CN111939594B - Stirring method of adsorption tank - Google Patents
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- CN111939594B CN111939594B CN202010726243.9A CN202010726243A CN111939594B CN 111939594 B CN111939594 B CN 111939594B CN 202010726243 A CN202010726243 A CN 202010726243A CN 111939594 B CN111939594 B CN 111939594B
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Abstract
The invention relates to the technical field of biological medicines, in particular to a stirring method of an adsorption tankTaking a stirrer, determining stirring parameters, and stirring; the method for adjusting the stirring height comprises the steps of determining the gel dosage M; determination of the minimum swelling ratio K of the gel in solutionm(ii) a Calculating the minimum sedimentation thickness h after the gel in the adsorption tank is settled; calculating the total height H of the liquid level in the adsorption tank; and adjusting the stirrer to ensure that the distance D between the lower edge of the stirrer and the filter plate is between H-5cm and H-2 cm. The method can quickly calculate the stirring height, limits parameters such as stirring time, rotating speed and the like, and can effectively avoid the deformation and the breakage of gel and the extension or the interruption of operation time caused by the deformation and the breakage of the gel on the premise of ensuring the stirring effect.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a stirring method of an adsorption tank.
Background
Human Prothrombin Complex (PCC) is a concentrated preparation of four blood coagulation factors ii, vii, ix, x in Human plasma, and is used clinically to treat deficiencies of congenital and acquired blood coagulation factors ii, vii, ix, x. The existing equipment for separating and purifying PCC mainly comprises an adsorption tank, an expanded bed and a chromatographic column, and the separation medium is DEAE Sephadex A-50 gel.
The gel is a weak anion exchange gel, the diameter of dry gel particles is 40-125 mu m, the state of the gel in the solution is influenced by the pH and ionic strength of the solution, and the gel has different expansion ratios, for example, the diameter of the gel particles is 214 mu m in 0.15mol/L sodium chloride solution, and the diameter of the gel particles is 182 mu m in 0.50mol/L sodium chloride solution. The mechanical strength of the gel is low, and the gel is easy to deform and break.
The chinese patent application CN104328036A discloses a human prothrombin complex adsorption separation device, the stirring part of the adsorption separation device comprises an upper stirrer, a lower stirrer and other structures, the lower end of the lower stirrer is also provided with a scraper, and the gap between the scraper and the filter plate is 1-5 mm. Although the destruction of scraper blade to the filter plate has been avoided in above design, because scraper blade and filter plate distance are too little, the gel can be in the extrusion state among the stirring process, and the mechanical strength of gel is low, easily takes place deformation and breakage under the effect of scraper blade, and these gel get into and cause the jam in the middle of the filter plate hole to influence the speed that liquid passes through the filter plate, "increase adsorption medium's on the filter plate thickness, increase the resistance that liquid passes through the filter plate, extension operating time" compare, because the extension operating time that the jam caused is more obvious.
The Chinese patent application CN106881058A discloses a full-automatic solid-liquid separation system and a preparation method thereof, wherein the top of a reaction tank of the separation system is provided with a stirrer, and the stirrer extends to the upper part of a sieve plate of the reaction tank. When using the retort to carry out gel absorption and separation simultaneously, the agitator need all play the stirring effect to the solution volume of difference, because the solution volume that the relative adsorption process of separation process used is few, so the setting of stirring is particularly critical, it can not be deformed and broken to both protect the gel, prevent the jam of bottom filter plate, the stirring effect of separation process is guaranteed again, in case the agitator is misused, or lead to gel deformation and breakage, block up the filter plate, operating time prolongs greatly, seriously influence the smooth and easy nature of technology, or stirring effect is not good, influence the yield and the quality of product.
Disclosure of Invention
The stirring method of the adsorption tank can quickly calculate the stirring height, limits the stirring time, the rotating speed and other parameters, and can effectively avoid the deformation and the crushing of the gel and the prolonging or the interruption of the operation time on the premise of ensuring the stirring effect.
A stirring method of an adsorption tank comprises the steps of (I) adjusting the stirring height according to the gel dosage and (II) selecting a stirrer, determining stirring parameters and stirring;
the method for adjusting the stirring height comprises the following steps:
(1) determining the gel dosage M according to the treated plasma amount;
(2) taking a small amount of gel with mass m, and determining the minimum swelling ratio K of the gel in the solution according to the mass m of the gel before treatment and the volume V of the settled gel after treatmentm；
(3) According to KmCalculating the minimum sedimentation thickness h after the gel in the adsorption tank is settled, wherein the minimum sedimentation thickness h refers to the distance between a gel interface and a filter plate;
(4) according to the volume V of the solution in the adsorption tanklAnd calculating the total height H of the liquid surface in the adsorption tank by the volume V of the settled gel, wherein the total height H of the liquid surface refers to the height from the top surface of the liquid surface to the filter plate;
(5) and adjusting the stirrer to ensure that the distance D between the lower edge of the stirrer and the filter plate is between H-5cm and H-2 cm.
Furthermore, the dosage M of the gel is 1.0-2.0 g/kg of blood plasma.
Further, the minimum expansion coefficient K in the step (2)mThe determination method comprises the following steps:
(21) judging the swelling proportion of the gel in each solution according to the pH and ionic strength of the solution used for gel treatment, and calculating the minimum swelling proportion K according to the stage S corresponding to the solution with the minimum swelling of the gel mWherein, in the step (A),
the solution used for the gel treatment comprises swelling solution, equilibrium solution, washing solution, eluent, regeneration solution and preservation solution,
the stage S comprises swelling, balancing, washing, eluting, regenerating and storing;
(22) get the mass asm gel, treating the gel according to the sequence of gel swelling → balancing → washing → eluting → regenerating → storing, discharging the solution until no visible liquid exists in the gel interface after the stage S treatment is finished, reading the volume V of the settled gel, and calculating the minimum expansion ratio Km，KmWherein, the ratio of (i) to (ii),
the absence of a visible liquid at the gel interface means a state in which the liquid cannot be naturally discharged within 1min of the gel, and is preferably a state before the gel interface is cracked.
Furthermore, the mass M of the gel taken in the step (22) is 1/100-1/1000 of the dosage M of the gel in the step (1).
Further, the method for determining the minimum sedimentation thickness h in the step (3) comprises the following steps:
(31) according to the minimum expansion ratio KmCalculating the volume V of the settled gel corresponding to the gel dosage MM，VM＝Km×M；
(32) Then according to the radius r of the filter plate, the minimum sedimentation thickness h, h ═ V of the gel is calculatedM/(πr2)。
Further, the method for determining the total height H of the liquid level in the adsorption tank in the step (4) comprises the following steps:
(41) Determining the volume V of the solution added into the adsorption tanklAnd calculating the total volume V of the solution and the gel in the adsorption tankt；
(42) Then according to the radius r of filter plate calculating total height of liquid level in the adsorption tank, H ═ Vt/(πr2)。
Further, the distance D between the lower edge of the stirrer and the filter plate in the step (5) is h +/-2 cm.
Furthermore, the distance D between the lower edge of the stirrer and the filter plate in the step (5) also needs to be larger than or equal to 1cm, and preferably, the distance D is larger than or equal to 2 cm.
Further, the gel is DEAE Sephadex A-50 gel.
Further, the stirring is carried out by adopting a propeller stirrer, a turbine stirrer or a paddle stirrer, preferably by adopting a propeller stirrer and a turbine stirrer;
the stirring parameters include: the stirring time is more than or equal to 2min, the stirring speed is more than or equal to 5r/min, preferably, the stirring time is more than or equal to 5min, and the stirring speed is more than or equal to 10 r/min.
Further, the amount of the solution used for the gel other than the solution having the smallest gel swelling ratio is calculated based on the swelling volume of the gel in the solution used, and is preferably 2 times the volume of the gel after swelling.
The beneficial effect of the invention is that,
the invention provides a stirring method of an adsorption tank, which can quickly set the position and the stirring parameters of a stirrer;
The stirring method is beneficial to protecting the gel form, and prevents the filter plate from being blocked due to the deformation and the breakage of the gel, and the interruption or the time extension of the production operation caused by the blockage;
the stirring method of the invention ensures that the stirring effect is not affected, so as to maintain the stability of the process and ensure the stability of the yield and the quality of the product.
Drawings
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
Fig. 1 is a schematic structural view of an adsorption tank stirrer and a filter plate.
In the figure, 1-filter plate, 2-settled gel, 3-stirrer lower edge, 4-stirrer, 5-gel interface.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A stirring method of an adsorption tank comprises the steps of (I) adjusting the stirring height according to the gel dosage and (II) selecting a stirrer 4, determining stirring parameters and stirring;
the method for adjusting the stirring height comprises the following steps:
(1) adsorbing 1000kg of plasma according to gel, using 1.5g of gel per kg of plasma, and determining the gel dosage M to be 1500 g;
(2) determining the minimum swelling ratio K of the gel in the solutionmThe method comprises the following steps:
(21) the solution used for gel treatment comprises swelling solution, equilibrium solution, washing solution, eluent, regeneration solution and preservation solution, wherein the swelling solution is 0.075mol/L sodium chloride solution,
the balance liquid is 0.08mol/L sodium chloride, 0.015mol/L sodium citrate, pH7.2 +/-0.1,
the washing solution is 0.18mol/L sodium chloride, 0.015mol/L sodium citrate, pH7.2 + -0.1,
the eluent is 1mol/L sodium chloride and 0.025mol/L sodium citrate, the pH value is 7.2 plus or minus 0.1,
the regeneration liquid is 2mol/L sodium chloride solution,
the preservation solution is 20 percent of ethanol,
from the pH and ionic strength of the above solutions, it is found that the gel has the smallest swelling ratio in the regenerating solution, and the smallest swelling ratio K is calculated from the regeneration stage corresponding to the regenerating solution in which the gel has the smallest swellingm；
(22) According to the amount of 1/1000 used of the gel M, 1.5g of the gel was taken and the gel was treated in the order of gel swelling → equilibration → washing → elution → regeneration, in the state of the solution used, including pH, conductivity, temperature and the like, in accordance with the state used in the production,
The regeneration stage is carried out twice, 40mL of regeneration liquid is used each time, the stirring time is 10min, the rotating speed is 10r/min, the regeneration liquid is discharged after the treatment is finished until no visible liquid exists on a gel interface 5, the volume V of the settled gel 2 is read to be 32mL, and the minimum expansion ratio K is calculatedm＝32/1.5＝21mL/g；
(3) Calculating the minimum sedimentation thickness h after the gel sedimentation, comprising the following steps:
(31) according to the minimum expansion ratio KmCalculating the volume V of the settled gel 2 corresponding to the gel dosage MM＝21×1500＝32L；
(32) Calculating the minimum sedimentation thickness h of the gel to be 320/(3.14 multiplied by 9) to be 11.3cm according to the radius r of the filter plate 1 to be 30 cm;
(4) calculating the total height H of the liquid level in the adsorption tank, and comprising the following steps:
(41)1500g of gel requires 40L of regeneration liquid to be added at a time, so the total volume V of the solution and gel in the adsorption tankt＝32+40＝72L；
(42) Using an adsorption tank with a filter plate 1 diameter of 60cm, calculating the total height H of the liquid surface in the adsorption tank to be 720/(3.14 × 9) to be 25.5 cm;
(5) obtaining that the distance D between the lower edge 3 of the stirrer and the filter plate 1 is 6.3-23.5 cm, wherein the distance D between the lower edge 3 of the stirrer and the filter plate 1 meets the requirement that D is more than or equal to 1cm, adjusting the position of the stirrer 4 to the distance D between the lower edge 3 of the stirrer and the filter plate 1 to be 11cm according to the preferable range of 9.3-13.3 cm, and fixing the stirrer 4 to be in a horizontal state;
The stirrer 4 is of a folding flat paddle type, the stirring time is 10min, the stirring speed is 10r/min, and the volume of the solution used in the swelling, balancing, washing, eluting and storing stages is 2 times of the volume of the gel expanded in each solution.
Example 2
A stirring method of an adsorption tank comprises the steps of (I) adjusting the stirring height according to the gel dosage and (II) selecting a stirrer 4, determining stirring parameters and stirring;
the method for adjusting the stirring height comprises the following steps:
(1) adsorbing plasma amount by gel 1500kg, using 1.2g gel per kg plasma, and determining gel dosage M as 1800 g;
(2) determining the minimum swelling ratio K of the gel in the solutionmThe method comprises the following steps:
(21) the solution used for gel treatment comprises swelling solution, equilibrium solution, washing solution, eluent, regeneration solution and preservation solution, wherein the swelling solution is 0.075mol/L sodium chloride solution,
the balance liquid is 0.08mol/L sodium chloride, 0.015mol/L sodium citrate, pH7.2 +/-0.2,
the washing solution is 0.18mol/L sodium chloride, 0.015mol/L sodium citrate, pH7.2 + -0.2,
the eluent is 2mol/L sodium chloride, 0.015mol/L sodium citrate, pH7.2 + -0.2,
the regeneration liquid is 2mol/L sodium chloride solution,
the solution for preservation is 20% ethanol,
From the pH and ionic strength of the above solution, it can be seen that the gel has the smallest swelling ratio in the eluent, and the smallest swelling ratio K is calculated from the elution stage corresponding to the eluent with the smallest swelling of the gelm；
(22) According to the amount of 1/1000 used of the gel, 1.8g of the gel was taken, and the gel was treated in the order of gel swelling → equilibration → washing → elution, and the state of the solution used was the same as that used in the production including pH, conductivity, temperature, etc.,
the elution stage is carried out for three times, 50mL of eluent is used each time, the stirring time is 10min, the rotating speed is 10r/min, the eluent is discharged after the treatment is finished until no visible liquid exists on a gel interface 5, the volume V of the settled gel 2 is read to be 36mL, and the minimum expansion ratio K is calculatedm＝36/1.8＝20mL/g；
(3) Calculating the minimum sedimentation thickness h after the gel sedimentation, comprising the following steps:
(31) according to the minimum expansion ratio KmCalculating the volume V of the settled gel 2 corresponding to the gel dosage MM＝20×1800＝36L；
(32) Using an adsorption tank with a filter plate 1 diameter of 80cm, calculating the minimum sedimentation thickness h of the gel to be 360/(3.14 × 16) to be 7.2 cm;
(4) calculating the total height H of the liquid level in the adsorption tank, and comprising the following steps:
(41)1800g of gel elution requires 50L of eluent to be added at a time, so the total volume V of solution and gel in the adsorption tank t＝36+50＝86L；
(42) Then, according to the radius r of the filter plate 1 being 40cm, the total height H of the liquid level in the adsorption tank being 860/(3.14 × 16) being 17.1 cm;
(5) obtaining that the distance D between the lower edge 3 of the stirrer and the filter plate 1 is between 2.2 and 15.1cm, the distance satisfies that the distance D between the lower edge 3 of the stirrer and the filter plate 1 is more than or equal to 1cm, adjusting the position of the stirrer 4 to the distance D between the lower edge 3 of the stirrer and the filter plate 1 to be 8cm according to the preferable range of 5.2 to 9.2cm, and fixing the stirrer 4 to be in a horizontal state;
the stirrer 4 is in a push type, the stirring time is 10min, the stirring speed is 10r/min, and the volume of the solution used in the swelling, balancing, washing, regenerating and storing stages is 2 times of the volume of the gel after swelling in each solution.
Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. A stirring method of an adsorption tank is characterized by comprising the steps of (I) adjusting the stirring height according to the gel dosage and (II) selecting a stirrer, determining stirring parameters and stirring;
the method for adjusting the stirring height comprises the following steps:
(1) determining the gel dosage M according to the treated plasma amount;
(2) taking a small amount of gel with mass m, and determining the minimum swelling ratio K of the gel in the solution according to the mass m of the gel before treatment and the volume V of the settled gel after treatmentm；
(3) According to KmCalculating the minimum sedimentation thickness h after the gel in the adsorption tank is settled, wherein the minimum sedimentation thickness h refers to a gel interfaceDistance to the filter plate;
(4) according to the volume V of the solution in the adsorption tanklAnd calculating the total height H of the liquid surface in the adsorption tank by the volume V of the settled gel, wherein the total height H of the liquid surface refers to the height from the top surface of the liquid surface to the filter plate;
(5) and adjusting the stirrer to ensure that the distance D between the lower edge of the stirrer and the filter plate is between H-5cm and H-2 cm.
2. A method of mixing as claimed in claim 1, wherein the gel is present in an amount M of 1.0 to 2.0g/kg of plasma.
3. A method of mixing as defined in claim 1 wherein, in step (2), the minimum coefficient of expansion K is mThe determination method comprises the following steps:
(21) judging the swelling proportion of the gel in each solution according to the pH and ionic strength of the solution used for gel treatment, and calculating the minimum swelling proportion K according to the stage S corresponding to the solution with the minimum swelling of the gelmWherein, in the process,
the solution used for the gel treatment comprises swelling solution, equilibrium solution, washing solution, eluent, regeneration solution and preservation solution,
the stage S comprises swelling, balancing, washing, eluting, regenerating and storing;
(22) taking gel with mass m, treating the gel according to the sequence of gel swelling → balance → washing → elution → regeneration → preservation, discharging the solution until no visible liquid exists on the gel interface after the treatment of the stage S is finished, reading the volume V of the settled gel, and calculating the minimum expansion ratio Km，Km＝V/m。
4. A stirring method as claimed in claim 3, wherein the mass M of the gel taken in step (22) is 1/100-1/1000 of the amount M of gel used in step (1).
5. A stirring method according to claim 1, wherein said determination of the minimum sedimentation thickness h in step (3) comprises the steps of:
(31) according to the minimum expansion ratio KmCalculating the volume V of the settled gel corresponding to the gel dosage M M，VM＝Km×M；
(32) Then according to the radius r of the filter plate calculating the minimum sedimentation thickness h, h ═ V of the gelM/(πr2)。
6. The stirring method according to claim 1, wherein the method for determining the total height H of the liquid surface in the adsorption tank in the step (4) comprises the steps of:
(41) determining the volume V of the solution added into the adsorption tanklAnd calculating the total volume V of the solution and the gel in the adsorption tankt；
(42) Then according to the radius r of filter plate calculating total height of liquid level in the adsorption tank, H ═ Vt/(πr2)。
7. A method of agitation according to claim 1 wherein the distance D between the lower edge of the agitator and the filter plate in step (5) is between h ± 2 cm.
8. A stirring method according to claim 1, wherein in step (5), the distance D between the lower edge of the stirrer and the filter plate is equal to or greater than 1 cm.
9. A method of agitation according to claim 1 wherein said gel is DEAE Sephadex a-50 gel.
10. The stirring method according to claim 1, wherein the stirring is performed by a propeller stirrer, a turbine stirrer or a paddle stirrer; the stirring parameters include: the stirring time is more than or equal to 2min, and the stirring speed is more than or equal to 5 r/min.
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