阅读说明：本技术 层析柱延长装置、系统和利用其装柱的方法 (Chromatographic column extension device, system and method for packing column by using same ) 是由 臧美翎 董翠 花季 毛晟峰 于 2021-06-30 设计创作，主要内容包括：本发明属于生物设备领域,具体的,提出了一种层析柱延长装置,包括：自上而下设置的容纳区、第一收缩区和分布区；所述分布区开口端具有至少一个分布角,所述分布角的端面具有高度差。利用该层析柱延长装置可以减小填料装填时产生的表面张力,填料快速均匀沉降,获得较优柱效,无需缓冲液逐步置换出填料,无需反复贴壁加入填料等繁琐步骤,提高装柱效率,尤其适用于小规格层析柱,适于广泛推广应用。(The invention belongs to the field of biological equipment, and particularly provides a chromatographic column lengthening device, which comprises: the containing area, the first contraction area and the distribution area are arranged from top to bottom; the open end of the distribution area is provided with at least one distribution angle, and the end surface of the distribution angle is provided with a height difference. The chromatographic column lengthening device can reduce the surface tension generated during filling of the filler, the filler is quickly and uniformly settled, a better column effect is obtained, the filler is not required to be gradually replaced by buffer solution, complicated steps of adding the filler by repeatedly adhering the wall are not required, the column filling efficiency is improved, and the device is particularly suitable for small-specification chromatographic columns and is suitable for wide popularization and application.)

1. A chromatography column extension device, comprising: the containing area, the first contraction area and the distribution area are arranged from top to bottom;

the open end of the distribution area is provided with at least one distribution angle, and the end surface of the distribution angle is provided with a height difference.

2. The chromatography column extension device of claim 1, wherein a connection surface formed between the highest point and the lowest point of the end surface of the distribution angle is an upward arc surface;

optionally, the radian of the arc surface is 0.15-0.5 degrees, and the height difference between the highest point and the lowest point is 1-1.5 cm;

optionally, the height ratio of the containing zone, the first contraction zone and the distribution zone is (10-15): (1-2): (2-5).

3. The chromatography column extension device of claim 1, wherein the distribution zone is removably connected with the first constriction zone;

the opening end of the containing area is provided with a detachable cover body, and the distribution area is provided with a valve.

4. The chromatography column extension device of claim 1, wherein the containing zone is provided with a first zone, a second constriction zone and a third zone from top to bottom, and the third zone is connected with the first constriction zone;

optionally, the height ratio between the first, second and third zones is (8-10): (1-1.5): (2-3.5);

the ratio of the inner diameters of the first area to the third area is (2-3): (1-1.5);

preferably, the height of the accommodating area is 10-15 cm, and the inner diameter of the opening end is 2-3 cm;

the height of the first contraction zone is 1-2 cm;

the height of the distribution area is 2-5 cm, the inner diameter is 4-5 mm, and the outer diameter is 5.5-6.0 mm.

5. The chromatographic column extension device of claim 1, wherein the distribution area is provided with a fixing area, and the outer wall of the fixing area is provided with a fixing part which can be connected with the chromatographic column;

optionally, the securing means comprises a thread, a rubber plug or a snap.

6. A chromatography system, comprising:

the chromatographic column comprises a feeding hole and a discharging hole;

the chromatography column extension fixture of any one of claims 1 to 5, wherein the distribution region of the chromatography column extension fixture is connected with the feed inlet of the chromatography column.

7. The chromatography system of claim 6, wherein the inner diameter of the chromatography column is 5-10 mm, the outer diameter of the distribution area of the chromatography column extension device is smaller than the inner diameter of the chromatography column, and the difference is not more than 1-1.5 mm.

8. Use of a chromatography column extension device according to any one of claims 1 to 5 or a chromatography system according to claim 6 or 7 in a chromatography column.

9. A method of packing a chromatography column using the chromatography system of claim 6 or 7, comprising:

and injecting column filling liquid containing a filler and a column filling buffer solution into the open end of the containing area of the chromatographic column extension device, wherein the filler is filled in the chromatographic column, and at least part of liquid flows out from the discharge hole of the chromatographic column.

10. The method according to claim 9, wherein the column buffer comprises 10-30 vol% of ethanol and 0.1-0.2 mol/L of sodium chloride;

optionally, the method further comprises:

when at least part of liquid flows out of a discharge hole of the chromatographic column, the chromatographic column extension device is disassembled, balance liquid is injected into a feed hole of the chromatographic column, and at least part of liquid flows out of the discharge hole of the chromatographic column;

optionally, the equilibrium solution comprises 100-200 mmol/L sodium chloride buffer solution.

Technical Field

The present invention relates to the field of biological devices. In particular, the present invention relates to chromatography column extension devices, systems, methods of packing columns using the same, and column packing buffers suitable for use in chromatography systems.

Background

Column chromatography is widely used for extracting, separating and purifying nucleic acid, protein, organic compounds and natural products in the fields of chemistry, chemical engineering and pharmacy due to its advantages of simplicity, economy, good separation effect and the like.

The most critical ring in the development of column chromatography purification process is the screening of the filler, and the filler is used as a carrier of the filler, so that the filling of a small-specification chromatographic column is particularly important. For each process step, refinement of process development generally focuses on 1-3 candidate fillers. These fillers were tested under dynamic flow rate conditions using basic retention time, flow rate, pH and ionic strength process conditions. The process development chromatographic column needs to be filled with the same filler to the same column bed height, and the filled column bed has the same quality in the aspects of detection indexes such as column efficiency (number of tower plates) and asymmetry. In the early stages of development, the number of samples is limited and a certain number of samples are required for multi-conditional screening. The use of a small-sized chromatographic column can save samples and reduce the process development cost.

At present, two options are mainly available for small-size chromatographic columns, one is a pre-packed column, such as a mintuofan 1ml micro-chromatographic column pre-packed column, which is convenient to use and low in price, but is not suitable for more detailed chromatographic evaluation (because the chromatographic evaluation requires accurate simulation of dynamic liquid flow conditions), and cannot finally represent the chromatography of production scale.

The other is self-packing with empty columns, but the challenge is how to efficiently pack multiple chromatography columns for the process that meet the column efficiency requirements. Depending on the amount of packing and conditions to be evaluated, 30 or more columns may be required at this stage. These columns need to be reproducible to pack and may represent the final production scale. Conventional methods of column packing often result in column packing failures due to failure to meet column efficiency requirements, and higher column packing failure rates place additional time and resource pressure on process development teams. Before the conventional column filling, the filler needs to be gradually replaced into a salt solution from 20% ethanol preservation solution, generally more than three times of replacement is needed, the process needs longer time, and the operation is relatively complex. The prior related patent CN109589644B mentions that a 20% ethanol solution is directly used for a column packing method of a reduced scale, and a chromatographic column with qualified column efficiency can be obtained. However, because the inner pipe wall of a chromatography column with a small specification (for example, 6.6mm inner diameter) is small, the packing cannot be attached to the column body efficiently at one time, and when the packing is added to the column rapidly, an elastic membrane is easily formed at the column port due to liquid tension, so that the packing is easily overflowed, and loss is caused. Moreover, if the filler is directly filled into the column by using 20% ethanol solution, the settling time of the filler is longer, and the chromatographic column needs to be balanced by using 0.15mol of sodium chloride buffer solution before the column is filled for measuring the column effect, the column filling efficiency is not high. Therefore, manual column packing also restricts the wide application of such small-format chromatography columns.

In view of the above, the method for packing the chromatography column (especially, the small-scale column) still needs to be studied.

Disclosure of Invention

The present invention aims to solve, at least to some extent, the technical problems of the prior art. Therefore, the invention provides a chromatographic column extension device, a chromatographic system, application thereof and a method for packing the chromatographic column by using the chromatographic column extension device, which can reduce the surface tension generated during packing of the filler, quickly and uniformly settle the filler, obtain better column effect, do not need complicated steps of gradually replacing the filler by buffer solution, repeatedly adding the filler along the wall and the like, improve the column packing efficiency, are particularly suitable for small-specification chromatographic columns and are suitable for wide popularization and application.

In one aspect of the invention, a chromatography column extension device is provided. According to an embodiment of the present invention, the chromatography column extension device comprises: the device comprises an accommodating area, a first contraction area and a distribution area which are arranged from top to bottom, wherein the open end of the distribution area is provided with at least one distribution angle, and the end surface of the distribution angle is provided with a height difference.

According to the chromatographic column extending device provided by the embodiment of the invention, the containing area is used for containing the filler homogenate injected into the containing area, and then the filler homogenate flows into a chromatographic column connected with the chromatographic column extending device through the first contraction area and the distribution area so as to achieve the purpose of column loading. Because the internal diameter of holding the district is greater than the internal diameter of distribution district, the district can hold a large amount of filler homogenate, can once only with whole or most filler homogenate pour into the holding the district into, improve dress post efficiency. Also, since a large amount of the filler slurry exists in the holding section, hydraulic pressure is generated, and the flow rate of the liquid can be further increased. However, the conventional direct-connection device column needs to drop the filler homogenate a small amount of times, so that the filler is not uniformly filled, and the conventional direct-connection device column needs to shake the drop to avoid filler precipitation, so that the workload is increased, and for a small-specification chromatographic column, because the settling speed of the filler is low, and the filler homogenate needs to be dropped for many times, the whole column filling time is long, and the efficiency is low.

Through set up distribution angle (oblique angle) at distribution district open end (being feed liquid outflow end), the design of this distribution angle can make to pack and once only adhere to the wall and add into the chromatography column body high-efficiently, produces liquid tension when avoiding simultaneously adding the filler fast and leads to the column mouth to form the elastic membrane for the feed liquid overflows. Therefore, the filler is gathered and drained, the wall attaching effect of the filler is improved, and the distribution uniformity of the filler is improved.

According to an embodiment of the present invention, the above-mentioned chromatography column extension device may further have the following additional technical features:

according to the embodiment of the invention, the connecting surface formed between the highest point and the lowest point of the end surface of the distribution angle is an upward cambered surface.

According to the embodiment of the invention, the radian of the cambered surface is 0.15-0.5 degrees, and the height difference between the highest point and the lowest point is 1-1.5 cm.

According to an embodiment of the invention, the distribution area is detachably connected with the first constriction area.

According to the embodiment of the invention, the height ratio of the containing area, the first contraction area and the distribution area is (10-15): (1-2): (2-5).

According to the embodiment of the invention, the accommodating area is provided with a first area, a second contraction area and a third area from top to bottom, and the third area is connected with the first contraction area.

According to an embodiment of the invention, the height ratio between the first zone, the second constriction zone and the third zone is (8-10): (1-1.5): (2-3.5); the ratio of the inner diameters of the first area to the third area is (2-3): (1-1.5).

According to the embodiment of the invention, the distribution area is provided with a fixing area, and the outer wall of the fixing area is provided with a fixing part which can be connected with the chromatographic column.

According to an embodiment of the invention, the fixation means comprises a thread, a rubber plug or a snap.

According to the embodiment of the invention, the height of the containing area is 10-15 cm, and the inner diameter of the opening end is 2-3 cm; the height of the first contraction zone is 1-2 cm; the height of the distribution area is 2-5 cm, the inner diameter is 4-5 mm, and the outer diameter is 5.5-6.0 mm.

According to the embodiment of the invention, the opening end of the containing area is provided with a detachable cover body, and the distribution area is provided with a valve.

In another aspect of the invention, the invention features a chromatography system. According to an embodiment of the invention, the chromatography system comprises: the chromatographic column comprises a feeding hole and a discharging hole; the chromatographic column lengthening device is characterized in that the distribution area of the chromatographic column lengthening device is connected with the feed inlet of the chromatographic column. Therefore, the chromatographic system provided by the embodiment of the invention can reduce the surface tension generated during filling of the filler, the filler is quickly and uniformly settled, a better column effect is obtained, the filler is not required to be gradually replaced by a buffer solution, complicated steps of adding the filler by means of repeated adherence and the like are not required, the column filling efficiency is improved, and the chromatographic system is particularly suitable for small-specification chromatographic columns and is suitable for wide popularization and application.

Chromatography systems according to embodiments of the invention may also have the following additional technical features:

according to the embodiment of the invention, the inner diameter of the chromatographic column is 5-10 mm, the outer diameter of the distribution area of the chromatographic column extension device is smaller than the inner diameter of the chromatographic column, and the difference is not more than 1-1.5 mm.

In a further aspect of the invention, the invention provides the use of a chromatography column extension device or chromatography system as described above in a chromatography column pack. As described above, the chromatographic column extension device or the chromatographic system according to the embodiment of the present invention can reduce the surface tension generated during packing of the filler, and the filler is rapidly and uniformly settled, so as to obtain a better column effect, without the need of gradually replacing the filler with a buffer solution, without the need of complicated steps such as repeated wall-sticking addition of the filler, and the like, thereby improving the column packing efficiency.

In yet another aspect of the invention, the invention provides a method of packing a chromatography column using the chromatography system described above. According to an embodiment of the invention, the method comprises: and injecting column filling liquid containing a filler and a column filling buffer solution into the open end of the containing area of the chromatographic column extension device, wherein the filler is filled in the chromatographic column, and at least part of liquid flows out from the discharge hole of the chromatographic column. Therefore, the method provided by the embodiment of the invention can reduce the surface tension generated during filling of the filler, the filler is quickly and uniformly settled, a better column effect is obtained, the filler is not required to be gradually replaced by a buffer solution, the complicated steps of adding the filler by means of repeated adherence and the like are not required, the column filling efficiency is improved, and the method is particularly suitable for small-specification chromatographic columns and is suitable for wide popularization and application.

According to the embodiment of the invention, the method for packing the chromatographic column by using the chromatographic system can further have the following additional technical characteristics:

according to the embodiment of the invention, the column buffer solution contains 10-30 vol% of ethanol and 0.1-0.2 mol/L of sodium chloride.

According to an embodiment of the invention, the method further comprises: and when at least part of liquid flows out from the discharge hole of the chromatographic column, the chromatographic column extension device is detached, balance liquid is injected into the feed hole of the chromatographic column, and at least part of liquid flows out from the discharge hole of the chromatographic column.

According to the embodiment of the invention, the equilibrium solution comprises 100-200 mmol/L sodium chloride buffer solution.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a schematic view of a chromatography column extension device according to an embodiment of the invention;

FIG. 2 shows a schematic diagram of a chromatography system according to an embodiment of the invention;

FIG. 3 shows a column efficiency test chart of a reduced-size model chromatography column according to example 1 of the present invention;

FIG. 4 shows a column efficiency test chart of a reduced-size model chromatography column according to example 2 of the present invention;

FIG. 5 shows a column efficiency test chart of a reduced-size model chromatography column according to example 3 of the present invention;

FIG. 6 shows a column efficiency test chart of a reduced-size model chromatography column according to example 4 of the present invention;

FIG. 7 is a graph showing column efficiency test of a reduced-size model chromatography column according to comparative example 1 of the present invention;

FIG. 8 is a graph showing column efficiency test of a reduced-size model chromatography column according to comparative example 2 of the present invention;

FIG. 9 shows a column efficiency test chart of a reduced-size model chromatography column according to comparative example 3 of the present invention.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.

The present invention provides a chromatographic column lengthening device, a chromatographic system, applications thereof and a method for packing columns by using the same, which will be described in detail below.

Chromatographic column extension fixture

In one aspect of the invention, a chromatography column extension device is provided. According to an embodiment of the present invention, referring to fig. 1, the chromatography column extension device comprises: the containing region 100, the first contraction region 200 and the distribution region 300 are arranged from top to bottom. The holding area has a larger opening, so that the filling material can be conveniently injected. The first constriction region has a reduced diameter and the distribution region has an average inner diameter that is lower than the average inner diameter of the containment region. The containing region is used for containing the filler homogenate injected into the containing region, and then the filler homogenate flows into the chromatographic column connected with the chromatographic column extension device through the first contraction region and the distribution region, so as to achieve the purpose of column packing. Because the internal diameter of holding the district is greater than the internal diameter of distribution district, the district can hold a large amount of filler homogenate, can once only with whole or most filler homogenate pour into the holding the district into, improve dress post efficiency. Also, since a large amount of the filler slurry exists in the holding section, hydraulic pressure is generated, and the flow rate of the liquid can be further increased. However, the conventional direct-connection device column needs to drip filler homogenate for a small amount of times, and the filler that can produce is filled unevenly, and the conventional direct-connection device column still needs to shake to drip while avoiding filler to precipitate, has increased work load, and to small-specification chromatography column, because filler settling velocity is slower, need to drip filler homogenate for a plurality of times in addition, lead to holistic dress post time longer, efficiency is lower.

According to the embodiment of the present invention, the open end of the distribution area 300 has at least one distribution angle, and the end surface of the distribution angle has a height difference. The design of distribution angle can make filler once only add to the chromatography column body adherent to the wall high-efficiently, avoids producing liquid tension when adding filler fast simultaneously and leads to the column mouth to form the elastic membrane for the feed liquid spills over. Therefore, the filler is gathered and drained, the wall attaching effect of the filler is improved, and the distribution uniformity of the filler is improved.

According to the embodiment of the invention, the connecting surface formed between the highest point A and the lowest point B of the end surface of the distribution angle is an upward cambered surface 10. The design of distribution angle can make the filler adherent downward flow, and special cambered surface design can play the effect that further improves filler and gathers together drainage effect, improves the degree of consistency that the filler distributes on the whole. In particular, there may be two, four, six, eight, etc., arranged in a symmetrical manner in the main manner.

According to the embodiment of the invention, the radian of the cambered surface 10 is 0.15-0.5 degrees, and the height difference between the highest point and the lowest point is 1-1.5 cm. The cambered surface design of this size can play the effect of gathering together the drainage with packing, improves the packing adherence effect, improves the degree of consistency that the packing distributes.

According to an embodiment of the present invention, the distribution area 300 is detachably connected with the first contraction area 200. Therefore, the distribution area is convenient to replace, and the use efficiency is improved. When the pipe diameter of the joint is reduced slightly during the replacement process, the flow rate of local filling is accelerated, but the filling effect is not affected.

According to the embodiment of the present invention, the height ratios H1, H2 and H3 of the containing zone 100, the first contraction zone 200 and the distribution zone 300 are (10-15): (1-2): (2-5). From this, the filler can once only be added through holding the district, through the even distribution of first constriction region control velocity of flow, makes the filler wall flow in the post through the distribution region.

According to an embodiment of the present invention, the receiving region 100 is provided with a first region 110, a second contraction region 120, and a third region 130 from the top, the third region 110 being connected to the first contraction region 200. The average inner diameter of the first zone, the second contraction zone and the third zone is in a decreasing trend, and the design is helpful for controlling the filler to be at a proper flow rate, so that the filler is uniformly distributed, and the filling effect of the filler is improved. Specifically, the first zone and the third zone may independently be cylinders of uniform diameter from top to bottom.

According to an embodiment of the present invention, the height ratios H4, H5, and H6 between the first zone 110, the second constriction zone 120, and the third zone 130 are (8-10): (1-1.5): (2-3.5); the first area and the third area are both columnar, and the inner diameter ratio is (2-3): (1-1.5). Therefore, the flow rate of the filler is controlled properly, the filler is distributed uniformly, and the filling effect of the filler is improved.

According to the embodiment of the present invention, the distribution area 300 is provided with the fixing area 310, and the fixing part 20 connectable to the chromatography column is provided on the outer wall of the fixing area 310. The region at the lower end of the fixing region in the distribution region can extend into the cavity of the chromatographic column and then be fixed by the fixing part, so that the extension device can be stably and firmly fixed on the chromatographic column. Specifically, the type of the fixing component is not strictly limited, and the fixing component can be a thread, a rubber plug, a buckle and the like.

According to the embodiment of the invention, the height of the accommodating area is 10-15 cm, and the inner diameter of the opening end is 2-3 cm; the height of the first contraction zone is 1-2 cm; the height of the distribution area is 2-5 cm, the inner diameter is 4-5 mm, and the outer diameter is 5.5-6.0 mm. From this, the filler can once only be added through holding the district, through the even distribution of first constriction region control velocity of flow, makes the filler wall flow in the post through the distribution region.

According to an embodiment of the present invention, a detachable cover is provided on the open end of the receiving area 100, and a valve is provided on the distribution area 300. The cover body arranged at the opening end can be connected with a pressurizing device (such as an air pump with a sterile filter) to accelerate the sedimentation of the filler by pressurization, so that the filling efficiency is improved. The valve design can serve the purpose of controlling the flow rate.

Chromatography system

In another aspect of the invention, the invention features a chromatography system. According to an embodiment of the invention, referring to fig. 2, the chromatography system comprises: a chromatography column 1000 and a chromatography column extension device 2000. The chromatographic column lengthening device can enable the filler to be rapidly and uniformly settled, and the success rate and the efficiency of column packing are improved.

According to the embodiment of the invention, the inner diameter of the chromatographic column is 5-10 mm. At present, a liquid-transfering gun is generally adopted to fill filler for a small-specification chromatographic column with an inner diameter of 5-10 mm, and the gun head needs to be partially cut in advance. Because the inner diameter of the chromatographic column is smaller, the settling speed of the filler is very slow, the filler liquid is added after the filler is completely settled every time, and the filler liquid is added for a few times, so the time for using is very long. In addition, in order to prevent the uneven column filling caused by the uneven slurry of the sucked filler due to the sedimentation of the filler, the filler slurry needs to be stirred and shaken continuously, so that the overall workload is increased. Filling 9mL of filler homogenate usually consumes about 1 hour, the filling efficiency is low, and the column efficiency is not high.

By adopting the chromatographic column lengthening device, the surface tension generated during filling of the filler can be reduced, the filler is quickly and uniformly settled, a better column effect is obtained, the filler is not required to be gradually replaced by buffer solution, complicated steps of adding the filler by attaching the filler to the wall repeatedly and the like are not required, the column filling efficiency is improved, and the device is particularly suitable for small-specification chromatographic columns and is suitable for wide popularization and application.

In some embodiments, a column is used having a theoretical plate height of 0.05cm or less and a column symmetry of 0.8 to 1.4. The inner diameter of the chromatography column was 6.6mm, the outer diameter was 1.4cm, and the length of the chromatography column was 250 mm. The filler includes any one of protein a affinity filler, cation exchange filler, anion exchange filler and hydrophobic interaction filler. The filler may be selected from AF-rProtein A HC-650F, MabSelect LX, prismA, Eshmuno A, POROS 50XS, POROS 50HQ, Capto Q, Capto S Impact. The chromatographic column is Omnifit series chromatographic column. More preferably, the chromatography cartridge is selected from any one of Omnifit EZColumn and Omnifit EZ SOLVENTLUS Column.

According to the embodiment of the invention, the outer diameter of the distribution area of the chromatographic column extension device is smaller than the inner diameter of the chromatographic column, and the difference is not more than 1-1.5 mm. The inner diameter of the distribution area is designed to keep a certain gap with the inner wall of the chromatographic column, but the inner diameter of the distribution area can not be in interference fit, so that the adherence and the stable fluidity of the filler are improved, and the uniform and stable distribution is facilitated.

It will be appreciated by those skilled in the art that the features and advantages described above in relation to the column extension device apply equally to the chromatography system and will not be described in further detail herein.

Application of chromatographic column lengthening device or chromatographic system in chromatographic column packing

In a further aspect of the invention, the invention provides the use of a chromatography column extension device or chromatography system as described above in a chromatography column pack. As described above, the chromatographic column extension device or the chromatographic system according to the embodiment of the present invention can reduce the surface tension generated during packing of the filler, and the filler is rapidly and uniformly settled, so as to obtain a better column effect, without the need of gradually replacing the filler with a buffer solution, without the need of complicated steps such as repeated wall-sticking addition of the filler, and the like, thereby improving the column packing efficiency.

It will be appreciated by those skilled in the art that the features and advantages described above in relation to the column extension device or chromatography system are equally applicable to this application and will not be described in further detail herein.

Method for packing chromatographic column by using chromatographic system

In yet another aspect of the invention, the invention provides a method of packing a chromatography column using the chromatography system described above. According to an embodiment of the invention, the method comprises: and injecting column filling liquid containing a filler and a column filling buffer solution into the open end of the containing area of the chromatographic column extension device, wherein the filler is filled in the chromatographic column, and at least part of liquid flows out from the discharge hole of the chromatographic column. Therefore, the method provided by the embodiment of the invention can reduce the surface tension generated during filling of the filler, the filler is quickly and uniformly settled, a better column effect is obtained, the filler is not required to be gradually replaced by a buffer solution, the complicated steps of adding the filler by means of repeated adherence and the like are not required, the column filling efficiency is improved, and the method is particularly suitable for small-specification chromatographic columns and is suitable for wide popularization and application.

According to the embodiment of the invention, the column buffer solution contains 10-30 vol% of ethanol and 0.1-0.2 mol/L of sodium chloride.

At present, some fillers are stored in an ethanol solution, and before the fillers are used, the fillers are required to be mixed and washed by sodium chloride and the ethanol solution, then a part of the solution is poured out, and the ethanol content in the system is gradually diluted, so that the purpose of replacing the fillers in the sodium chloride solution is achieved. If the ethanol is not replaced, the ethanol has low density, so that the filler is slowly settled due to the existence of the ethanol in the filler, large particles of the filler with different particles are firstly settled due to different particle sizes, and small particles of the filler are later settled, so that the final filler is unevenly distributed in the whole column.

As mentioned above, the design of the chromatographic column lengthening device can improve the flow velocity of the filler, accelerate sedimentation, improve the adherence and stable fluidity of the filler and facilitate uniform distribution. Therefore, the ethanol is not required to be gradually replaced by the sodium chloride solution, the sodium chloride can be directly added into the preservation solution, and then the column is directly installed, so that the workload is reduced, and the column installing efficiency is improved. The filler homogenate processed by the method is beneficial to accelerating the uniform sedimentation of the filler and can quickly reach the balance of salt concentration before column packing.

According to an embodiment of the invention, the method further comprises: when at least part of liquid flows out from the discharge hole of the chromatographic column, the chromatographic column extension device is detached, balance liquid is injected into the feed hole of the column, and at least part of liquid flows out from the discharge hole of the chromatographic column. The column wall effect can be effectively relieved by using the equilibrium liquid to wash the chromatographic column, and better column effect is obtained.

According to the embodiment of the invention, the equilibrium solution comprises 100-200 mmol/L sodium chloride buffer solution. The column wall effect can be effectively relieved, and a better column effect is obtained.

It will be appreciated by those skilled in the art that the features and advantages described above for a chromatography system apply equally to this application and will not be described in further detail herein.

Has the advantages that:

(1) the invention provides a chromatographic column lengthening device, which can add filler to be filled at one time, the filler can flow into a column body along the wall of a column tube wall in an adherence manner, the column filling efficiency is improved, and the device is particularly suitable for a small-specification chromatographic column (such as a chromatographic column with the inner diameter less than 10mm for protein purification) and can achieve better column effect.

(2) According to the preparation method of the chromatographic column, the sodium chloride can be directly mixed with the ethanol and then loaded into the column, and the step of gradually replacing the ethanol is not required to be additionally added, so that the preparation method is simple to operate and easy to fill.

(3) The chromatographic column of the invention has high column packing efficiency and good column efficiency, and can lead the manual chromatographic column with small specification (reduced model) to be widely popularized.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

In this example, column chromatography using a reduced-size column chromatography includes the following steps:

(1) taking an Omnifit EZ Column with the inner diameter of 6.6mm, the outer diameter of 1.4cm and the height of 250mm, preparing a corresponding chromatographic Column extension device, and cleaning; detaching the telescopic column head at one end of the Omnifit chromatography column body, and then connecting the Omnifit chromatography column body with a chromatography column extension device;

the structure of the adopted chromatographic column lengthening device is shown in figure 1, a first area and a third area of an accommodating area are cylinders with the same upper and lower diameters, the inner diameter of the first area is 2cm and the height is 8cm, the height of a second contraction area is 1cm, and the inner diameter of the third area is 1cm and the height is 3 cm. The height of the first constriction region is 1.5 cm. The maximum height of the distribution area is 3.5cm, the inner diameter is 4mm, the outer diameter is 5.5mm, the radian of the cambered surface of the distribution angle is 0.17 degree, and the height difference between the highest point and the lowest point is 1 cm.

(2) Vertically fixing a chromatographic column, adding 1mol of sodium chloride into a filler homogenate (AF-rProtein A HC-650F containing 20% ethanol) with the calculated volume until the concentration of the sodium chloride is 0.15mol/L, adding the mixture into the chromatographic column through a chromatographic column extension device once after heavy suspension, detaching the chromatographic column extension device when the filler settlement liquid level reaches the upper part of the column, filling the chromatographic column with a column-containing balance liquid (0.15mol/L sodium chloride), recording the height of the column bed after the filler is settled until the height of the column bed is stable, and installing a telescopic column head at one end to the marked height of the column bed. Finally, column effect measurement is carried out, and the result of the column effect measurement is shown in figure 3.

Example 2

In this example, column chromatography using a reduced-size column chromatography includes the following steps:

(1) taking an Omnifit EZ Column with the inner diameter of 6.6mm, the outer diameter of 1.4cm and the height of 250mm, preparing a corresponding chromatographic Column extension device (the structure is the same as that of the embodiment 1) and cleaning; detaching the telescopic column head at one end of the Omnifit chromatography column body, and then connecting the Omnifit chromatography column body with a chromatography column extension device;

(2) vertically fixing a chromatographic column, adding 1mol of sodium chloride into a filler homogenate (AF-rProtein A HC-650F containing 20% ethanol) with the calculated volume until the concentration of the sodium chloride is 0.2mol/L, adding the mixture into the chromatographic column through a chromatographic column extension device once after heavy suspension, detaching the chromatographic column extension device when the filler settlement liquid level reaches the upper part of the column, filling the chromatographic column with a column-containing balance liquid (0.15mol/L sodium chloride), recording the height of the column bed after the filler is settled until the height of the column bed is stable, and installing a telescopic column head at one end to the marked height of the column bed. Finally, column effect measurement is carried out, and the result of the column effect measurement is shown in figure 4.

Example 3

In this example, column chromatography using a reduced-size column chromatography includes the following steps:

(1) taking an Omnifit EZ Column with the inner diameter of 6.6mm, the outer diameter of 1.4cm and the height of 250mm, preparing a corresponding chromatographic Column extension device (the structure is the same as that of the embodiment 1) and a Column loader, and cleaning; the telescopic column head at one end of the Omnifit chromatography column body is disassembled, and then the Omnifit chromatography column body is connected with a chromatography column extension device through a column installing device;

(2) vertically fixing a chromatographic column, adding 1mol of sodium chloride into a filler homogenate (AF-rProtein A HC-650F containing 20% ethanol) with the calculated volume until the concentration of the sodium chloride is 0.1mol/L, adding the mixture into the chromatographic column through a chromatographic column extension device after heavy suspension, removing the extension device when the sedimentation liquid level of the filler reaches the upper part of the column, filling the chromatographic column with a column-loading balancing liquid (0.15mol/L sodium chloride), recording the height of the column bed after the filler is settled until the height of the column bed is stable, and installing a telescopic column head at one end to the marked height of the column bed. Finally, column effect measurement is carried out, and the result of the column effect measurement is shown in figure 5.

Example 4

In this example, column chromatography using a reduced-size column chromatography includes the following steps:

(1) taking an Omnifit EZ Column with the inner diameter of 6.6mm, the outer diameter of 1.4cm and the height of 250mm, preparing a corresponding chromatographic Column extension device (the structure is the same as that of the embodiment 1) and cleaning; detaching the telescopic column head at one end of the Omnifit chromatography column body, and then connecting the Omnifit chromatography column body with a chromatography column extension device;

(2) vertically fixing a chromatographic column, adding 1mol of sodium chloride into filler homogenate (POROS 50XS containing 20% ethanol) with the calculated volume until the concentration of the sodium chloride is 0.15mol/L, adding the mixture into the chromatographic column through a chromatographic column extension device at one time after heavy suspension, detaching the chromatographic column extension device when the filler settlement liquid level reaches the position above the column, filling the chromatographic column with column-loading balance liquid (0.15mol/L of sodium chloride), recording the height of the column bed after the filler is settled until the height of the column bed is stable, and installing a telescopic column head at one end to the marked height of the column bed. Finally, column effect measurement is carried out, and the result of the column effect measurement is shown in figure 6.

Comparative example 1

In this comparative example, column chromatography was performed using a reduced-size column, comprising the following steps:

(1) taking a chromatography Column with an Omnifit EZ Column with the inner diameter of 6.6mm, the outer diameter of 1.4cm and the height of 250 mm;

(2) vertically fixing a chromatographic column, adding a filler homogenate (AF-rProtein A HC-650F containing 20% ethanol) after volume calculation into the chromatographic column after resuspension without adding sodium chloride, removing an extension device after the filler settlement liquid level reaches the upper part of the column, filling the chromatographic column with a column-loading balance liquid (0.15mol/L sodium chloride), recording the height of the column bed after the filler is settled to the height stability of the column bed, and installing a telescopic column head at one end to the marked height of the column bed. Finally, column effect measurement is carried out, and the result of the column effect measurement is shown in figure 7.

Comparative example 2

In this comparative example, column chromatography was performed using a reduced-size column, comprising the following steps:

(1) taking an Omnifit EZ Column with the inner diameter of 6.6mm, the outer diameter of 1.4cm and the height of 250 mm;

(2) vertically fixing a chromatographic column, homogenizing the filler (POROS 50XS, containing 20% ethanol) after the volume is calculated, adding the filler into the chromatographic column after the filler is resuspended without adding sodium chloride, filling the chromatographic column with a column-loading balance liquid (0.15mol/L sodium chloride) when the filler is settled to the position above the column, recording the height of the column bed after the filler is settled to the height of the column bed and is stable, and installing a telescopic column head at one end to the marked height of the column bed. Finally, column effect measurement is carried out, and the result of the column effect measurement is shown in figure 8.

Comparative example 3

In this comparative example, column chromatography was performed using a reduced-size column, comprising the following steps:

(1) taking an Omnifit EZ Column with the inner diameter of 6.6mm, the outer diameter of 1.4cm and the height of 250 mm;

(2) adding 1mol of salt into filler homogenate (POROS 50XS containing 20% ethanol) with the calculated volume until the salt concentration is 0.15mol, adding into a chromatographic column after heavy suspension, filling the chromatographic column with column-loading balance liquid (0.15mol/L sodium chloride) when the filler settlement liquid level reaches the upper part of the column, recording the height of the column bed after the filler is settled to the height of the column bed and installing a telescopic column head at one end to the marked height of the column bed. Finally, column effect measurement is carried out, and the result of the column effect measurement is shown in figure 9.

In order to comparatively illustrate the column efficiency of the columns of the reduced models of the examples of the present invention and the comparative examples, the column of the reduced models of the examples 1 to 4 of the present invention and the comparative examples 1 to 3 was tested for the packing settling time (min), symmetry (As) and height of theoretical plate (HETP) cm.

The column efficiency determination solution was as follows:

balance liquid: 0.15mol of sodium chloride

Sample loading liquid: 1mol of sodium chloride.

The column effect determination process comprises the following steps: the chromatographic column is balanced by a balance liquid until the effluent current is conducted off flatly (not less than 1CV) -the sample is loaded and balanced, the column effect is measured, and the test result is shown in table 1.

TABLE 1 column efficiency index of different chromatographic columns

The height of the theoretical plate of the chromatographic column is less than or equal to 0.05 cm. The symmetry of the column is 0.8-1.8.

According to the test results, the column packing efficiency of the chromatography of the reduced model can be improved by using the small-size chromatographic column extension device and the column packing buffer solution. The used column loading buffer solution and the salt added to the filler are both solutions for column effect measurement, no new solution is introduced, the operation is simple, the filler is not required to be added into the salt solution by a replacement method, and the column effect measurement can obtain the column effect which is better than that of the column loading directly by using 20% ethanol. The chromatographic column of the comparative example has low efficiency, the operation of the filler adding mode is inconvenient in the column filling process, the filler adding and settling time is long, the equilibrium liquid needs to be used for balancing for a long time during the column effect measurement, and the relative efficiency is low.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.