CN117126228A - Efficient protein recovery process - Google Patents

Abstract

The application belongs to the technical field of protein preparations, and discloses a protein efficient recovery process which comprises the following steps: collecting chromatographic solution with absorbance reading value of more than 40mAU at 280nm by gel filtration chromatography, and respectively collecting and storing according to the front peak, peak tip and peak tail; and taking the chromatographic solution before and/or at the peak tip as a first part of recovered product, further ultrafiltering the chromatographic solution at the peak tail collected after the conductivity change to obtain a second part of recovered product, and mixing the first part of recovered product and the second part of recovered product to obtain a recovered final product. Under the general condition of adopting the recovery process, the total recovery rate can reach more than 98 percent, the protein recovery rate is high, the time is short, the efficiency is high, the cost is low, and for some special proteins with biological activity, the recovery process can better retain the activity of the proteins.

Description

Efficient protein recovery process

Technical Field

The application belongs to the technical field of protein preparations, and particularly relates to a high-efficiency protein recovery process.

Background

In the preparation process of protein preparation, in particular to the preparation of antibody, in order to meet the subsequent requirements, a buffer system needs to be replaced and sometimes needs to be concentrated, and a replacement recovery process is needed, wherein the following three methods are adopted in the prior art:

1. dialysis method

In the dialysis, a protein solution is wrapped by a semipermeable membrane and immersed in a dialysis buffer solution, and the concentration of the buffer solution is lower than that in the membrane, so that substance exchange can occur on the semipermeable membrane; but the protein cannot pass through the semipermeable membrane, and only small molecular substances such as salt ions and the like can pass through the semipermeable membrane; the salt impurities in the protein solution are thus exchanged to the outside through the semipermeable membrane.

Dialysis is a relatively long period of buffer replacement, and requires a large amount of buffer replacement, which greatly limits its application, and is less industrially used, mainly in laboratory.

2. Gel filtration chromatography

The gel filtration medium is widely applied to desalting and buffer replacement of biomacromolecules, and is widely applied to biopharmaceutical technology due to mild process, in particular to desalting and buffer replacement after centrifugal exchange and affinity.

3. Ultrafiltration

The ultrafiltration method is a membrane filtration method, which uses a porous membrane as a separation medium and uses the pressure difference at two sides of the membrane as a driving force to separate substances with different molecular weights in the solution, thereby playing roles of replacement, concentration, purification and the like. It has the advantages of no phase conversion, no need of heating, low energy consumption, no need of adding chemical reagent, no damage to thermosensitive medicine, etc. Ultrafiltration ranges from laboratory micro-scale ultrafiltration tubes, to small scale ultrafiltration cups, to industrial ultrafiltration system technology.

The main drawbacks of these existing solutions are:

gel filtration chromatography in the protein recovery process, if it is desired to achieve higher recovery, there is a limit to the loading, typically a single loading within 1/5 of the column volume. If the column volume exceeds 1/5, the displacement becomes incomplete, and a part of the product with incomplete displacement needs to be discarded, which is a great waste for a protein having a relatively high value, particularly a recombinant monoclonal antibody. In addition, gel filtration chromatography has a dilution effect, and is difficult to be applied if the purpose of concentrating proteins is to be achieved.

The common method for concentrating the protein is ultrafiltration, but when the protein content is high, the ultrafiltration method is adopted to easily block the membrane, repeated washing and centrifugal filtration are needed, the consumption time is long, more ultrafiltration tubes are needed if the time is saved, and the cost of materials, energy and manpower is increased finally no matter which way is adopted. In addition, there are some special proteins which are more seriously wasted due to irreversible denaturation and inactivation after ultrafiltration.

Disclosure of Invention

In order to solve at least one of the above problems, the present application provides a process for efficiently recovering proteins, which has high recovery rate and short time-consuming period for a large amount of proteins, is efficient, and has low cost, and which can better retain the activity of proteins for some specific proteins.

In order to achieve the above purpose, the present application provides the following technical solutions:

the application provides a protein efficient recovery process, which comprises the following steps: the sample to be recovered is subjected to gel filtration chromatography, and the chromatographic solution with complete buffer solution replacement is collected as a first-step recovered product; collecting the chromatographic solution with incomplete replacement of the residual buffer solution as an ultrafiltration sample for ultrafiltration, and completely replacing the buffer solution to obtain a second-step recovered product; and mixing the recovered product in the first step and the recovered product in the second step to obtain a recovered final product.

In some embodiments, the gel filtration chromatography process is: after the purifier is pre-cleaned, the pipeline is washed to be balanced by a replacement buffer solution, a sample is loaded for chromatography, and the chromatographic solution is respectively collected according to the peak front, peak tip and peak tail according to a 280nm absorbance curve; when the absorbance reading value at 280nm starts to rise, buffer solutions before and at the peak tip are collected respectively to replace the complete chromatographic solution; the buffer solution at the tail of the collection peak starts to replace the incomplete chromatographic solution when the conductivity starts to change, and the collection is stopped when the absorbance reading at 280nm is reduced to the curve approaching level, and the protein content in the uncollected chromatographic solution is not more than 0.1%.

In some embodiments, collection of pre-peak and peak-tip buffer displacement complete chromatographic solutions begins when the 280nm absorbance reading rises to 40mAU, collection of peak-tail buffer displacement incomplete chromatographic solutions begins when conductivity begins to change, and collection is stopped when the 280nm absorbance reading falls to 40 mAU.

In some embodiments, the loading flow rate is 5ml/min. In some embodiments, 5-15ml of chromatographic solution are collected before peak, at peak tip and at peak tail, respectively. In some embodiments, the displacement eluate is a phosphate buffer solution.

In some embodiments, the ultrafiltration process is: adding an ultrafiltration sample into an ultrafiltration tube, adding a replacement buffer solution to complement the volume, centrifuging to ensure that about 1ml of liquid on the membrane is obtained, blowing and uniformly mixing by using a liquid transfer device, and adding the replacement buffer solution into the ultrafiltration tube to complement the volume; repeating the centrifugation and blowing process for multiple times, blowing and mixing with a liquid transfer device after the last centrifugation, sucking out all the liquid, adding a small amount of replacement buffer solution into the ultrafiltration tube, blowing and mixing, sucking out all the liquid, and mixing to obtain the product recovered in the second step.

In some embodiments, the centrifugation conditions are 4300rpm, and centrifugation is performed for 5 minutes; the times of repeated centrifugation and blowing are not less than 4 times. In some embodiments, a plurality of ultrafiltration tubes may be used, each ultrafiltration tube having no more than 3/5 of the amount of sample added to the tube.

In some embodiments, when the chromatographic solution before the peak is mixed with the chromatographic solution at the peak tip, the protein concentration does not meet the set requirement, and the chromatographic solution before the peak and the chromatographic solution at the peak tail are mixed into an ultrafiltration sample for ultrafiltration.

In some embodiments, the sample to be recovered meets at least one of the following characteristics:

(1) The sample size is large and is not less than 10mg;

(2) The concentration of the recovered final product has the set requirement, and the single concentration method cannot reach the high concentration of the set requirement;

(3) And recovering the activity loss of the final product after ultrafiltration and concentration.

In some embodiments, the sample to be recovered is a protein sample having biological activity. In some embodiments, the biologically active protein sample is a monoclonal antibody.

The beneficial effects of the application are that

The application has the following beneficial effects: the process adopts a mode of combining the post-flux and ultrafiltration of gel filtration chromatography: the gel filtration chromatography needs to be pre-cleaned for 30min, the sample is collected for 8-15min, and then the gel filtration chromatography is cleaned for 30min, and the cleaning process is automatic cleaning without being attended by people. The method comprises the steps of firstly recovering 5-15ml of most protein at peak tips, and the residual part, generally 10-30ml, adopting 1-2 ultrafiltration tubes to centrifuge for 5-8min each time, at least 4 times including intermediate flushing of a filter membrane, and finishing about half an hour, wherein the gel filtration chromatographic column can finish cleaning at the same time, the total time is about 1.5 hours, and the pre-cleaning is not required to be attended for half an hour. The general overall recovery rate can reach more than 98 percent. Wherein, the buffer solution in the chromatographic solution before the peak is replaced, if the protein concentration can meet the set requirement after the chromatographic solution with the peak tip is mixed, the chromatographic solution before the peak is not ultrafiltered.

The gel filtration chromatography and ultrafiltration are combined, the once loading amount can be increased in the gel filtration chromatography, the times of chromatography are reduced, the times of chromatographic column cleaning and balancing and loading are reduced, and the time is saved.

When the protein content in the sample is more than 10mg, particularly more than 25mg, the centrifugation time is 30min or more, the replacement buffer solution is centrifuged at least 4 times, namely more than 2 hours, and the filter membrane is washed after being taken out in the middle for the next centrifugation. Or more ultrafiltration tubes are adopted, so that the centrifugation time is properly shortened, but the consumable materials are more to use and are troublesome to operate, and the experimenter is required to keep on duty all the time.

In general, the present application provides a protein recovery process that is high and short in time, efficient and low in cost for large amounts of protein, and that can better retain protein activity in addition to achieving high recovery for some specific proteins.

Drawings

FIG. 1 is a graph showing absorbance readings at 280nm and conductivity readings for the pD-L1 rabbit monoclonal antibody of example 1;

FIG. 2 is a graph showing absorbance readings at 280nm and conductivity for the MSH6 rabbit monoclonal antibody of example 2;

FIG. 3 is a schematic representation of the recovery of protein from scheme 1 of example 2 in a conventional immunohistochemical assay method;

FIG. 4 is a schematic representation of the recovery of protein in accordance with scheme 2 of example 2 in a conventional immunohistochemical assay method;

FIG. 5 is a graph showing absorbance readings at 280nm and conductivity for the CD15 murine monoclonal antibody of example 3;

FIG. 6 is a schematic diagram of the recovery of protein in scheme 1 of example 3 in a conventional immunohistochemical detection method;

FIG. 7 is a schematic diagram of the recovery of protein in conventional immunohistochemical detection method in scheme 2 of example 3;

wherein fig. 3 and 4 are esophageal tissue slices, and fig. 6 and 7 are spleen tissue slices.

Detailed Description

Unless otherwise indicated, implied from the context, or common denominator in the art, all parts and percentages in the present application are based on weight and the test and characterization methods used are synchronized with the filing date of the present application. Where applicable, the disclosure of any patent, patent application, or publication referred to in this disclosure is incorporated herein by reference in its entirety, and the equivalent patents are incorporated herein by reference, especially with respect to the definitions of synthetic techniques, product and process designs, polymers, comonomers, initiators or catalysts, etc. in the art, as disclosed in these documents. If the definition of a particular term disclosed in the prior art is inconsistent with any definition provided in the present application, the definition of the term provided in the present application controls.

The numerical ranges in the present application are approximations, so that it may include the numerical values outside the range unless otherwise indicated. The numerical range includes all values from the lower value to the upper value that increase by 1 unit, provided that there is a spacing of at least 2 units between any lower value and any higher value. For example, if a component, physical or other property (e.g., molecular weight, melt index, etc.) is recited as being 100 to 1000, it is intended that all individual values, e.g., 100, 101, 102, etc., and all subranges, e.g., 100 to 166, 155 to 170, 198 to 200, etc., are explicitly recited. For ranges containing values less than 1 or containing fractions greater than 1 (e.g., 1.1,1.5, etc.), then 1 unit is suitably considered to be 0.0001,0.001,0.01, or 0.1. For a range containing units of less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are merely specific examples of what is intended to be provided, and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

As used with respect to chemical compounds, the singular includes all isomeric forms and vice versa unless explicitly stated otherwise (e.g., "hexane" includes all isomers of hexane, either individually or collectively). In addition, unless explicitly stated otherwise, the use of the terms "a," "an," or "the" include plural referents.

The terms "comprises," "comprising," "including," and their derivatives do not exclude the presence of any other component, step or process, and are not related to whether or not such other component, step or process is disclosed in the present application. For the avoidance of any doubt, all use of the terms "comprising", "including" or "having" herein, unless expressly stated otherwise, may include any additional additive, adjuvant or compound. Rather, the term "consisting essentially of … …" excludes any other component, step or process from the scope of any of the terms recited below, as those out of necessity for operability. The term "consisting of … …" does not include any components, steps or processes not specifically described or listed. The term "or" refers to the listed individual members or any combination thereof unless explicitly stated otherwise.

In order to make the technical problems, technical schemes and beneficial effects solved by the application more clear, the application is further described in detail below with reference to the embodiments.

Examples

The following examples are presented herein to demonstrate preferred embodiments of the present application. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the application, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the application.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. Such equivalents are intended to be encompassed by the claims.

EXAMPLE 1 gel filtration chromatography parameter exploration

In this example, we increased the loading to 25ml and used 1ml per tube of continuous collection chromatography solution to explore the optimal gel filtration chromatography parameters as follows:

1. sample of

pD-L1 Rabbit monoclonal antibody (clone number BP6099, hangzhou Bailing Biotechnology Co., ltd.) protein solution, 30.83mg,25 ml, original buffer was conventional citrate-sodium citrate elution buffer.

2 reagent and consumable

2.1, desalting column: ezLoad 26/10G 25F: EG026, bogurone

2.2, substitution buffer: DPBS: b210, source cultivation;

2.3, 20% ethanol: laboratory preparation, and filtering with 0.45 μm filter membrane;

2.4, purified water: the laboratory is routinely equipped.

3. Instrument and equipment

3.1, purifying instrument: AKTA explorer.

The experimental operation steps are as follows:

(1) Pre-cleaning: starting the purifier, and pre-cleaning according to the operation of the instruction book; flushing the pipeline to balance by adopting a replacement buffer solution;

(2) Loading: 25 The ml sample pipeline S1 is placed into a sample bottle, and the flow rate is set to be 5ml/min; immediately transferring the sample into a replacement buffer solution after the sample is completely sucked up;

(3) And (3) collecting: setting collection options: and collecting a solution with an absorbance reading value of 280nm being greater than or equal to 40mAU, wherein 1 ml/tube, the target protein can flow out before the original buffer solution, and the target protein is replaced by the replacement buffer solution. The sample loading amount of gel filtration chromatography is increased to 1/2 of the column volume, namely 25ml is tested, and the replacement product is collected according to 1ml of each tube; the purifier shows a curve as shown in fig. 1.

(4) Post-cleaning: and (5) performing post-cleaning according to the operation of the specification, and shutting down after cleaning.

The results illustrate: observing absorbance readings and conductivity readings at 280nm in the graph; the loss of the part with the absorbance reading value of 280nm lower than 40mAU accounting for 0.05 percent of the total protein amount can be calculated and can be abandoned; the amount of the partially effectively replaced protein with the conductivity variation amplitude less than 5 percent is 66.5 percent of the total protein amount, and the partially effectively replaced protein can be collected as a recovery product of gel filtration chromatography; when the conductivity changes, which means that the buffer solution is not completely replaced, the partial protein with the conductivity change amplitude of more than 5% is calculated to account for 33.45% of the total protein, about 1/3, and the sample size of the partially recovered product is obviously more than that collected by adopting the conventional sample size because the sample size is increased in the experiment, and the recovered product with incomplete replacement needs to be additionally ultrafiltered.

The recovery process combines the ultrafiltration process, so that the incomplete replacement amount in the gel filtration chromatography can be properly increased.

EXAMPLE 2 comparison of MSH6 Rabbit monoclonal antibody purification

1. Sample of

MSH6 rabbit monoclonal antibody (Hangzhou Bailing Biotechnology Co., ltd., clone number BP 6007) protein solution, 48.99mg,46 ml, original buffer is conventional citric acid-sodium citrate elution buffer; two protocol experiments were performed with 13ml (13.85 mg) samples each.

2. Reagent and consumable

2.1 ultrafiltration tube (15 ml): amicon cube Ultra-15 Centrifugal Filter device (50 KD): UFC905024, millipore;

2.2, desalting column: ezLoad 26/10G 25F: EG026, bognong;

2.3, substitution buffer: DPBS: b210, source cultivation;

2.4, 20% ethanol: laboratory preparation, and filtering with 0.45 μm filter membrane;

2.5, purified water: the laboratory is routinely equipped.

3. Instrument and equipment

3.1, thermo bench type high capacity refrigerated centrifuge: multifuge X3R, thermo;

3.2, purifying instrument: AKTA explorer.

4. Experimental procedure

Experimental protocol 1

I. Gel filtration chromatography

(1) Pre-cleaning: starting the purifier, and pre-cleaning according to the operation of the instruction book; flushing the pipeline to balance by adopting a replacement buffer solution;

(2) Loading: 13ml of sample pipeline S1 is placed in a sample bottle, and the flow rate is set to be 5ml/min; immediately transferring the sample into a replacement buffer solution after the sample is completely sucked up;

(3) And (3) collecting: setting collection options: collecting a solution with an absorbance reading value of 280nm being greater than or equal to 40mAU, wherein 5 ml/tube of the solution, the target protein flows out before the original buffer solution and is replaced by the replacement buffer solution, a curve displayed by a purifier is shown as a graph in FIG. 2, the absorbance reading value and the conductivity reading value of 280nm are observed, when the absorbance reading value of 280nm rises to 40mAU, the curve change is obvious, and a peak appears, namely, the collection is started; stopping collecting after the reading value is reduced to 40mAU, and at the moment, a large amount of original buffer solution flows out, and the 280 curve is obviously reduced until the reading value is close to 0; collecting 6 tubes of A1-A6, wherein the concentration of the A1 is 5 ml/tube, the A2 and the A3 are peak tips before the peak, and the A4-6 is the peak tail;

(4) And (3) recycling: the peak tip solution is the first part of the desired product: 2 tubes A2, A3, 10ml. Directly preserving, namely preserving a front-peak solution and a rear-peak solution 4 pipe for standby;

(5) Post-cleaning: and (5) performing post-cleaning according to the operation of the specification, and shutting down after cleaning.

II. Ultrafiltration

(1) Sample adding: mixing 4 tubes of solution at the front and tail of the peak to obtain about 18ml of ultrafiltration sample; taking 2 ultrafiltration tubes, adding the samples into the ultrafiltration tubes on average, and adding a replacement buffer solution to complement 15ml;

(2) And (3) centrifuging: 4300rpm, centrifuging for 5min, observing the sample volume in the ultrafiltration tube, and ensuring that the liquid on the membrane is about 1mL.

(3) Blowing: the suction head is extended to the bottom of the filter element by a liquid-transfering device, the filter membranes at the two sides are blown up, down, left and right for 3-5 times, and then the replacement buffer solution is added into the ultrafiltration tube to be complemented to 15ml.

(4) The centrifugation and blowing process was repeated for a total of 4 times;

(5) And (3) recycling: after the last centrifugation is finished, the suction head of a liquid transfer device is extended to the bottom of the filter membrane to blow the filter membranes on the two sides for 3-5 times, the majority of liquid is sucked out, about 0.8ml of residual liquid is left in each tube, 0.5ml of replacement buffer solution is added into the ultrafiltration tube, the suction head is extended to the bottom of the filter membrane to blow the filter membranes on the two sides for 3-5 times, and all the liquid is sucked out; all the liquids recovered from the ultrafiltration tubes were mixed to give a second portion of the desired product: 2.1ml.

III, mixing

And mixing the two products obtained by the two steps of gel filtration chromatography and ultrafiltration, thus obtaining the final product.

IV, results

The final product was found to be 13.66mg,12.1ml total and 98.63% recovery.

In FIG. 2, the initial conductivity curve of A4 is significantly increased, indicating that the buffer composition is changed and that the original elution buffer has been mixed. As can be seen from the peak area calculation of fig. 2: the amount of protein contained in the A1-3 tube is 83.15% of the total amount of the 6 tubes, namely the amount of protein contained in the A4-6 tube is 16.85% of the total amount of the 6 tubes; the amount of protein contained in the A1 tube was 11.27% of the total amount of 6 tubes. From the change in the conductivity curve, the original buffer was mixed from the 4 th tube solution, and if gel filtration chromatography was used alone, the previous 3-4 tube solution was recovered; if the protein needs to be recovered as much as possible, taking the first 4 tubes, changing the solution from 15ml to 20ml for loading, adding the loss of the protein, obviously reducing the concentration, and incompletely replacing the buffer solution; if the buffer solution is to be completely replaced, taking the first 3 tubes, the protein loss is increased to 16.85%, the volume is unchanged, and the concentration is reduced; if high concentration is to be ensured, the loss of the 2 tubes with peak tips is further increased to be about 30%. Whereas the cost of 1mg of commercially available monoclonal antibodies, particularly antibodies for immunohistochemical related detection, is often in the hundreds or even thousands of yuan.

Experimental scheme 2:

(1) Sample adding: taking 1 ultrafiltration tube, adding 13ml of sample into the ultrafiltration tube, and adding replacement buffer solution to make up to 15ml;

(2) And (3) centrifuging: 4300rpm, centrifuging for 25min, observing the sample volume in the ultrafiltration tube, and ensuring that the liquid on the membrane is about 1mL.

(3) Blowing: the suction head is extended to the bottom of the filter element by a liquid-transfering device, the filter membranes at the two sides are blown up, down, left and right for 3-5 times, and then the replacement buffer solution is added into the ultrafiltration tube to be complemented to 15ml.

(4) The centrifugation and blowing process was repeated for a total of 4 times;

(5) And (3) recycling: after the last centrifugation is finished, the suction head of the liquid-transfering device is extended to the bottom of the filter membrane to blow the filter membrane on both sides for 3-5 times, the majority of liquid is sucked out, about 1ml of residual liquid is left in the tube, 10ml of replacement buffer solution is added into the ultrafiltration tube, the suction head is extended to the bottom of the filter membrane to blow the filter membrane on both sides for 3-5 times, and all the liquid is sucked out; all the liquid recovered by the ultrafiltration tube is mixed to obtain the final product.

The results show that: the final product measured 11.98mg,11ml total, recovery 86.50%.

From a comparison of experimental scheme 1 and experimental scheme 2, it can be seen that: the protein recovery of protocol 1 was significantly higher than that of protocol 2, and in addition, protocol 1 took less than 1.5 hours total, and protocol 2 took about 2 hours.

5. Antibody Activity detection

Esophageal tissue sections were taken, the primary antibody was the antibody obtained in the above-mentioned experiment scheme 1 and experiment scheme 2, the final concentration was adjusted to 0.003mg/ml, the incubation amount was 100ul, and the test was carried out using a conventional immunohistochemical detection reagent and method, and the experimental results are shown in FIG. 3 and FIG. 4.

The results show that experiment 1 was not only high in recovery but also slightly higher in antibody titer than experiment 2.

EXAMPLE 3 comparison of purification of CD15 murine monoclonal antibodies

1. Sample of

CD15 murine monoclonal antibody (clone MMA, hangzhou Biotechnology Co., ltd.) protein solution, 11.53mg,29 ml, original buffer was conventional citrate-sodium citrate elution buffer. Two protocol experiments were performed with 10ml (3.98 mg) samples each. The sample has high concentration, easy precipitation and denaturation, and can not be concentrated to an excessive degree.

2. Reagent and consumable:

2.1 ultrafiltration tube (15 ml): amicon cube Ultra-15 Centrifugal Filter device (50 KD): UFC905024, millipore;

2.2, desalting column: ezLoad 26/10G 25F: EG026, bognong;

2.3, substitution buffer: DPBS: b210, source cultivation;

2.4, 20% ethanol: laboratory preparation, and filtering with 0.45 μm filter membrane;

2.5, purified water: the laboratory is routinely equipped.

3. Instrument and equipment

3.1, thermo bench type high capacity refrigerated centrifuge: multifuge X3R, thermo;

3.2, purifying instrument: AKTA explorer.

4. Experimental procedure

Experimental protocol 1

I. Gel filtration chromatography

(1) Pre-cleaning: starting the purifier, and pre-cleaning according to the operation of the instruction book; flushing the pipeline to balance by adopting a replacement buffer solution;

(2) Loading: 10ml of sample pipeline S1 is put into a sample bottle, and the flow rate is set to be 5ml/min; immediately transferring the sample into a replacement buffer solution after the sample is completely sucked up;

(3) And (3) collecting: setting collection options: collecting a solution with an absorbance reading value of 280nm being greater than or equal to 40mAU, wherein the solution flows out of a tube by 4 ml/tube before the original buffer solution, and is replaced by the replacement buffer solution, a curve displayed by a purifier is shown as a graph in FIG. 5, the absorbance reading value and the conductivity reading value of 280nm in the lower graph are observed, when the absorbance reading value of 280nm rises to 40mAU, the curve change is obvious, and a peak appears, namely, the collection is started; stopping collecting after the reading value is reduced to 40mAU, and at the moment, a large amount of original buffer solution flows out, and the 280 curve is obviously reduced until the reading value is close to 0; collecting 5 tubes of A1-A5, wherein the concentration of A1 is 4 ml/tube, A2 and A3 are peak tips before peak, and A4 and A5 are peak tails;

(4) And (3) recycling: the peak tip portion solution is the first part of the desired product: 2 tubes of A2 and A3 are totally 8ml, are directly stored, and 3 tubes of solutions of the front peak part, the tail peak part and A1, A4 and A5 are reserved for standby;

(5) Post-cleaning: and (5) performing post-cleaning according to the operation of the specification, and shutting down after cleaning.

II. Ultrafiltration

(1) Sample adding: the front and tail of the peak is 3 pipe solution, about 9 ml is ultrafiltration sample; taking an ultrafiltration tube, adding the sample into the ultrafiltration tube, and adding a replacement buffer solution to make up to 15ml;

(2) And (3) centrifuging: 4300rpm, centrifuging for 5min, observing the sample volume in the ultrafiltration tube, and ensuring that the liquid on the membrane is about 1mL.

(3) Blowing: the suction head is extended to the bottom of the filter element by a liquid-transfering device, the filter membranes at the two sides are blown up, down, left and right for 3-5 times, and then the replacement buffer solution is added into the ultrafiltration tube to be complemented to 15ml.

(4) The centrifugation and blowing process was repeated for a total of 4 times;

(5) And (3) recycling: after the last centrifugation is completed, the pipette tip is extended to the bottom of the filter membrane to blow the filter membranes on the two sides for 3-5 times, the majority of liquid is sucked out, about 0.7ml of residual liquid is remained in the tube, 0.5ml of replacement buffer solution is added into the ultrafiltration tube, the pipette tip is extended to the bottom of the filter membrane to blow the filter membranes on the two sides for 3-5 times, all the liquid is sucked out, and the second part of required products are obtained: 1.2 ml.

III, mixing

And mixing the two products obtained in the two steps of gel filtration chromatography and ultrafiltration to obtain the final product.

IV, results

The final product amounted to 3.19mg,9.2 ml and recovery was 80.15%.

In addition, fig. 6 is obtained by peak area calculation: the amount of protein contained in the A1-3 tube is 94.73% of the total amount of the 5 tubes, namely, the amount of protein contained in the A4-5 tube is 5.27% of the total amount of the 5 tubes; the amount of protein contained in the A1 tube was 18.53% of the total amount of 5 tubes. If gel filtration chromatography is used singly, the solution of the front 3 tubes is recovered; from the change of the conductivity curve, the conductivity curve is obviously increased from A4 to indicate that the buffer composition is changed and the original elution buffer is mixed; the concentration of the solution is obviously reduced from 10ml to 12ml, and the loss of protein is added; if high concentration is to be ensured, the loss of the 2 tubes with peak tips is further increased to be approximately 25%.

Experimental protocol 2

(1) Sample adding: taking 1 ultrafiltration tube, adding 10ml of sample into the ultrafiltration tube, and adding replacement buffer solution to make up to 15ml;

(2) And (3) centrifuging: 4300rpm, centrifuging for 30min, observing the volume of the sample in the ultrafiltration tube, and ensuring that the liquid on the membrane is about 1 mL; at this point, it was found that the particulate precipitate adhered to the film;

(3) Blowing: extending the suction head to the bottom of the filter element by using a liquid-transfering device, blowing the filter membranes at the two sides for 3-5 times respectively, and adding a replacement buffer solution into the ultrafiltration tube to make up to 15ml;

(4) The centrifugation and blowing process was repeated for a total of 4 times;

(5) And (3) recycling: after the last centrifugation is finished, the pipette tips are extended to the bottom of the filter membrane to blow the filter membranes on the two sides for 3-5 times, the majority of liquid is sucked out, about 1ml of liquid remains in the tube, 7.5ml of replacement buffer solution is added into the ultrafiltration tube, the tips are extended to the bottom of the filter membrane to blow the filter membranes on the two sides for 3-5 times, and all the liquid is sucked out; all the liquid recovered by the ultrafiltration tube is mixed to obtain the final product.

The results show that: the final product measured a total of 2.25mg,8.5ml, recovery of 56.53%.

From the comparison of the results of experiment 1 and experiment 2, it is apparent that the protein recovery rate of experiment 1 is much higher than that of experiment 2, and that the total time of experiment 1 is less than 1.5 hours, and that the time of experiment 2 is about 2.5 hours.

5. Antibody Activity detection

Spleen tissue sections were taken, the primary antibody was the antibody obtained in the above-mentioned experiment scheme 1 and experiment scheme 2, the final concentration was adjusted to 0.0045mg/ml, the incubation amount was 100ul, and the results of the experiments were confirmed by using a conventional immunohistochemical detection reagent and method, and the experimental results are shown in FIG. 6 and FIG. 7.

The comparison result shows that: not only was the recovery significantly higher in experiment 1 than in experiment 2, but also the antibody titer was significantly higher in experiment 2, and there was evidence of inactivation in addition to the loss of antibody in experiment 2.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, after reading the above teachings of the present application, those skilled in the art may make various changes or modifications to the present application, which equivalent forms also fall within the scope of the present application as defined in the appended claims.

Claims (9)

1. The efficient protein recovery process is characterized by comprising the following steps of: the sample to be recovered is subjected to gel filtration chromatography, and the chromatographic solution with complete buffer solution replacement is collected as a first-step recovered product; collecting the chromatographic solution with incomplete replacement of the residual buffer solution as an ultrafiltration sample for ultrafiltration, and completely replacing the buffer solution to obtain a second-step recovered product; and mixing the recovered product in the first step and the recovered product in the second step to obtain a recovered final product.

2. The efficient protein recovery process according to claim 1, wherein: the gel filtration chromatography process is as follows: after the purifier is pre-cleaned, the pipeline is washed to be balanced by a replacement buffer solution, a sample is loaded for chromatography, and the chromatographic solution is respectively collected according to the peak front, peak tip and peak tail according to a 280nm absorbance curve; when the absorbance reading value at 280nm starts to rise, buffer solutions before and at the peak tip are collected respectively to replace the complete chromatographic solution; the buffer solution at the tail of the collection peak starts to replace the incomplete chromatographic solution when the conductivity starts to change, and the collection is stopped when the absorbance reading at 280nm is reduced to the curve approaching level, and the protein content in the uncollected chromatographic solution is not more than 0.1%.

3. The efficient protein recovery process according to claim 1, wherein: the ultrafiltration process comprises the following steps: adding an ultrafiltration sample into an ultrafiltration tube, adding a replacement buffer solution to complement the volume, centrifuging to ensure that about 1ml of liquid on the membrane is obtained, blowing and uniformly mixing by using a liquid transfer device, and adding the replacement buffer solution into the ultrafiltration tube to complement the volume; repeating the centrifugation and blowing process for multiple times, blowing and mixing with a liquid transfer device after the last centrifugation, sucking out all the liquid, adding a small amount of replacement buffer solution into the ultrafiltration tube, blowing and mixing, sucking out all the liquid, and mixing to obtain the product recovered in the second step.

4. The efficient protein recovery process according to claim 2, wherein: when the protein concentration of the chromatographic solution before and at the peak tip does not meet the set requirement after mixing, the chromatographic solution before and at the peak tail are ultrafiltered together.

5. The efficient protein recovery process according to claim 2, wherein: the loading flow rate in the gel filtration chromatography is 5ml/min.

6. The efficient protein recovery process according to claim 2, wherein: when the absorbance reading value at 280nm rises to 40mAU, collecting the buffer solution before and at the peak tip respectively to replace the complete chromatographic solution, when the conductivity starts to change, collecting the buffer solution at the peak tail to replace the incomplete chromatographic solution, and when the absorbance reading value at 280nm falls to 40mAU, stopping collecting.

7. A process for the efficient recovery of protein according to claim 3, characterized in that: in the ultrafiltration process, the centrifugation and blowing are repeated for at least 4 times.

8. A process for the efficient recovery of proteins according to any one of claims 1 to 7, characterized in that: the sample to be recovered satisfies at least one of the following characteristics:

(1) The sample size is large and is not less than 10mg;

(2) The concentration of the recovered final product has the set requirement, and the single concentration method cannot reach the high concentration of the set requirement;

(3) And recovering the activity loss of the final product after ultrafiltration and concentration.

9. The efficient protein recovery process according to claim 8, wherein: the sample to be recovered is a protein sample with biological activity.