and the thickness is 10 mm.
  • the length is 100 mm.
  • the roughness of the inner surface of the column of the chromatographic medium should be less than 1.6 ⁇ m, in order to seal and reduce the irreversible adsorption of biomacromolecules.
  • the chromatographic medium column includes upper and lower platens 3, 4 with mobile phase inlet and outlet holes 1, 2, and a column 5 with a side hole 9, and the upper and lower platens 3, 4, and column 5 surround the chromatographic medium.
  • the inner cavity of the column; the side hole 9 on the column 5 is blocked with a plug after filling the chromatographic medium 10.
  • a sealing ring 8 of corrosion-resistant engineering plastic is installed between the upper and lower pressure plates 3, 4 and the cylinder 5.
  • the upper and lower pressure plates 3 and 4 are respectively equipped with stainless steel sieve plates 6 with sieve plate holes on the sides close to the inner cavity of the chromatography medium column.
  • the diameter of the sieve plate 6 is larger than the diameter of the inner cavity of the chromatography medium column. Between the diameter of the particles of the chromatographic medium 10 and the diameter of the biomacromolecules. '
  • a distributor 7 made of acid- and alkali-resistant engineering plastic is installed between the upper and lower pressure plates 3, 4 and the sieve plate 6, respectively.
  • the distributor 7 has the same cross-sectional shape as the inner cavity of the chromatography medium column.
  • the flat plate has radial and annular diversion grooves 11 on both sides of the surface.
  • the cross section of the diversion groove 11 is triangular.
  • a circular distribution hole 12 is provided at the intersection of the radial and annular diversion grooves 11, The pore diameter of the distribution holes increases gradually as the distance from the center of the distributor increases.
  • the shape of the cross section of the inner cavity of the chromatography medium column is various, and may be circular, polygonal, elliptical, or the like.
  • the ratio of the thickness of the inner cavity of the chromatography medium column to the radial length should be less than or equal to 1, and the preferred thickness is 0.2 to 50 mm and the radial length is 5.0 to 1000 mm.
  • Chromatographic media columns can be made of various materials such as acid and alkali resistant, pressure resistant stainless steel, titanium alloys or various engineering plastics.
  • the chromatographic media filled can also be diverse.
  • the number of holes 9 on the upper side of the chromatographic medium column 5 can be determined according to the actual situation, and it can be one or more.
  • Example 3 Filling-preparing the chromatographic cake of the present invention with a diameter of less than 50 mm by radial packing
  • the ordinary axial packing method and the radial packing method of the present invention were used to pack a small particle hydrophobic chromatography packing with a diameter of 5 ⁇ into 10 X.
  • cytochrome-C, ribonuclease A, lysozyme, ⁇ -amylase were separated under the same chromatographic conditions at a flow rate of 5.0 mL / min, a gradient of 0-100% B, and 25 minutes. And insulin five proteins. Repeat the above operations five times each.
  • the chromatograms of the two loading methods are shown in Figures 5 and 6.
  • 1 is cytochrome-C
  • 2 is ribonuclease A
  • 3 is lysozyme
  • 4 is ⁇ -starch.
  • Enzyme 5 is insulin.
  • Cytochrome-C Cyt-C
  • Myo myoglobin
  • Lysozyme Lys
  • a-Amy ⁇ -amylase
  • the packed packing is refilled into the chromatographic cake from the side holes without removing the upper and lower platens, which has no significant effect on the resolution, indicating that
  • the side holes of the chromatographic cake can be used to easily drive out the failed packing, and it can also be easily filled with new or supplemental packing.
  • Figure 12 shows the separation of 1 cytochrome-C; 2 myoglobin at a flow rate of 100 ml / min, a damping coefficient of 0.05 AUFS, a gradient of 100% A to 100% B, and 40 minutes of chromatographic conditions; 3 Lysozyme; 4 ⁇ -amylase; 5 insulin.
  • Figure 13 shows the separation of 1 cytochrome-C; 2 myoglobin at a flow rate of 120.0 ml / min, a damping coefficient of 0.1 AUFS, a gradient of 100% A to 100% B, and 60 minutes of chromatography; Ribonuclease A; 4 lysozyme; 5 ⁇ -amylase. It can be seen from the figure that the chromatographic cakes of different specifications have a good separation effect.
  • Example 8 Refolding efficiency of chromatographic cake for urea-denatured and guanidine hydrochloride-denatured lysozyme
  • Figure 14 shows the renaturation and purification of recombinant human interferon- ⁇ (rhINF- ⁇ ) in a 10 X 50 band I. D. chromatographic cake.
  • the sample was a rhINF- ⁇ solution extracted from E. coli cell disruption solution with 7. Omol / L guanidine hydrochloride.
  • the method for measuring the activity of rhINF- ⁇ is a cytopathic inhibition method. Operating conditions: Inject 1 ml of rhINF- ⁇ 7. Omol / L guanidine hydrochloride from the E. coli extract into a chromatographic cake equilibrated with mobile phase A at a flow rate of 3.0-7.0 ml / min. The mobile phase composition was gradually changed from 100% A to 100% B in 25-45 minute intervals. The effluent components were collected and measured for activity. The results showed that the activity recovery was 1774. 57%, which was more than 17 times higher than the conventional method.
  • Chromatographic cake present invention can withstand a pressure of more than 20M P, does not deform during use, to ensure flatness of the injection end of the chromatographic medium surface, help to improve the separation efficiency. 'The separation speed is fast and the system pressure is low (generally less than 5.0 Mpa). From the point of view of good separation results under high flow conditions, its performance is similar to perfusion chromatography, which is conducive to industrial production.
  • the chromatographic cake of the present invention allows the introduction of high viscosity samples and produces a small amount of precipitation. When performing chromatographic separations, fixed inlets and outlets are not required. The irreversible adsorption of the target product is small, which significantly improves the recovery of the target product.
  • the chromatographic cake of the present invention Using the chromatographic cake of the present invention, separation, renaturation, and purification can be completed at one time, which is at least three times simplified than the conventional renaturation and purification processes, shortening the production cycle associated with it by several times, and significantly reducing equipment investment. At the same time, the denaturant can be recycled, which can not only make the denaturant reuse, but also reduce the environmental pollution of the denaturant.
  • the chromatographic cake of the present invention has the function of "one stone and four birds" as shown in FIG. 15 (fast and complete dedenaturing agent, convenient for denaturing agent recovery, protein refolding, and separation of heteroproteins). At the same column volume, it is similar to a general column. In comparison, the method of the present invention can load more fillers, and has a larger mass load and volume load.
  • the chromatographic cake of the present invention will be widely used in biological macromolecule separation, renaturation and purification.

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Description

一种色谱饼及其制造方法与应用
技术领域
本发明涉及一种色谱饼及其制造方法与应用， 特别是涉及一种用于生物 大分子分离或同时复性及纯化的色谱饼及其制造方法与应用。
背景技术
色谱法和毛细管电泳法是常用的两种生物大分子分离手段。 由于毛细 管电泳法处理流动相的量有限， 因此只能用于分析规模。 色谱法是目前生 物大分子分离、 复性、 纯化最重要， 效果最好的分离手段， 既可用于分析 规模， 又可用于生产规模。
在使用色谱法进行分离时， 被人们普遍接受的观点是分离效果与柱塔板 数即柱长成正比。 然而， 柱子太长不仅价格昂贵， 而且导致柱压升高， 必须 使用高效液相色谱仪进行控制。 有文献报道， 由液相色谱中的溶质计量置换 模型可获得使用短柱子进行生物大分子有效分离的证据， 并采用柱长仅为 2 毫米的装填柱和从连续棒上切割的 2毫米厚的薄片形成的膜色谱进行了生物 大分子分离， 效果很好， 但却只能用于分析规模。 如何得到效果稳定可用于 的工业化生产的短柱子， 柱子可以短到什么程度仍然是未知数。
目前，用于生产规模的高效液相色谱柱一般装填直径为 20— 30 μ ιη的粒状 填料， 且柱子的长 /径比以 10为佳， 以避免在高流速条件下柱压过高， 降低分 离效果。 为了不因柱长增加而使软基质填料本身因受压使柱床体积变得愈来 愈小， 导致柱压愈来愈高， 以生产常压和中压色谱介质著名于世的 Phamacia 公司提供了一种厚度较小， （但柱长至少大于 lOCm)直径较大的饼型色谱柱， 但使用时需将几个这样的饼型色谱柱串联在一起， 因此实际上这些串联在一 起的柱长之和还是比柱径大许多倍， 使用时流速不能高。
在生物工程中用大肠杆菌表达的许多蛋白质因其疏水性太强， 在大肠杆 菌中常以包含体的形式存在。 虽然蛋白质的一级结构是正确的， 然而其三级 或四级结构基本是错误的。 由于包含体的疏水性一般都很强， 因此要用高浓 度变性剂， 如 7. 0mol/L盐酸胍（GuHCl )或 8. Omol/L脲溶解包含体， 然后再进 行蛋白复性。 现有技术中常用透析法和稀释法来进行蛋白质复性， 但是这两 种方法不仅复性效率较低 （一般为 5 %— 20% )， 而且耗时且不能达到分离杂 蛋白的目的。有人曾提出用高效液相疏水色谱（HPHIC)实现变性蛋白同时复 性及纯化的工艺， 但是， 在进样时如果遇到蛋白质发生沉淀， 色谱柱就会被 堵塞或毁坏。目前使用的变性蛋白复性及同时纯化装置或多功能蛋白纯化器， 效果并不理想。
归纳起来， 当前生物工程下游产物的分离纯化主要存在的问题是： 1、生 物大分子的分离纯化通常是在装填有大颗粒软基质色谱介质的玻璃柱、 塑料 柱或不锈钢柱中进行的， 其缺点是柱效低， 需要填料多， 消耗流动相的量大， 生产周期长。 2、 当用色谱柱分离小分子物质时， 其分离度与柱长成正比， 柱 径与柱长比一般为 1 : 10。生物大分子的分离度受柱长的影响较小，通常选用 柱长为 5厘米的柱子， 当其装填小颗粒填料后， 色谱系统压力显著增大， 需要 高压液相色谱仪与之配套， 使生产成本增大， 影响充分发挥小颗粒填料柱效 高， 容量大， 重现性好的优点。 3、通常的色谱柱不允许高粘度的样品进入柱 子， 也不许少量样品沉淀在柱头， 并且在使用中要固定流动相的进出口， 给 操作带来不便。 4、通常的分离纯化工艺要经过复性、除去变性剂、粗纯化和 多步精纯化才能获得较纯的产品， 工艺路线长， 质量和活性损失大， 回收率 低（一般为 5— 20% )。 5、通常的复性方法采用稀释法或透析法， 而稀释法又 得采用逐渐降低浓度的分步稀释步骤， 对样品几十倍， 甚至上百倍的稀释会 给随后的分离纯化带来许多困难； 透析法耗时较长（一步透析通常需要 24小 时)， 而且需多次更换透析液。另外， 这两种方法均容易使目标产物在复性过 程中发生聚合沉淀而影响复性效率。 6、 目前的分离纯化方法步骤多， 含目标 产品的溶液体积不断增加， 而且每一步都需要与之配套的设备， 因此设备投 资大， 生产成本高。
目前， 对于装填制造生产型的色谱柱来说， 用轴向加压和径向加压技术 装填色谱填料均可获得满意的柱效， 但这两种方法仅适用于柱长 /径比大于 1 的长色谱柱的装填， 对于装填柱长 /径比小于或等于 1的色谱饼， 还没有理想 的装填制造方法。如果釆用传统工艺装填制造这种色谱饼，会出现以下问题： 1、装填色谱柱的传统工艺是从轴向进出，一般仅适用于柱长 /径比〉 10的色谱 柱。 2、 由于对装柱技术要求很高，稍有不慎很容易出现出口紧密进口疏松等 装填不均匀的情况， 装柱重现性较差。 3、在通常装填色谱柱的过程中， 装完 柱后很容易就可用刀片将填料进口端，也就是流动相的进口端表面填料刮平， 但对于色谱饼而言，没有简单有效的方法使这么大的填料表面平整而又光滑。
4、通常色谱柱在承受相当长时间的高压后才需重新装柱，或者在重新装柱以 前， 修补凹陷的色谱柱进口端可以延长其使用寿命， 而修补凹陷的色谱柱进 口端时必须拆卸柱头， 拿下筛板才能实现。 对于小直径的柱子， 因柱筛板面 积较小， 较容易取下， 而 "色谱饼"筛板的面积一般是通常色谱柱的 1一 100 倍， 而且分布器与筛板嵌合紧密， 并较死地压入柱体的凹槽内， 很难取下， 如果强行取下， 常常会破坏分布器。 5、 当采用高压匀浆法将小颗粒介质轴向 装入色谱饼时， 由于色谱饼的直径较大， 密封比较困难， 必须先试漏， 再装 柱。装好色谱填料后，仍然存在漏液的问题，给装柱工作带来极大的困难。 6、 对于较大的色谱饼需要较大的匀浆罐和辅助设备， 增大生产成本。
发明公开
本发明的目的是提供一种用于生物大分子分离或同时复性及纯化，效果 较好的色谱饼。
一种色谱饼 ，它包括带有流动相进出孔的色谱介质柱及填充于色谱介质 柱内腔中的色谱介质，所述色谱介质柱内腔的厚度与径向长度之比小于或等 于 1。
其中， 所述色谱介质柱内腔较优选的尺寸是： 厚度为 0. 2- 50毫米， 径向 长度为 5. 0-1000毫米。
所述色谱介质柱包括组合在一起的分别带有所述流动相进出孔的上下压 板及带有至少一个侧孔的柱体。
为了防止色谱介质流失， 所述色谱介质柱上下压板靠近色谱介质柱内腔 的一侧分别装设有筛板。 筛板上的筛板孔径应大于流动相中的生物大分子， 小于色谱介质。
为了使流动相均匀分布， 溶质均匀， 提高分离效果， 所述色谱介质柱上 下压板与筛板之间分别装设有分布器。
所述分布器为与所述色谱介质柱内腔截面形状相同的平板， 所述分布器 的至少一侧表面设有放射状和环状的导流槽， 所述放射状和环状导流槽的交 汇处设有分布孔。 所述导流槽的横截面为三角形或弦弧形。
所述分布孔的孔径按与分布器中心距离的增加而逐渐增加。
为了使色谱介质柱的密封性能更好， 所述上下压板及柱体之间装设有密 封圈。
利用本发明的色谱饼， 在生物工程下游的分离纯化过程中， 能在中常压 力下充分利用和发挥小颗粒填料柱效高、 容量大、 重现性好等优点， 降低成 本， 提高产量， 并且将生物大分子的分离纯化及复性中除去变性剂、 粗纯化 和多步精纯化合并为一步完成。 本发明的色谱饼可在 l-200kg/cm2条件下工 作。
本发明的第二个目的是提供一种速度快、 成本低柱效高的色谱饼制备方 法。
为实现这一目的， 本发明釆用以下技术方案： 一种制备色谱饼的方法， 包括以下步骤：
1 )制造色谱介质柱，该色谱介质柱包括组合在一起的分别带有流动相进 出孔的上下压板及带有至少一个侧孔的柱体， 由所述上下压板及柱体围成的 色谱介质柱内腔的厚度与径向长度之比小于或等于 1；
2 )将色谱介质自色谱介质柱的侧孔加入色谱介质柱内腔中。
所述色谱介质柱的侧孔可以与高压匀桨装柱机上的匀浆罐直接相连通进 亍装柱。
制备直径小于 50毫米的色谱饼时， 可以直接将色谱饼装于高压匀浆装柱 机上， 按照一般的装柱方法将色谱介质匀桨液从色谱介质柱的侧孔加入色谱 介质柱内腔中。
当制备直径大于 50毫米的色谱饼时， 为了使色谱介质装填的更紧密， 应 先用抽滤法装柱， 然后将色谱饼装于高压匀浆装柱机上， 将色谱介质匀浆液 从色谱介质柱的侧孔加入色谱介质柱内腔中。
通过色谱介质柱的侧孔来进行补加色谱介质， 是为了达到消除色谱饼中 死体积的目的， 因为色谱饼使用一段时间后， 由于色谱介质的流失死体积会 增加， 影响分离效果。 补加色谱介质的方法与装柱方法相同。
通过色谱介质柱的侧孔来进行冲洗色谱饼， 是为了将分布器和筛板上 积聚的杂质和未进到色谱饼中的生物大分子物质去除掉， 因为这些积聚物 一方面会增加色谱系统的压力， 另一方面会污染后续待分离的样品。 冲洗 色谱饼的方法是将侧孔作为进口， 将色谱饼上下压板上的进出口作为出 口， 通入缓冲液或水进行冲洗。
通过色谱介质柱的侧孔来进行更换失效色谱介质， 是为了将导致系统压 力显著升高， 柱效变得很低的失效色谱介质去除， 装填新的介质。 更换的方 法是将色谱饼上下两个压板上的进出口作为进口， 侧孔作为出口， 通入水冲 出失效的色谱介质， 并用本发明上述装柱法重新装填色谱介质。
用本发明的方法装柱操作非常方便容易， 省时省力； 由于检漏后不需打 开两端的压板而直接装柱， 能确保色谱饼不漏液及介质表面的平整， 有利于 提高柱效； 在装填较大的色谱饼时不需要更大的匀浆罐和另外的辅助设备， 减少了生产成本。
下面结合附图及具体实施例对本发明做进一步说明。
附图说明
图 1为本发明色谱饼的结构示意图。
图 2为图 1的 AA剖视图。
图 3为本发明色谱饼分布器的结构示意图。
图 4为图 3的 BB剖视图。
图 5为径向装填色谱饼的色谱图。
图 6为轴向装填色谱饼的色谱图。
图 7径向装柱色谱饼的色谱图。
图 8为重新径向装柱色谱饼的色谱图。
图 9为色谱饼对目的物的分离色谱。
图 10为色谱柱对目的物的分离色谱。
图 11为 10x50 讓 I. D.色谱饼对七种标准蛋白的色谱分离图。
图 12为 10 X 200mml. D.色谱饼对五种标准蛋白的分离图。
图 13为 10 X 300mml. D.色谱饼对五种标准蛋白的分离图。
图 14为 10X 50ramI. D.色谱饼对重组人干扰素- Y的复性与纯化图。
图 15为本发明色谱饼的多项功效示意图。 实施发明的最佳方式
实施例 1、 本发明色谱饼的结构
如图 1、 图 2所示， 本发明色谱饼包括不锈钢色谱介质柱及填充于色谱介 质柱内腔中的色谱介质 10，色谱介质柱内腔的横截面为圆形， 厚度为 10毫米， 径向长度为 100毫米。色谱介质柱内腔表面的粗糙度应小于 1. 6 μ πι， 以便密封 和减少对生物大分子的不可逆吸附。
色谱介质柱包括组合在一起的分别带有流动相进出孔 1、 2的上下压板 3、 4及带有一个侧孔 9的柱体 5， 上下压板 3、 4及柱体 5围成了色谱介质柱内腔； 柱体 5上的侧孔 9在填充完色谱介质 10后即用堵头堵住。 为了防止泄漏， 上下 压板 3、 4及柱体 5之间装设有耐腐蚀工程塑料的密封圈 8。 上下压板 3、 4靠近 色谱介质柱内腔的一侧分别装设有带有筛板孔的不锈钢筛板 6， 筛板 6的直径 大于色谱介质柱内腔的直径， 筛板孔的孔径介于色谱介质 10颗粒的直径和生 物大分子的直径之间。 '
如图 3、 图 4所示， 上下压板 3、 4与筛板 6之间分别装设有耐酸碱工程塑料 制成的分布器 7， 分布器 7是与色谱介质柱内腔截面形状相同的平板， 它的两 侧表面均设有放射状和环状的导流槽 11， 导流槽 11的横截面为三角形， 放射 状和环状导流槽 11的交汇处设有圆形的分布孔 12， 分布孔的孔径按与分布器 中心距离的增加而逐渐增加。
在该实施例中， 色谱介质柱内腔横截面的形状是多种多样的， 可以是圆 形、 多边形、 椭圆形等。 但色谱介质柱内腔的厚度与径向长度之比应小于或 等于 1， 其中较优选的厚度为 0. 2- 50毫米， 径向长度为 5. 0-1000毫米。色谱介 质柱可选用耐酸碱、 耐压力的不锈钢、 钛合金或各种工程塑料等多种材料制 成， 所填充的色谱介质也可以是多种多样的。 色谱介质柱柱体 5上侧孔 9的数 量可以根据实际情况来确定， 可以是一个或多个。
实施例 2、 用径向装柱法装填制备大于 50毫米的本发明色谱饼
1、 按照实施例 1的方法制造色谱介质柱；
2、 用普通的抽滤法装柱；
3、将色谱介质柱的侧孔与高压匀浆装柱机上的勾浆罐直接相连色谱介质 自色谱介质柱的侧孔加入色谱介质柱内腔中。 实施例 3、 用径向装柱法装填制-备小于 50毫米的本发明色谱饼
1、 按照实施例 1的方法制造色谱介质柱；
2、将色谱介质柱的侧孔与高压匀浆装柱机上的勾浆罐直接相连色谱介质 自色谱介质柱的侧孔加入色谱介质柱内腔中。
实施例 4、 两种方法装填色谱饼的性能比较
在装柱压力为 40MPa和装柱时间为 30min相同的装柱条件下， 分别用通常 的轴向装柱法与本发明径向装柱法将直径为 5 μ πι小颗粒疏水色谱填料装于 10 X 50mmI. D.的色谱饼中， 在流速为 5. 0mL/min， 梯度为 0-100%B， 25min相同的 色谱条件下分离细胞色素- C、核糖核酸酶 A、溶菌酶、 α-淀粉酶和胰岛素五种 蛋白。 重复上述操作各五次， 两种装填方法的色谱图如图 5、 图 6所示， 图中， 1为细胞色素 - C; 2为核糖核酸酶 Α; 3为溶菌酶； 4为 α-淀粉酶； 5为胰岛素， 从图中的结果可以看出， 两种装柱方法所装填的色谱饼对标准蛋白的分离都 取得了好的分离效果，而且径向装填方法比轴向装柱方法具有更好的重现性。
实施例 5、 径向打出及重新装填色谱饼的分离性能
用径向高压匀浆法从色谱饼的侧孔将疏水填料装入其中， 用堵头封好侧 孔， 在流速为 5ml/min和梯度为 100%A〜100%B， 25min条件下， 分离细胞色素 - C (Cyt- C)、 肌红蛋白 （Myo)、 溶菌酶 （Lys ) 和 α-淀粉酶 （a- Amy), 结果 如图 7所示， 图中 1为细胞色素 -C; 2为核糖核酸酶 A; 3为溶菌酶； 4为 α-淀粉 酶。 然后， 将该色谱饼充分冲洗后， 以侧孔为出口， 以色谱饼的进出口为进 口， 用水作流动相， 开启色谱泵， 打出填料。 在打出的填料中补加少许填料 并经超声除气及匀浆后， 以同样装柱方法重新装填该色谱饼， 再在上述色谱 条件下， 分离这四种蛋白， 结果如图 8所示， 图中 1为细胞色素 -C; 2为核糖核 酸酶 Α; 3为溶菌酶； 4为 α-淀粉酶。 由图 7和图 8看出， 通过色谱饼的侧孔打 出填料后， 在不拆卸上下压板的情况下， 再从侧孔将打出的填料重新装入色 谱饼， 对分离度没有显著影响， 表明利用色谱饼的侧孔可以将失效的填料方 便的打出， 也可以再方便地装入新填料或补加填料。
实施例 6、 色谱柱与色谱饼分离性能的比较
规格为 5 X 50顧 I. D.的色谱饼和 200 X 7. 9匪 I. D.的色谱柱， 二者的内腔 体积为 9. 9±0. 2mL， 并在 40MPa压力条件下装填同一批 HPHIC填料。 在进样量 相同和流速均为 4. 0mL/min条件下，对六种标准蛋白进行了分离。结果如图 9、 图 10所示，图中 1为细胞色素 -C; 2为肌红蛋白； 3为核糖核酸酶 A; 4为溶菌酶； 5为 a-淀粉酶； 6为胰岛素， 从图中可以看出， 色谱饼和色谱柱对六种蛋白的 色谱分离度基本相同， 但色谱饼厚度只有柱床几何体积相同的色谱柱长度的 1/40。 表明与色谱柱几何体积相同， 直径大、 柱长短的色谱饼用于蛋白的液 相色谱分离时， 可以满足对分离度的要求。
实施例 7、 不同规格色谱饼的分离性能
用 10x50 mm I. D. 、 10x200 mm I. D.和 10x300 mm I. D.三种用本发明方 法制备的色谱饼对标准蛋白进行分离， 结果如图 11、 图 12和图 13所示， 图 11 所示的是在流速为 5. Oml/min,阻尼系数为 0. 08AUFS,梯度为 100%A变成 100%B， 40πήη色谱条件下分离 1细胞色素 - C; 2肌红蛋白； 3核糖核酸酶 A; 4溶菌酶； 5α-糜原蛋白酶 （cx-Chy) ; 6cc-淀粉酶； 7胰岛素。 图 12所示的是在流速为 100. Oml/min, 阻尼系数为 0. 05AUFS, 梯度为 100%A变成 100%B， 40min色谱条 件下分离 1细胞色素 -C; 2肌红蛋白； 3溶菌酶； 4α-淀粉酶； 5胰岛素。 图 13 所示的是在流速为 120. 0ml/min， 阻尼系数为 0. 1AUFS， 梯度为 100%A变成 100%B， 60min色谱条件下分离 1细胞色素 - C; 2肌红蛋白； 3核糖核酸酶 A; 4 溶菌酶; 5α-淀粉酶。从图中可以看出，不同规格的色谱饼均具有很好的分离 效果。
实施例 8、 色谱饼对脲变性和盐酸胍变性的溶菌酶的复性效率
在所设定的两种色谱条件下， 先用 Α液平衡 5 X 50腿 I. D.色谱饼后， 分别 进样脲变性和盐酸胍变性的溶菌酶溶液，收集它们经过色谱饼复性后的组分， 测定活性回收率如表 1所示。 所用梯度为 100%A变成 100%B。 从表 1的结果可以 看出， 本发明色谱饼对脲变性和盐酸胍变性的溶菌酶的复性效果显著。 表 1 不同色谱条件下溶菌酶的活性回收率
样品 流速 4mL/rain, 线性 流速 2mL/min， 线性梯度
梯度时间 25min 时间 50min
脲变性溶菌酶 102. 9% 104. 7%
盐酸胍变性溶菌酶 103..7 % 104. 0% 实施例 9、 色谱饼对重组人干扰素- Y的复性与纯化
图 14为重组人干扰素 - Υ ( rhINF- γ ) 在 10 X 50匪 I. D.色谱饼中的复 性纯化图。 样品为用 7. Omol/L盐酸胍从大肠杆菌细胞破碎液中提取的 rhINF- γ溶液。 rhINF- γ的活性测定方法为细胞病变抑制法。 操作条件： 将 1ml rhINF- γ的 7. Omol/L盐酸胍从大肠杆菌提取液进样到已用流动相 A 平衡的色谱饼中， 流速为 3. 0— 7. 0ml/min。 在 25— 45分钟的间隔内使流动 相组成逐渐由 100%A变成 100%B。 分别收集流出的组分并测定其活性， 结果 表明活性回收率为 1774. 57%, 比常规方法提高了 17倍多。
工业应用
本发明的色谱饼可以耐受 20MPa以上的压力，在使用过程中不变形，可确 保进样端色谱介质表面的平整性， 有利于提高分离效率。 '分离速度快， 系统 压力低（一般小于 5. 0Mpa)， 从能在高流速条件下有好的分离结果来看， 其性 能与灌注色谱相似， 有利于工业化生产。 本发明的色谱饼允许进高粘度的样 品和产生少许沉淀。 在进行色谱分离时， 不需要固定进、 出口。 对目标产物 的不可逆吸附小， 使目标产物的回收率显著提高。 使用本发明的色谱饼， 可 以使分离、复性、纯化一次完成， 较通常的复性及纯化工艺至少简化了三倍， 使与之相关的生产周期缩短数倍，设备投资显著减少， 并可同时回收变性剂， 既可以使变性剂重复使用， 又可减少变性剂对环境的污染。 本发明的色谱饼 有如图 15所示的 "一石四鸟"的作用 （快速而完全的除变性剂， 便于变性剂 回收， 蛋白复性， 杂蛋白分离）， 在相同柱体积时， 与一般柱子比较， 用本发 明的方法可以装入较多的填料， 具有较大的质量负载和体积负载。
本发明的色谱饼在生物大分子分离、 复性及纯化中将得到广泛应用。

Claims

权利要求书
1、 一种色谱饼 ， 它包括带有流动相进出孔的色谱介质柱及填充于色谱 介质柱内腔中的色谱介质， 其特征在于： 所述色谱介质柱内腔的厚度与径向 长度之比小于或等于 1。
2、 根据权利要求 1所述的色谱饼， 其特征在于： 所述色谱介质柱内腔厚 度为 0. 2-50毫米， 径向长度为 5. 0-1000毫米。
3、根据权利要求 1或 2所述的色谱饼， 其特征在于： 所述色谱介质柱包括 组合在一起的分别带有所述流动相进出孔的上下压板及带有至少一个侧孔的 柱体。
4、 根据权利要求 3所述的色谱饼， 其特征在于： 所述色谱介质柱上下压 板靠近色谱介质柱内腔的一侧分别装设有筛板。
5、 根据权利要求 4所述的色谱饼， 其特征在于： 所述色谱介质柱上下压 板与筛板之间分别装设有分布器。
6、 根据权利要求 5所述的色谱饼， 其特征在于： 所述分布器为与所述色 谱介质柱内腔截面形状相同的平板， 所述分布器的至少一侧表面设有放射状 和环状的导流槽， 所述放射状和环状导流槽的交汇处设有分布孔。
7、 ·根据权利要求 6所述的色谱饼， 其特征在于： 所述导流槽的横截面为 三角形或弦弧形。
8、 根据权利要求 6所述的色谱饼， 其特征在于： 所述分布孔的孔径按与 分布器中心距离的增加而逐渐增加。
9、 根据权利要求 3所述的色谱饼， 其特征在于： 所述上下压板及柱体之 间装设有密封圈。
10、 一种制备色谱饼的方法， 包括以下步骤：
1 )制造色谱介质柱， 该色谱介质柱包括组合在一起的分别带有流动相进 出孔的上下压板及带有至少一个侧孔的柱体， 由所述上下压板及柱体围成的 色谱介质柱内腔的厚度与径向长度之比小于或等于 1;
2 ) 将色谱介质自色谱介质柱的侧孔加入色谱介质柱内腔中。
11、 根据权利要求 10所述的制备色谱饼的方法， 其特征在于： 所述色谱 介质柱上下压板靠近色谱介质柱内腔的一侧分别装设有筛板。
12、 根据权利要求 11所述的制备色谱饼的方法， 其特征在于： 所述色谱 介质柱上下压板与筛板之间分别装设有分布器； 所述分布器为与所述色谱介 质柱内腔截面形状相同的平板， 所述分布器的至少一侧表面设有放射状和环 状的导流槽， 所述放射状和环状导流槽的交汇处设有分布孔； 所述导流槽的 横截面为三角形或弦弧形； 所述分布孔的孔径按与分布器中心距离的增加而 逐渐增加。
13、 根据权利要求 10所述的制备色谱饼的方法， 其特征在于： 所述上下 压板及柱体之间设有密封圈。
14、 根据权利要求 10或 11或 12或 13所述的制备色谱饼的方法， 其特征在 于- 所述色谱介质柱的侧孔与高压匀浆装柱机上的匀浆罐直接相连通进行装 柱。
15、 根据权利要求 10或 11或 12或 13所述的制备色谱饼的方法， 其特征在 于： 制备直径小于 50毫米的色谱饼时，直接将色谱饼装于高压勾浆装柱机上， 将色谱介质匀浆液从色谱介质柱的侧孔加入色谱介质柱内腔中。
16、 根据权利要求 10或 11或 12或 13所述的制备色谱饼的方法， 其特征在 于： 制备直径大于 50毫米的色谱饼时， 先用抽滤法装柱， 然后将色谱饼装于 高压匀浆装柱机上， 将色谱介质匀浆液从色谱介质柱的侧孔加入色谱介质柱 内腔中。
17、 根据权利要求 10或 11或 12或 13所述的制备色谱饼的方法， 其特征在 于： 通过色谱介质柱的侧孔来进行补加色谱介质、 冲洗色谱饼、 更换失效色 谱介质。
18、 权利要求 1一 9中所述的色谱饼在生物大分子分离中的应用。
19、 权利要求 1一 9中所述的色谱饼在生物大分子复性及纯化中的应用。
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