CN109206508B - Method for screening affinity chromatography packing - Google Patents
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- 238000011068 loading method Methods 0.000 claims abstract description 104
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- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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Abstract
The invention relates to a screening method of a filler for affinity chromatography, which comprises the following steps: under the same flow rate and retention time, continuously loading the sample loading liquid containing the monoclonal antibody into a plurality of affinity chromatographic columns respectively to overload each affinity chromatographic column, wherein each affinity chromatographic column contains one affinity chromatographic filler, and the affinity chromatographic fillers in at least two affinity chromatographic columns are different; subpackaging the flow-through liquid of each affinity chromatography column into a plurality of liquids to be detected according to the penetrating sequence; detecting the concentration of the monoclonal antibody in each solution to be detected; and determining the dynamic binding capacity of various affinity chromatography fillers to the monoclonal antibody according to the concentration of the monoclonal antibody in each solution to be detected, and screening the affinity chromatography fillers according to the dynamic binding capacity. According to the screening method of the filler for affinity chromatography, the filler for affinity chromatography suitable for the monoclonal antibody can be rapidly screened.
Description
Technical Field
The invention relates to the field of biology, in particular to a screening method of affinity chromatography packing.
Background
Since the first murine monoclonal antibody, muromonab-CD3(OKT3), which is directed against post-transplant immune rejection, was approved by the united states Food and Drug Administration (FDA) for marketing, monoclonal antibodies are increasingly being used in the therapeutic pharmaceutical field.
In the production and development of monoclonal antibodies, the development of a monoclonal antibody purification process is an extremely important loop. The most common platform purification technology at present is: the cell culture fluid containing the antibody is subjected to clarification treatment, affinity chromatography, virus inactivation, intermediate purification of ion exchange chromatography, fine purification of ion exchange chromatography, virus removal filtration, ultrafiltration concentration and buffer solution replacement to obtain the purified monoclonal antibody.
In the development process of affinity chromatography process, how to quickly and efficiently select one to two fillers meeting the process requirements from a great variety of chromatography fillers is challenging, and mainly relates to the evaluation of the dynamic adsorption capacity of the chromatography fillers. The traditional screening method is determined by setting different flow rates and retention times, running the adsorption/elution process of the sample and the chromatography medium for multiple times, monitoring the penetration position and the penetration ratio of the target antibody in the flow-through by using a protein purifier, and fitting a flow-through curve. The disadvantages of this screening method are the long process time and the high material and sample consumption.
Disclosure of Invention
Based on the method, a rapid screening method for the filler of the affinity chromatography is provided.
A method for screening a filler for affinity chromatography, comprising the steps of:
under the same flow rate and retention time, continuously loading a loading solution containing the monoclonal antibody into a plurality of affinity chromatography columns respectively to overload each affinity chromatography column, wherein each affinity chromatography column contains one affinity chromatography filler, and the affinity chromatography fillers in at least two affinity chromatography columns are different;
subpackaging the flow-through liquid of each affinity chromatography column into a plurality of liquids to be detected according to the penetrating sequence;
detecting the concentration of the monoclonal antibody in each solution to be detected;
determining the dynamic binding capacity of various affinity chromatography fillers to the monoclonal antibody according to the concentration of the monoclonal antibody in the solution to be detected; and
screening said affinity chromatography packing material according to said dynamic binding capacity.
The screening method of the filler for affinity chromatography comprises the steps of continuously loading sample loading liquid containing the monoclonal antibody into a plurality of affinity chromatography columns respectively to overload the affinity chromatography columns, subpackaging flow-through liquid into a plurality of liquid to be detected according to the penetrating sequence, and rapidly analyzing the dynamic binding capacity of each affinity chromatography filler to the monoclonal antibody by comparing the concentration of the monoclonal antibody in the liquid to be detected, so that the affinity chromatography filler suitable for the monoclonal antibody is screened and obtained. In addition, in the whole screening process, the binding capacity of the affinity chromatography filler can be evaluated by one-time continuous sample loading, the consumption of the monoclonal antibody is less, and the consumption of related consumables is less.
In one embodiment, the product of the volume of the loading solution added to each of the affinity layer columns and the mass concentration of the monoclonal antibody in the loading solution is greater than the product of the inherent loading of the affinity chromatography packing and the volume of the affinity packing.
In one embodiment, the step of detecting the concentration of the monoclonal antibody in each of the test solutions comprises:
filling each affinity chromatography filler on a porous filter plate respectively, and balancing each affinity chromatography filler by using a balancing solution to obtain a microporous chromatography plate;
adding the same volume of the multiple solutions to be detected of each affinity chromatography column into the corresponding holes of the microporous chromatography plate to obtain multiple solutions to be incubated;
incubating, centrifuging and eluting the solutions to be incubated to obtain a plurality of eluents; and
and detecting the concentration of the monoclonal antibody in each eluent to obtain the concentration of the monoclonal antibody in each solution to be detected.
In one embodiment, the step of incubating, centrifuging, and eluting each of the solutions to be incubated to obtain a plurality of eluents comprises:
incubating and centrifuging the solutions to be incubated to obtain a plurality of centrifugates;
detecting the concentration of the monoclonal antibody in each centrifugate, if the ratio of the concentration of the monoclonal antibody in the centrifugate to the concentration of the monomer-bound antibody in the sample solution is more than 5%, adding the centrifugate into the corresponding holes of the microporous chromatography plate again for incubation, then centrifuging and detecting the concentration of the monoclonal antibody until the ratio of the concentration of the monoclonal antibody in each centrifugate to the concentration of the monomer-bound antibody in the sample solution is not more than 5%, and obtaining the microporous chromatography plate absorbed with the monoclonal antibody; and
and washing the microporous chromatography plate adsorbed with the monoclonal antibody by using an elution buffer solution to obtain the eluent.
In one embodiment, the loading solution contains the monoclonal antibodies at the same concentration, and the step of determining the dynamic binding capacity of each affinity chromatography packing material for the monoclonal antibodies according to the concentration of the monoclonal antibodies in the test solution comprises:
calculating the total sample loading volume corresponding to each liquid to be detected; and
and calculating the ratio of the concentration of the monoclonal antibody in each liquid to be detected to the concentration of the monoclonal antibody in the sample loading liquid, wherein the affinity chromatography filler with the large sample loading total volume has large dynamic binding capacity under the same ratio.
In one embodiment, in the plurality of test solutions of each affinity chromatography column, the ratio of the concentration of the monoclonal antibody in at least one of the test solutions to the concentration of the monoclonal antibody in the loading solution is between 0.05 and 0.15.
In one embodiment, the sample solution is a supernatant of a fermentation broth containing the monoclonal antibody.
In one embodiment, before the step of loading the monoclonal antibody-containing loading solution into the affinity chromatography columns continuously at the same flow rate and retention time, respectively, to overload each of the affinity chromatography columns, the method further comprises the following steps:
the static binding capacity of each of the affinity chromatography fillers to the monoclonal antibody was confirmed.
In one embodiment, the step of confirming the static binding capacity of each of the affinity chromatography fillers for the monoclonal antibody comprises:
configuring the monoclonal antibody into loading samples with different concentrations;
respectively filling each affinity chromatography filler with the same volume on a porous filter plate, and balancing each affinity chromatography filler by using an affinity chromatography balancing solution to obtain a microporous chromatography plate;
respectively adding the sample loading samples with different concentrations to the microporous chromatography plate in equal volume, incubating, centrifuging, collecting supernatant, and detecting the concentration of the monoclonal antibody in the supernatant; and
confirming the static binding capacity of each of the affinity chromatography fillers for the monoclonal antibody based on the concentration of the monoclonal antibody in the supernatant of each of the affinity chromatography fillers.
In one embodiment, the monoclonal antibody is prepared into loading samples with different concentrations by using a gradient dilution method.
Drawings
FIG. 1 is a graph showing the number of penetration times at a loading concentration of 14.9mg/m L as a function of the concentration of unbound monoclonal antibody in the loading concentration in example 1;
FIG. 2 is a graph showing the number of penetration times at a loading concentration of 29.1mg/m L in example 1 as a function of the concentration of unbound monoclonal antibody in the loading concentration;
FIG. 3 is a graph showing the number of penetration times at a loading concentration of 43.7mg/m L as a function of the concentration of unbound monoclonal antibody in the loading concentration in example 1;
FIG. 4 is a graph showing the number of penetration times at a concentration of 57.2mg/m L in example 1 as a function of the concentration of unbound monoclonal antibody in the sample;
FIG. 5 is a graph showing the number of penetration times at a loading concentration of 69.9mg/m L as a function of the concentration of unbound monoclonal antibody in the loading concentration in example 1;
FIG. 6 is a graph showing the number of penetration times at a concentration of 79.2mg/m L in example 1 as a function of the concentration of unbound monoclonal antibody in the loaded concentration;
FIG. 7 is a graph of the number of breakthrough times at a loading concentration of 84.7mg/m L as a function of the concentration of unbound monoclonal antibody in the loading concentration in example 1;
FIG. 8 is a graph showing the number of breakthrough times at a loading concentration of 88.4mg/m L as a function of the concentration of unbound monoclonal antibody in the loading concentration in example 1;
FIG. 9 is a chromatogram of Resin D in example 2;
FIG. 10 is a chromatogram of Resin E in example 2;
FIG. 11 is a chromatogram of Resin B in example 2;
FIG. 12 is a graph showing the change in the content of monoclonal antibodies in the flow-through of Resin D, Resin E and Resin B in example 2.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Some embodiments of the invention are presented in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The method for screening affinity chromatography packing of an embodiment comprises the following steps:
s110, confirming the static binding capacity of each affinity chromatography filler to the monoclonal antibody. And (3) primarily screening affinity chromatography fillers suitable for the monoclonal antibody.
Specifically, step S110 includes steps S111 to S117.
S111, preparing the micropore chromatography plate.
Specifically, each affinity chromatography filler is filled on a porous filter plate in equal volume, and each affinity chromatography filler is balanced by an affinity chromatography balancing solution to obtain the microporous chromatography plate.
Further, each affinity chromatography filler is prepared into a colloidal suspension, and then the colloidal suspension is paved, cleaned and balanced to obtain the microporous chromatography plate.
Further, the operation of preparing the gum suspension comprises: mixing the affinity chromatography filler and the filler preserving fluid respectively. Specifically, the filler preservation solution is an ethanol solution with the volume fraction of 10-30%. The volume ratio of each affinity chromatography filler to the filler preservation solution is 1: 0.5 to 2. Preferably, the filler preservation solution is an ethanol solution with 20 percent of volume fraction. The volume ratio of the affinity chromatography filler to the filler preservation solution is 1: 1.
further, the plating operation comprises adding each colloidal suspension made of affinity chromatography packing separately to a different well of the porous filter plate, specifically, the volume of the added colloidal suspension is 10 μ L/well to 40 μ L/well, preferably, the volume of the added colloidal suspension is 15 μ L/well to 25 μ L/well, more preferably, the volume of the added colloidal suspension is 20 μ L/well.
Further, the operations of cleaning and balancing include: the filter plates after the plate spreading are washed with ultrapure water and then with a balance buffer solution. And further, cleaning the filter plate after the plate paving for 3-5 times by using ultrapure water and a balance buffer solution, directly performing suction filtration to remove clear liquid after each cleaning, and centrifuging and removing the clear liquid after the final balancing to obtain the microporous chromatography plate. In this embodiment, the equilibration buffer is 1 × PBS (10mM PB, 150mM NaCl, ph 7.4). Of course, in other embodiments, the equilibration buffer may also be selected according to the specification for the affinity chromatography packing.
S113, preparing the monoclonal antibody into loading samples with different concentrations.
Specifically, samples containing monoclonal antibodies are prepared into sample loading samples with different concentrations according to the inherent loading capacity of each affinity chromatography filler, so as to observe the adsorption condition of each affinity chromatography filler on the monoclonal antibody under different concentrations. Wherein the concentration of the sample comprises at least a concentration capable of overloading the affinity chromatography packing. Overload refers to a sample load greater than the intrinsic load. That is, the product of the volume of the loading solution added to each affinity layer column and the mass concentration of the monoclonal antibody in the loading solution is larger than the product of the inherent loading of the affinity chromatography packing in the affinity chromatography column and the volume of the affinity packing. Of course, the volume of the sample solution at this time corresponds to the unit of the volume of the affinity filler, and the mass concentration of the monoclonal antibody corresponds to the unit of the inherent loading amount. The sample loading is equal to the product of the mass concentration of monoclonal antibody in the loaded sample and the loading volume divided by the packing volume of the affinity chromatography column. The intrinsic loading can be obtained according to the specification for the affinity chromatography packing. The volume of the affinity filler can be calculated according to the volume of the added colloidal suspension and the proportion of the affinity chromatography filler in the colloidal suspension.
In the process of affinity chromatography purification of the monoclonal antibody, different affinity chromatography fillers have different binding capacities to different monoclonal antibodies, and the purification effects are different, so that the affinity chromatography purification of the monoclonal antibody needs to screen proper affinity chromatography fillers for purification.
Further, the monoclonal antibody is prepared into sample loading samples with different concentrations by adopting a gradient dilution method.
Further, protein concentration detection is respectively carried out on the sample loading samples with different concentrations after gradient dilution, and initial sample loading concentration is obtained. Specifically, the samples after gradient dilution are respectively placed in different wells of a multi-well UV non-absorption plate, and the concentration of the monoclonal antibody in the samples is detected by using a Take3&96-well optical path correction A280 method (Epoch, BioTeK) to obtain the initial sample concentration of the samples.
And S115, incubation.
Specifically, equal volumes of samples at different concentrations were applied to the microplate and incubated. Further, incubation is performed by intermittent shaking. Further, oscillating for 30min to 60min at the rotating speed of 337.5rpm to 675rpm (Ribel, QB-9001), pausing for 10s to 60s, then oscillating and pausing again, and repeating for 3 to 4 times.
And S117, detecting the content of the monoclonal antibody.
Specifically, samples of different concentrations after incubation are centrifuged, the supernatant is collected, and the supernatant is then assayed for the amount of monoclonal antibody. And preliminarily judging the static binding capacity of the monoclonal antibody and each affinity chromatography filler according to the content of the monoclonal antibody in the supernatant. The higher the content of monoclonal antibody in the supernatant of the monoclonal antibody at the same loading concentration indicates that the lower the static binding capacity of the monoclonal antibody to the affinity chromatography packing at the loading concentration, the less suitable it is as an affinity chromatography packing for purifying the monoclonal antibody.
Further, the steps of loading, incubating, centrifuging and detecting the content of the monoclonal antibody are repeated for a plurality of times until the detected data are stable. And repeating the processes of loading, incubating, centrifuging and detecting until the detection data is stable, so that the adsorption quantity of different fillers in different incubation batches can be determined, and the loading capacity and the adsorption speed can be compared.
S130, screening the affinity chromatography filling material suitable for the monoclonal antibody according to the dynamic binding capacity of the affinity chromatography filling material and the monoclonal antibody.
Specifically, step S130 includes steps S131 to S135.
S131, sampling and collecting flow-through liquid.
Specifically, the loading solution containing the monoclonal antibody is continuously loaded into a plurality of affinity chromatography columns respectively at the same flow rate and retention time, so that each affinity chromatography column is overloaded. Each affinity chromatographic column contains one affinity chromatographic filler, and at least two affinity chromatographic columns have different affinity chromatographic fillers. And (3) subpackaging the flow-through liquid of each affinity chromatography column into a plurality of liquids to be detected according to the sequence of penetration.
Further, the volume of the sample loading solution at the time of overload was calculated from the inherent amount of each affinity chromatography packing and the concentration of the monoclonal antibody at the time of loading. In each affinity chromatography column, the product of the volume of the loading solution and the concentration of monoclonal antibody in the loading solution is greater than the product of the inherent loading of the affinity chromatography packing and the volume of the affinity packing, thereby overloading the affinity chromatography column. Of course, the volume of the sample loading solution at this time is in accordance with the unit of the filler for affinity chromatography, and the concentration of the monoclonal antibody is in accordance with the unit of the amount of the specific load for affinity chromatography. Then the sample loading is stopped when the sample loading is continued to the volume of the sample solution. Collecting the flow-through liquid from the sample loading, and subpackaging into a plurality of liquids to be detected according to the sequence of penetration. Further, a plurality of flow-through liquids close to the sample loading stop are collected as the liquid to be detected. Furthermore, the ratio of the concentration of the monoclonal antibody of at least one of the multiple solutions to be detected in each affinity chromatography column to the concentration of the monoclonal antibody in the loading solution is between 0.05 and 0.15.
Further, the concentration of monoclonal antibody in the loading solution was the same. Furthermore, the sample solution may be a supernatant of a fermentation broth containing the monoclonal antibody, or a solution prepared from a purified monoclonal antibody.
And S133, detecting the concentration of the monoclonal antibody in each solution to be detected.
Specifically, each affinity chromatography filler is filled on a porous filter plate, and each affinity chromatography filler is balanced by a balance liquid to obtain the microporous chromatography plate. And then adding the multiple solutions to be detected of each affinity chromatography column in equal volume into the corresponding holes of the microporous chromatography plate to obtain multiple solutions to be incubated. Then, each solution to be incubated is incubated, centrifuged and eluted to obtain a plurality of eluents. And then detecting the concentration of the monoclonal antibody in each eluent to obtain the concentration of the monoclonal antibody in each solution to be detected.
Further, incubating and centrifuging each solution to be incubated to obtain a plurality of centrifugates, detecting the concentration of the monoclonal antibody in each centrifugate, if the ratio of the concentration of the monoclonal antibody in the centrifugate to the concentration of the monomer-ron antibody in the sample loading solution is more than 5%, adding the centrifugates into the corresponding holes of the microporous chromatography plate again for incubation, and then centrifuging and detecting the concentration of the monoclonal antibody until the ratio of the concentration of the monoclonal antibody in each centrifugate to the concentration of the monomer-ron antibody in the sample loading solution is not more than 5%, thereby obtaining the microporous chromatography plate absorbed with the monoclonal antibody. Then, different wells of the microporous chromatography plate adsorbed with the monoclonal antibody are respectively washed with an elution buffer solution to obtain a plurality of eluents. And then detecting the concentration of the monoclonal antibody in each eluent to obtain the concentration of the monoclonal antibody in each solution to be detected.
Further, after the step of detecting that the ratio of the concentration of the monoclonal antibody in each of the centrifugations to the concentration of the monoclonal antibody in the sample solution is less than 5%, the step of washing the different wells of the microporous chromatography plate adsorbed with the monoclonal antibody with an elution buffer is preceded by the step of washing the microporous chromatography plate adsorbed with the monoclonal antibody with an equilibration buffer. Specifically, the microporous chromatography plate adsorbed with the monoclonal antibody is washed with an equilibrium buffer solution, the supernatant is filtered and discarded, the washing is continued with the equilibrium buffer solution after repeating for 1 to 3 times, and the supernatant is discarded after centrifugation. When the sample solution is a fermentation solution containing the monoclonal antibody, the fermentation solution usually contains other impure proteins, and at the moment, the microporous chromatography plate adsorbed with the monoclonal antibody is washed by the equilibrium buffer solution, so that the impure proteins can be removed, and the detection of the monoclonal antibody in the eluent is convenient. Of course, it is understood that, if the sample solution is a solution prepared from a pure monoclonal antibody, the step of washing the microplate having the monoclonal antibody adsorbed thereon with the equilibration buffer solution may not be performed.
Further, the concentration of the monoclonal antibody in the eluate was determined by the Take3&96-well optical path correction A280 method.
And S135, determining the dynamic binding capacity of various affinity chromatography fillers to the monoclonal antibody according to the concentration of the monoclonal antibody in each solution to be detected. Affinity chromatography packing was screened for dynamic binding capacity. Affinity chromatography packing with large dynamic binding capacity is used as the affinity chromatography packing of the monoclonal antibody.
Specifically, calculating the total sample loading volume corresponding to each liquid to be detected; calculating the ratio of the concentration of the monoclonal antibody in each solution to be detected to the concentration of the monoclonal antibody in the sample loading solution; wherein, under the same sample concentration and the same ratio, the affinity chromatography filler with large sample total volume corresponding to the affinity chromatography filler has large dynamic binding capacity.
Further, the ratio of the concentration of the monoclonal antibody in each solution to be tested to the concentration of the monoclonal antibody in the corresponding sample solution is used as a vertical coordinate, and the solutions to be tested collected according to the time sequence are used as horizontal coordinates to draw a graph of the content change of the monoclonal antibody in each affinity chromatography column. According to the content change diagram of the monoclonal antibody of the affinity chromatography filler, determining the binding capacity of each affinity chromatography filler to the monoclonal antibody, and screening the affinity chromatography filler suitable for the monoclonal antibody.
The screening method of the filler for affinity chromatography at least has the following advantages:
(1) is simple and convenient. The sample loading liquid containing the monoclonal antibody is continuously loaded into a plurality of affinity chromatographic columns respectively to overload the affinity chromatographic columns, the flow-through liquid is divided into a plurality of liquid to be detected according to the sequence of penetration, and the dynamic binding capacity of each affinity chromatographic filler to the monoclonal antibody can be rapidly analyzed by comparing the concentration of the monoclonal antibody in the liquid to be detected, so that the affinity chromatographic filler suitable for the monoclonal antibody is obtained by screening. Particularly, under the condition that the concentrations of the monoclonal antibodies contained in the sample liquid are the same, and the ratio of the concentration of the monoclonal antibody in the liquid to be detected to the concentration of the monoclonal antibody in the sample liquid is the same, the dynamic binding capacity of each affinity chromatography filler can be judged more quickly, and the appropriate affinity chromatography filler is screened out.
(2) For example, a 1m L system is used for at least 3 cycles for each chromatographic filler, and five chromatographic fillers are to be screened, wherein each sample is subjected to 50mg/m L loading, 5 times of 15 cycles are required, and 3 days or so are required to finish the whole process.
(3) The traditional screening method is characterized in that multiple cycles of sample loading detection are needed for each chromatography medium, the consumption of the samples is huge, taking 50 mg/15 times of consumed samples as an example of loading capacity of L mg/m for each time, the time cost and the material cost of the whole process development are undoubtedly increased for the early development that the samples are more precious or the stability of the samples has certain risk, the volume of the sample loaded under each experimental condition of the screening method for the affinity chromatography packing can be reduced to 200 mu L at minimum, only 0.8mg of protein is needed for the design loading capacity of L mg/m, the sample loading of 96 pore plates is less than 50mg, the sample consumption is reduced by more than 10 times, the buffer solution consumption is less, and the method is an extremely economic development method.
(4) The traditional evaluation method is adopted, the flow-through collected samples generated in the process are required to be sampled for a plurality of times, the number of the flow-through collected samples generated in the process reaches more than one hundred grades, then the collected samples are respectively detected, at the initial development stage of the purification process, the sample loading sample is often fermentation feed liquid, so that the A280 method cannot be directly adopted in the process of detecting the protein content, the A280 or HP L C detection process must be carried out after the purification, and the detection pressure and the challenge brought by the sample with the number of more than one hundred grades are imaginable.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
High-throughput antibody content detection was performed using 96-well filter plates (Pall) and corresponding closure membranes, shaking shaker (linbel), Epoch (BioTeK, with Take3 microplates), 96-well UV non-absorbing plates (Corning), vacuum pump module (Pall), ultrafiltration concentrator tubes (Millipore), stopwatch, etc., with 1 PBS (10mM PB, 150mM NaCl, pH7.4) as equilibration Buffer (Buffer A), 20mM NaAc, pH3.4 as elution Buffer (Buffer B1), and 0.1M NaOH (Buffer B2) as washing Buffer.
Example 1
(1) Sample pretreatment and gradient concentration sample dilution: 80mg of PD1 (monoclonal cell Deathprotein 1) monoclonal antibody (produced by Yusheng, Shenzhen Shenpeng biopharmaceutical Co., Ltd.) was collected, concentrated by ultrafiltration (Millipore,
ultra 15m L Centrifugal Filters, MWCO: 30KDa) for buffer replacement, and performing equal volume batch operation for more than 5 times (completing, changing the solution to 1 × PBS, adopting Take3&And (3) a 96-well optical path correction A280 method, which is used for accurately detecting the concentration of the sample after concentration and liquid change, and accurately diluting the sample to 4mg/m L according to the result of Take3A 280.
(2) Diluting a gradient concentration sample, namely taking a monoclonal antibody with the concentration of 4mg/m as SP 1, namely a sample with the load of 80mg/m 0, taking SP 11 with the volume of 11m 2, taking 2.625m 3 SP 41, adding 375 mu 5 PBS, uniformly mixing, taking SP 62 as SP 62, namely a sample with the load of 70mg/m 7, taking 2.25m 8 SP 91, adding 75 mu PBS, uniformly mixing, taking SP 03 as a sample with the load of 60mg/m 1, taking 1.875m 2 SP 31, adding 1125 mu 4 PBS, uniformly mixing, taking SP 54 as a sample with the load of 50mg/m 6, taking 3m 7 SP 81, adding 3m 9 PBS, uniformly mixing, taking SP 5 as a sample with the load of 40mg/m 0, taking 1.125m 1 SP 21, adding 1875 mu 3, uniformly mixing, taking SP 46 as a sample with the load of 30mg/m 5, taking 3m 6 SP 75 as a sample with 3m 8, uniformly mixing, taking PBS 97 as a sample with the load of 20mg/m 3, namely a sample with the load of SP 7, adding 10mg/m and uniformly mixing.
(3) The preparation of the gel suspension comprises the steps of taking 5 centrifuge tubes of 15m L, respectively filling 5 kinds of affinity chromatography fillers which are respectively from GE MabSelect Sure (hereinafter referred to as "Resin A"), MabSelect Sure L X (hereinafter referred to as "Resin B"), Merck EshMuno A (hereinafter referred to as "Resin C"), Tosoh TOYAPEAR L AF-rProtein AHC-650F (hereinafter referred to as "Resin D"), BestChrrom AT Protein A Diamond (hereinafter referred to as "Resin"), each affinity chromatography filler has a volume of 2m L, leveling, centrifuging (175g × 5min), standing for 3min to 5min, sucking out or continuously adding a certain volume of 20% EtOH according to the scales on the centrifuge tubes, so that the volume concentration of the affinity chromatography fillers in each centrifuge tube is 50%, and homogenizing, thus obtaining the gel suspension.
(4) Plating after resuspending the 50% gum suspension of step (3) with a calandria, at the position shown in Table 1, 20. mu. L/well was added to the corresponding 96-well filter plate (Pall) (20. mu. L/well of gum suspension corresponds to a volume of affinity chromatography packing of 10. mu. L/well).
TABLE 1
(5) And (3) cleaning and balancing, namely firstly ultra pure water, then 1 × PBS (10mM PB, 150mM NaCl, pH7.4) balance Buffer solution (Buffer A) for 3 times at a rate of 0.2m L/hole on all the holes of the 96-hole filter plate which is finished by the plate paving and obtained in the step (4), directly filtering and removing clear liquid after each cleaning, and centrifuging and removing the clear liquid after the last balancing to obtain the 96-hole filter plate.
(6) And (3) loading, namely transferring SP L1-SP L8 into corresponding holes of a clean 96-hole UV non-absorption plate (Corning) according to 230 mu L/hole, detecting by a 96-well optical path correction method A280 to obtain an initial loading sample concentration, and then transferring SP L1-SP L8 samples in the 96-hole plate into the 96-hole filter plate obtained in the step (5) at 200 mu L/hole by using a discharging gun.
(7) Incubation and flow-through antibody content detection: shaking at 675rpm to mix the sample and the filler thoroughly, incubating for 30min, suspending, centrifuging at 150g for 5min, collecting the centrifuged supernatant, measuring the concentration of unbound protein in the supernatant by 96-well light path correction and Take3A280, and checking the unbound protein in the supernatant. Then, the collected supernatant was further subjected to incubation, and the resulting mixture was shaken at 675rpm (shaking table, Linbel, QB-9001), suspended after 30min, centrifuged at 150g for 5min, collected and centrifuged, and the concentration of the collected supernatant was measured by 96-well optical path calibration and Take3A280 to examine the unbound protein in the supernatant. The above incubation-loading-concentration detection process was repeated five times, and the obtained supernatants were designated as FT1 to FT5 (numbers of the number of penetrations). The ratio of the concentration of the monoclonal antibody in each chromatographic packing to the concentration of the monoclonal antibody in the sample was plotted according to the unbound protein in the supernatant of each time, and the results are shown in FIGS. 1 to 8.
According to detection, the initial loading concentrations of SP L8-SP L1 are 0.749mg/m L0, 1.455mg/m L1, 2.188mg/m L2, 2.863mg/m L3, 3.496mg/m L4, 3.963mg/m L5, 4.238mg/m L6 and 4.423mg/m L7 respectively, the sample loading amounts are equal to the product of the initial loading concentration and the loading volume divided by the volume of the affinity chromatography filler, and the actual loading amounts corresponding to SP L88-SP L91 are 14.9mg/m L, 29.1mg/m L, 43.7mg/m L, 57.2mg/m L, 69.9mg/m L, 79.2mg/m L, 84.7mg/m L and 88.4mg/m L respectively.
As can be seen from FIGS. 1-8, both Resin E and Resin D showed greater adsorption capacity at various loading concentrations, indicating that both Resin E and Resin D had high adsorption efficiencies, Resin B was the slowest adsorbing filler at loadings < 60mg/m L, and Resin B was modified at high loadings.
Example 2
The following steps were performed on a 1m L column packed in Resin B affinity chromatography:
(1) and (2) carefully pumping the Sample into a Sample pump S under the conditions of low flow rate and By-pass of the chromatographic column until the purple peak appears, not performing wash on the Sample pump, and simultaneously performing the wash on the A pump again to ensure that the Sample pump is filled with the Sample liquid to be loaded, (2) setting the retention time to be 3min, and continuously loading the Sample at the flow rate of 0.33m L/min, wherein the flow-through liquid adopts a 96-pore plate to collect the Sample liquid from the beginning of loading By 1.5m L/tube, the loading is stopped when the Sample is loaded to the set volume (33.13m L), and the Sample pump Buffer pipeline mp rinsing and Sample pump are not performed, wherein the collected flow-through liquid is used as the liquid to be tested before the Sample loading is finished, and the collected flow-through liquid is respectively correspondingly numbered as Resin B1-FT30 after the Sample is loaded, and then the affinity chromatographic column is eluted By a conventional method.
(3) And (3) respectively carrying out the operations corresponding to the steps (1) to (2) on the 1m L columns of the two affinity chromatographic fillers of Resin D and Resin E, pushing 30 samples of the collected flow-through liquid forward from the end of sample loading as corresponding liquid to be detected, and respectively corresponding to the numbers of Resin D FT 1-FT 30 and Resin E FT 1-FT 30 from the beginning to the end.
(4) High throughput determination of antibody content in a test solution by plating, washing and equilibrating an empty 96-well filter plate according to steps (4) to (5) of example 1 to obtain a 96-well filter plate for measuring monoclonal antibodies in the test solution, wherein the 96-well filter plate is positioned as shown in table 2 (without duplicate wells), then 20 μ L steps (2) are performed to obtain transudates B FT1 to FT30 and Resin D FT1 to FT30 and Resin E FT1 to FT30 obtained in step (3) are added to the 96-well filter plate for measuring antibodies in the transudates, respectively, wherein 1.2mg/m L, 0.6mg/m L and 0.1mg/m L Protein a affinity elution sample (authigenic, Shenpeng biopharmaceutical corporation) is used as a positive control, then the monoclonal antibody concentration in the buffer binding assay is determined by incubating at 675rpm, 15s every 10min, 2h, 150g 5 h, and the monoclonal antibody concentration in the buffer is determined by the centrifugation method, and the monoclonal antibody concentration in the buffer is determined by the same washing method.
TABLE 2
And calculating the ratio of the concentration of the monoclonal antibody in each solution to be tested to the concentration of the monoclonal antibody in the sample loading solution according to the content and the volume of the monoclonal antibody in each solution to be tested of the Resin B, the Resin D and the Resin E and the corresponding total sample loading volume, and drawing a graph of the content change of the monoclonal antibody in the flow-through liquid of each affinity chromatography filler.
As a result: chromatograms of Resin D, Resin E and Resin B are shown in FIGS. 9-11, respectively. In FIGS. 9 to 11, UV 1-280 indicates ultraviolet absorption (mAU) at 280nm, Cond indicates conductivity (mS/cm), and pH indicates pH. The changes in the amounts of monoclonal antibodies in the flowthrough of Resin E, Resin D and Resin B are shown in FIG. 12.
As can be seen from FIG. 12, when the retention time is 3min, DBC of ResinD and ResinE10％Higher, about 70mg monoclonal antibody/m L filler, and DBC of Resin B10％Lower, about 31mg monoclonal antibody/m L filler.
Therefore, ResinD and ResinE are more suitable as the affinity chromatography purification filler of the target monoclonal antibody at the retention time.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (8)
1. A method for screening affinity chromatography packing is characterized by comprising the following steps:
1) confirming the static binding capacity of each affinity chromatography filler to be detected to the monoclonal antibody, primarily screening a plurality of affinity chromatography fillers suitable for the monoclonal antibody, and respectively loading each preliminarily screened affinity chromatography filler into different affinity chromatography columns;
2) under the same flow rate and retention time, continuously loading sample solutions containing monoclonal antibodies with the same concentration into each affinity chromatography column in the step 1) respectively, overloading the affinity chromatography columns, and subpackaging flow-through solutions of each affinity chromatography column into a plurality of solutions to be detected according to the penetrating sequence, wherein the ratio of the concentration of the monoclonal antibody of at least one solution to be detected to the concentration of the monoclonal antibody in the sample solution is 0.05-0.15 in the plurality of solutions to be detected of each affinity chromatography column;
3) detecting the concentration of the monoclonal antibody in each solution to be detected;
4) determining the dynamic binding capacity of various affinity chromatography fillers to the monoclonal antibody according to the concentration of the monoclonal antibody in each liquid to be detected, wherein the dynamic binding capacity of the affinity chromatography fillers with large sample loading total volume is large under the condition that the ratio of the concentration of the monoclonal antibody in the liquid to be detected to the concentration of the monoclonal antibody in the sample loading liquid is the same, or the dynamic binding capacity of the affinity chromatography fillers with small concentration of the monoclonal antibody in the liquid to be detected is large under the same time condition;
5) selecting an affinity chromatography packing for isolating the monoclonal antibody based on dynamic binding capacity.
2. The screening method according to claim 1, wherein the product of the volume of the loading solution added to each affinity layer column and the mass concentration of the monoclonal antibody in the loading solution is larger than the product of the inherent loading of the affinity chromatography packing and the volume of the affinity packing.
3. The screening method according to claim 1, wherein the specific steps of step 3) comprise:
filling each affinity chromatography filler on a porous filter plate respectively, and balancing each affinity chromatography filler by using a balancing solution to obtain a microporous chromatography plate;
respectively adding a plurality of solutions to be detected of each affinity chromatography column in equal volume into corresponding holes of the microporous chromatography plate to obtain a plurality of solutions to be incubated;
incubating each solution to be incubated, and carrying out centrifugal elution to obtain a plurality of eluents; and
and detecting the concentration of the monoclonal antibody in each eluent to obtain the concentration of the monoclonal antibody in each solution to be detected.
4. The screening method according to claim 3, wherein the step of incubating each incubation solution and performing centrifugal elution to obtain a plurality of eluents comprises:
incubating and centrifuging each solution to be incubated to obtain a plurality of centrifugates;
detecting the concentration of the monoclonal antibody in each centrifugate, if the ratio of the concentration of the monoclonal antibody in the centrifugate to the concentration of the monoclonal antibody in the sample loading liquid is more than 5%, adding the centrifugate into the corresponding hole of the microporous chromatography plate again for incubation, then centrifuging, and detecting the concentration of the monoclonal antibody until the ratio of the concentration of the monoclonal antibody in each centrifugate to the concentration of the monoclonal antibody in the sample loading liquid is not more than 5%, thus obtaining the microporous chromatography plate absorbed with the monoclonal antibody; and
and washing the microporous chromatography plate adsorbed with the monoclonal antibody by using an elution buffer solution to obtain a plurality of eluents.
5. The screening method of claim 1, wherein the step of determining the dynamic binding capacity of each affinity chromatography packing to be tested for monoclonal antibodies comprises:
calculating the total sample loading volume corresponding to each liquid to be detected; and
and calculating the ratio of the concentration of the monoclonal antibody in each solution to be detected to the concentration of the monoclonal antibody in the sample loading solution, wherein the affinity chromatography filler with large sample loading total volume has large dynamic binding capacity under the same ratio.
6. The screening method according to claim 1, wherein the supernatant is a supernatant of a fermentation broth containing the monoclonal antibody.
7. The screening method of claim 1, wherein the step of confirming the static binding capacity of each affinity chromatography packing for the monoclonal antibody comprises:
preparing the monoclonal antibody into sample loading samples with different concentrations;
respectively filling each affinity chromatography filler with the same volume on a porous filter plate, and balancing each affinity chromatography filler by using an affinity chromatography balancing solution to obtain a microporous chromatography plate;
respectively adding sample samples with different concentrations to a microporous chromatography plate in equal volume, incubating, centrifuging, collecting supernatant, and detecting the concentration of the monoclonal antibody in the supernatant; and
the static binding capacity of each affinity chromatography packing to the monoclonal antibody was confirmed according to the concentration of the monoclonal antibody in the supernatant of each affinity chromatography packing.
8. The screening method according to claim 7, wherein the monoclonal antibody is prepared into a sample to be loaded at different concentrations by a gradient dilution method.
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