CN109206508A - The screening technique of affinity chromatography filler - Google Patents
The screening technique of affinity chromatography filler Download PDFInfo
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- CN109206508A CN109206508A CN201810982583.0A CN201810982583A CN109206508A CN 109206508 A CN109206508 A CN 109206508A CN 201810982583 A CN201810982583 A CN 201810982583A CN 109206508 A CN109206508 A CN 109206508A
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- 238000001042 affinity chromatography Methods 0.000 title claims abstract description 114
- 238000000034 method Methods 0.000 title claims abstract description 57
- 238000012216 screening Methods 0.000 title claims abstract description 35
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- 238000001514 detection method Methods 0.000 claims description 20
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 claims description 16
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- 239000000047 product Substances 0.000 claims description 10
- 238000004587 chromatography analysis Methods 0.000 claims description 7
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- 238000010828 elution Methods 0.000 claims description 6
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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 present invention relates to a kind of screening techniques of the filler of affinity chromatography, the following steps are included: under identical flow velocity, retention time, sample solution containing monoclonal antibody is continuously loaded in multiple affinity columns respectively, make each affinity chromatography column overload, contain a kind of affinity chromatography filler in each affinity column, and at least there are two the affinity chromatography filler in affinity column is different；The liquid that flows through of each affinity column is packed as multiple prepare liquids according to the sequencing penetrated；Detect the concentration of monoclonal antibody in each prepare liquid；According to the concentration of monoclonal antibody in each prepare liquid, determine that various affinity chromatography fillers screen affinity chromatography filler to the Dynamic binding capacities of monoclonal antibody, and according to Dynamic binding capacities.According to the screening technique of the filler of above-mentioned affinity chromatography, can quickly screen to obtain the affinity chromatography filler for being suitble to the monoclonal antibody.
Description
Technical field
The present invention relates to biological fields, more particularly to a kind of screening technique of affinity chromatography filler.
Background technique
From the source of mouse monoclonal antibody muromonab-CD3 (OKT3) of immunological rejection after first anti-transplanting through beauty
Since food and medicine Surveillance Authority of state (Food and Drug Administration, FDA) approval listing, monoclonal antibody
Just it is applied to therapeutic pharmaceutical field more and more widely.
In the production development of monoclonal antibody, the exploitation of monoclonal antibody-purified technique is even more wherein extremely important one
Ring.Current most common hardware and software platform purification technique are as follows: by cell culture fluid antibody-containing by clarifying treatment, affinity chromatography,
Inactivation of virus, ion-exchange chromatography polishing purification, removes virus filtration and ultrafiltration concentration and delays at the purifying of ion-exchange chromatography moderate
Fliud flushing displacement, thus the monoclonal antibody purified.
In the development process of affinity chromatography technique, how quickly and efficiently to be picked out from the chromatographic stuffing of a great variety
One to two kind of filler for meeting process requirements be it is challenging, relate generally to commenting for the dynamic adsorption capacity of chromatographic stuffing
Valence.Traditional screening technique be run multiple times by the way that different flow velocity, retention time is arranged the absorption of sample and chromatography media/
Elution process, by monitoring the ratio for flowing through the penetration site of middle target antibody and penetrating using protein purification instrument, fitting is flowed through
Curve determines.The shortcomings that this screening technique is that the process time is long, and material and sample consumption are big.
Summary of the invention
Based on this, a kind of screening technique of the filler of quick affinity chromatography is provided.
A kind of screening technique of the filler of affinity chromatography, comprising the following steps:
Under identical flow velocity, retention time, the sample solution containing monoclonal antibody is continuously loaded to multiple parents respectively
In chromatographic column, make each affinity chromatography column overload, wherein contain a kind of affinity chromatography in each affinity column
Filler, and at least there are two the affinity chromatography filler in the affinity column is different；
The liquid that flows through of each affinity column is packed as multiple prepare liquids according to the sequencing penetrated；
Detect the concentration of monoclonal antibody described in each prepare liquid；
According to the concentration of monoclonal antibody described in the prepare liquid, determine various affinity chromatography fillers to the monoclonal
The Dynamic binding capacities of antibody；And
The affinity chromatography filler is screened according to the Dynamic binding capacities.
Sample solution containing monoclonal antibody is continuously loaded to more by the screening technique of the filler of above-mentioned affinity chromatography respectively
In a affinity column, make affinity chromatography column overload, and liquid will be flowed through according to the sequencing penetrated and be packed as multiple prepare liquids,
By comparing the concentration of the monoclonal antibody in prepare liquid, every kind of affinity chromatography filler can be quickly analyzed to the monoclonal antibody
Dynamic binding capacities, so that screening obtains being suitble to the affinity chromatography filler of the monoclonal antibody.In addition, entire screening process
In, one-time continuous loading can evaluate a kind of binding ability of affinity chromatography filler, and the dosage of monoclonal antibody is few, the consumption being related to
Material dosage is few.
It is added to the volume and the loading of the sample solution in each affine layer column in one of the embodiments,
The product of the mass concentration of monoclonal antibody described in liquid is greater than the intrinsic carrying capacity and the affine filler of the affinity chromatography filler
The product of volume.
The step of concentration of monoclonal antibody described in each prepare liquid of detection in one of the embodiments,
Include:
Each affinity chromatography filler is loaded on porous filter plate respectively, balances each affinity chromatography with equilibrium liquid
Filler obtains micropore chromatoplate；
It is corresponding that multiple prepare liquids of each affinity column are added to the micropore chromatoplate in equal volume respectively
Kong Zhong is obtained multiple to Incubating Solution；
Will be each described to Incubating Solution incubation, centrifugation and elution, obtain multiple eluents；And
It is anti-to obtain monoclonal described in each prepare liquid for the concentration for detecting monoclonal antibody described in each eluent
The concentration of body.
In one of the embodiments, it is described will it is each it is described be incubated for Incubating Solution, centrifugation, elution, obtain multiple eluents
The step of include:
Will be each described to Incubating Solution incubation, centrifugation, obtain multiple centrifugates；
The concentration of the monoclonal antibody in each centrifugate is detected, if the monoclonal in the centrifugate
The ratio of the concentration of the grand antibody of monomer described in the concentration of antibody and the sample solution is greater than 5%, then again by the centrifugate
Addition is incubated for into the corresponding hole of the micropore chromatoplate, is then centrifuged for, detects the concentration of the monoclonal antibody, until
The ratio of the concentration of the grand antibody of monomer described in the concentration of the monoclonal antibody in each centrifugate and the sample solution
Value is not more than 5%, obtains the micropore chromatoplate for being adsorbed with the monoclonal antibody；And
With elution buffer clean described in be adsorbed with the micropore chromatoplate of the monoclonal antibody, obtain the eluent.
The sample solution contains the monoclonal antibody of same concentrations in one of the embodiments, described according to institute
The concentration for stating monoclonal antibody described in prepare liquid determines every kind of affinity chromatography filler to the dynamic of the monoclonal antibody
The step of binding ability includes:
Calculate the corresponding loading total volume of each prepare liquid；And
The concentration and monoclonal antibody described in the sample solution for calculating monoclonal antibody described in each prepare liquid
Concentration ratio, under the identical ratio, the Dynamic binding capacities of the big affinity chromatography filler of the loading total volume
Greatly.
In one of the embodiments, in multiple prepare liquids of each affinity column, described at least one
The ratio of the concentration of monoclonal antibody in the concentration of the monoclonal antibody of prepare liquid and the sample solution is in 0.05~
Between 0.15.
The sample solution is the fermented liquid supernatant containing the monoclonal antibody in one of the embodiments,.
The upper of monoclonal antibody will be contained under identical flow velocity, retention time described in one of the embodiments,
Before the step of sample liquid is continuously loaded in multiple affinity columns respectively, makes each affinity chromatography column overload, further include
Following steps:
Confirm every kind of affinity chromatography filler to the static binding capacity of the monoclonal antibody.
Static state of the every kind of affinity chromatography filler of the confirmation to the monoclonal antibody in one of the embodiments,
The step of binding ability includes:
The monoclonal antibody is configured to the loading sample of various concentration；
Each affinity chromatography filler is loaded on porous filter plate in equal volume respectively, is balanced with affinity chromatography equilibrium liquid
Every kind of affinity chromatography filler, obtains micropore chromatoplate；
The loading sample of various concentration is added in equal volume on the micropore chromatoplate respectively, and be incubated for, from
The heart collects supernatant, detects the concentration of the monoclonal antibody in the supernatant；And
According to MAb concentration described in the supernatant of every kind of affinity chromatography filler, every kind of confirmation is described affine
Static binding capacity of the chromatographic stuffing to the monoclonal antibody.
The monoclonal antibody is configured to using gradient dilution method the loading of various concentration in one of the embodiments,
Sample.
Detailed description of the invention
Fig. 1 is to implement to penetrate number number and unbonded MAb concentration when sample concentration is 14.9mg/mL in 1
Account for sample concentration proportionate relationship figure；
Fig. 2 is to implement to penetrate number number and unbonded MAb concentration when sample concentration is 29.1mg/mL in 1
Account for sample concentration proportionate relationship figure；
Fig. 3 is to implement to penetrate number number and unbonded MAb concentration when sample concentration is 43.7mg/mL in 1
Account for sample concentration proportionate relationship figure；
Fig. 4 is to implement to penetrate number number and unbonded MAb concentration when sample concentration is 57.2mg/mL in 1
Account for sample concentration proportionate relationship figure；
Fig. 5 is to implement to penetrate number number and unbonded MAb concentration when sample concentration is 69.9mg/mL in 1
Account for sample concentration proportionate relationship figure；
Fig. 6 is to implement to penetrate number number and unbonded MAb concentration when sample concentration is 79.2mg/mL in 1
Account for sample concentration proportionate relationship figure；
Fig. 7 is to implement to penetrate number number and unbonded MAb concentration when sample concentration is 84.7mg/mL in 1
Account for sample concentration proportionate relationship figure；
Fig. 8 is to implement to penetrate number number and unbonded MAb concentration when sample concentration is 88.4mg/mL in 1
Account for sample concentration proportionate relationship figure；
Fig. 9 is the chromatography map of Resin D in embodiment 2；
Figure 10 is the chromatography map of Resin E in embodiment 2；
Figure 11 is the chromatography map of Resin B in embodiment 2；
Figure 12 be embodiment 2 in the monoclonal antibody of Resin D, Resin E and Resin B flowed through in liquid contain quantitative change
Change figure.
Specific embodiment
To facilitate the understanding of the present invention, a more comprehensive description of the invention is given in the following sections with reference to the relevant attached drawings.In attached drawing
Give section Example of the invention.But the invention can be realized in many different forms, however it is not limited to this paper institute
The embodiment of description.On the contrary, purpose of providing these embodiments is keeps the disclosure of invention more thorough and comprehensive.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.
The screening technique of the affinity chromatography filler of one embodiment, comprising the following steps:
S110, confirm every kind of affinity chromatography filler to the static binding capacity of monoclonal antibody.Preliminary screening is suitable for list
The affinity chromatography filler of clonal antibody.
Specifically, step S110 includes step S111~S117.
S111, micropore chromatoplate is prepared.
Specifically, each affinity chromatography filler is loaded on porous filter plate in equal volume respectively, with affinity chromatography equilibrium liquid
Every kind of affinity chromatography filler is balanced, micropore chromatoplate is obtained.
Further, every kind of affinity chromatography filler is prepared into plastic suspension, then by glue suspension bed board, cleaning and balance,
Obtain micropore chromatoplate.
Further, the operation for preparing glue suspension includes: that each affinity chromatography filler is saved liquid with filler respectively to mix.Tool
Body, filler saves the ethanol solution that liquid is volume fraction 10%~30%.Every kind of affinity chromatography filler and filler save liquid
The ratio between volume is 1:0.5~2.Preferably, filler saves the ethanol solution that liquid is volume fraction 20%.Affinity chromatography filler with fill out
It is 1:1 that material, which saves the ratio between volume of liquid,.
Further, the operation of bed board includes: that by every kind, the addition of the glue suspension made of affinity chromatography filler is porous respectively
In the different holes of filter plate.Specifically, the volume of the glue suspension of addition is 10 holes μ L/~40 holes μ L/.Preferably, the glue of addition is outstanding
The volume of liquid is 15 holes μ L/~25 holes μ L/.It is highly preferred that the volume for the glue suspension being added is 20 holes μ L/.
Further, the operation cleaned and balanced includes: first with ultrapure water, the filter completed again with equilibration buffer to bed board
Plate is cleaned.Further, it is cleaned 3~5 times with the filter plate that ultrapure water and equilibration buffer complete bed board, every time after cleaning
Removal clear liquid is directly filtered, last time is centrifuged after balancing, removes clear liquid, obtains micropore chromatoplate.In the present embodiment, balance is slow
Fliud flushing is 1*PBS (10mM PB, 150mM NaCl, pH7.4).Certainly, in other embodiments, equilibration buffer can also be with
It is selected according to the specification of affinity chromatography filler.
S113, the loading sample that monoclonal antibody is configured to various concentration.
Specifically, according to the intrinsic carrying capacity of every kind of affinity chromatography filler, the sample containing monoclonal antibody is configured different
The loading sample of concentration, to observe under various concentration each affinity chromatography filler to the absorption situation of the monoclonal antibody.Wherein, exist
The concentration that affinity chromatography filler can be made to overload is included at least in the concentration of loading sample.It is intrinsic that overload refers to that sample load is greater than
Carrying capacity.In other words, the quality for being added to the volume and monoclonal antibody in the sample solution of the sample solution in each affine layer column is dense
The product of degree is greater than the product of the volume of the intrinsic carrying capacity and affine filler of affinity chromatography filler in the affinity column.Certainly, this
When sample solution volume it is consistent with the unit of the volume of affine filler, the unit of the mass concentration of monoclonal antibody and intrinsic carrying capacity
Unanimously.Sample load is equal to the mass concentration of monoclonal antibody in loading sample and the product of loading volume divided by the affinity column
Packing volume.Intrinsic carrying capacity can be obtained according to the specification of affinity chromatography filler.The volume of affine filler can be according to addition
The accounting in glue suspension of volume and affinity chromatography filler of glue suspension calculate.
Because during the affinitive layer purification of monoclonal antibody, different affinity chromatography fillers is anti-to different monoclonals
The binding ability of body is different, and the effect of purifying is not also identical, so, the affinitive layer purification of monoclonal antibody, which needs to screen, to be suitble to
Affinity chromatography filler purified.
Further, monoclonal antibody is configured to the loading sample of various concentration using gradient dilution method.
Further, the loading sample of the various concentration after gradient dilution is subjected to protein concentration detection respectively, is obtained just
Beginning sample concentration.Specifically, the loading sample after gradient dilution is placed in porous UV without in the different holes of baffle respectively, used
Take3&96-well light path corrects the concentration of monoclonal antibody in A280 method (Epoch, BioTeK) detection loading sample, obtains
The initial sample concentration of loading sample.
S115, incubation.
Specifically, the loading sample of various concentration is added in equal volume on micropore chromatoplate respectively, is then incubated for.Further
Ground is incubated for using interval concussion mode.Further, it is shaken at 337.5rpm~675rpm revolving speed (its woods Bell, QB-9001)
30min~60min is swung, suspends 10s~60s, then shakes, suspends again, be repeated 3 times~4 times.
S117, the content for detecting monoclonal antibody.
Specifically, it will be incubated for the loading sample for the various concentration completed, centrifugation collects supernatant, then detects supernatant
The content of middle monoclonal antibody.By the content of monoclonal antibody in supernatant, monoclonal antibody and each affine layer are tentatively judged
Analyse the static binding capacity of filler.The monoclonal antibody of identical sample concentration, the content of monoclonal antibody is higher in supernatant,
Indicate that monoclonal antibody is lower with the static binding capacity of the affinity chromatography filler under the sample concentration, is more not suitable as pure
Change the affinity chromatography filler of this monoclonal antibody.
Further, the step of loading-incubation-centrifugation-detection monoclonal antibody content is repeated several times, until detecting
Data it is steady.Repetition loading-incubation-centrifugation-detection process is steady up to detection data, may thereby determine that different fillers
In the different adsorbances for being incubated for batch, and then its carrying capacity and absorption speed can be compared.
S130, according to the Dynamic binding capacities of affinity chromatography filler and monoclonal antibody, screening is suitable for monoclonal antibody
Affinity chromatography filler.
Specifically, step S130 includes step S131~S135.
S131, loading and collection flow through liquid.
Specifically, under identical flow velocity, retention time, the sample solution containing monoclonal antibody is continuously loaded to respectively
In multiple affinity columns, make each affinity chromatography column overload.Contain a kind of affinity chromatography filler in each affinity column, and
At least there are two the affinity chromatography filler in affinity column is different.Each affinity column is flowed through into liquid respectively according to penetrating
Sequencing be packed as multiple prepare liquids.
Further, first according to intrinsic carrying capacity, the loading of each affinity chromatography filler when monoclonal antibody concentration, calculate
Sample solution volume when overload.In each affinity column, the product of the concentration of monoclonal antibody in sample solution volume and sample solution
Greater than the intrinsic carrying capacity of affinity chromatography filler and the product of affine packing volume, so that affinity chromatography column overload.Certainly, at this time
Sample solution volume is consistent with the unit of affinity chromatography filler, the concentration of monoclonal antibody and the unit one of the intrinsic carrying capacity of affinity chromatography
It causes.Then stop loading when continuous loading to sample solution volume.It is collected since loading and flows through liquid, and is successive according to what is penetrated
Sequence is packed as multiple prepare liquids.Further, it collects and multiple stops close flowing through liquid as prepare liquid apart from loading.Progress one
Ground, Dan Ke in the concentration of the monoclonal antibody of at least one prepare liquid and sample solution in multiple prepare liquids of each affinity column
The ratio of the concentration of grand antibody is between 0.05~0.15.
Further, the concentration of monoclonal antibody is identical in sample solution.Further, sample solution can be including monoclonal
The fermented liquid supernatant of antibody is also possible to the solution prepared with monoclonal antibody sterling.
The concentration of monoclonal antibody in S133, each prepare liquid of detection.
Specifically, each affinity chromatography filler is loaded on porous filter plate respectively, is balanced with equilibrium liquid each described affine
Chromatographic stuffing obtains micropore chromatoplate.Then multiple prepare liquids of each affinity column are added to micropore chromatography in equal volume respectively
In the corresponding hole of plate, obtain multiple to Incubating Solution.Then it will respectively be incubated for, be centrifuged and elute to Incubating Solution, and obtain multiple eluents.
Then the concentration for detecting monoclonal antibody in each eluent obtains the concentration of monoclonal antibody in each prepare liquid.
Further, it will be respectively incubated for Incubating Solution, centrifugation, and obtain multiple centrifugates, detect the monoclonal in each centrifugate
The concentration of antibody, if the ratio of the concentration of the grand antibody of monomer is greater than in the concentration of the monoclonal antibody in centrifugate and sample solution
5%, then centrifugate is added into the corresponding hole of micropore chromatoplate again and is incubated for, is then centrifuged for, detects monoclonal antibody
Concentration, until the ratio of the concentration of the concentration and grand antibody of monomer in sample solution of the monoclonal antibody in each centrifugate is little
In 5%, the micropore chromatoplate for being adsorbed with monoclonal antibody is obtained.Then it is respectively washed that be adsorbed with monoclonal anti-with elution buffer
The different holes of the micropore chromatoplate of body, obtain multiple eluents.Then the concentration of the monoclonal antibody in each eluent is detected again,
Obtain the concentration of monoclonal antibody in each prepare liquid.
Further, the grand antibody of monomer in the concentration and sample solution for detecting the monoclonal antibody in each centrifugate
After the step of ratio of concentration is less than 5%, the micropore chromatoplate for being adsorbed with monoclonal antibody is respectively washed with elution buffer
Before the step of different holes, further include the steps that cleaning the micropore chromatoplate for being adsorbed with monoclonal antibody with equilibration buffer.Tool
Body, the micropore chromatoplate for being adsorbed with monoclonal antibody is cleaned with equilibration buffer, and filter abandoning supernatant, after being repeated 1 times~3 times
Continue to clean the micropore chromatoplate for being adsorbed with monoclonal antibody with equilibration buffer, is then centrifuged for discarding supernatant liquid.On
When sample liquid is the fermentation liquid containing monoclonal antibody, other foreign proteins are usually contained in fermentation liquid, at this point, clear with equilibration buffer
Wash be adsorbed with the micropore chromatoplate of monoclonal antibody can be except foreigh protein removing, convenient for the detection of monoclonal antibody in eluent.When
So, it is to be understood that, can be without clear with equilibration buffer if sample solution is the solution prepared with monoclonal antibody sterling
The step of washing the micropore chromatoplate for being adsorbed with monoclonal antibody.
Further, using the dense of the monoclonal antibody in Take3&96-well light path correction A280 method detection eluent
Degree.
S135, according to the concentration of monoclonal antibody in each prepare liquid, determine various affinity chromatography fillers to monoclonal antibody
Dynamic binding capacities.Affinity chromatography filler is screened according to Dynamic binding capacities.It is filled out with the affinity chromatography that Dynamic binding capacities are big
Expect the affinity chromatography filler as monoclonal antibody.
Specifically, the corresponding loading total volume of each prepare liquid is calculated；Calculate the dense of monoclonal antibody in each prepare liquid
The ratio of degree and the concentration of monoclonal antibody in sample solution；Wherein, under identical sample concentration, identical ratio, affine layer
The Dynamic binding capacities for analysing the big affinity chromatography filler of the corresponding loading total volume of filler are big.
Further, with the concentration of the concentration of monoclonal antibody in each prepare liquid and monoclonal antibody in corresponding sample solution
Ratio be ordinate, according to chronological order collect prepare liquid be abscissa draw monoclonal in every kind of affinity column
The content change diagram of antibody.According to the content change diagram of the monoclonal antibody of affinity chromatography filler, determine that every kind of affinity chromatography is filled out
Material is suitble to the affinity chromatography filler of the monoclonal antibody to monoclonal antibody binding ability, screening.
The screening technique of the filler of above-mentioned affinity chromatography has at least the following advantages:
(1) easy.Sample solution containing monoclonal antibody is continuously loaded in multiple affinity columns respectively, makes affine layer
Column overload is analysed, and liquid will be flowed through according to the sequencing penetrated and be packed as multiple prepare liquids, by comparing the Dan Ke in prepare liquid
The concentration of grand antibody can quickly analyze every kind of affinity chromatography filler to the Dynamic binding capacities of the monoclonal antibody, to sieve
Choosing obtains the affinity chromatography filler for being suitble to the monoclonal antibody.Especially, the monoclonal antibody contained in containing sample solution
Concentration is identical, the concentration of monoclonal antibody ratio identical with the ratio of the concentration of monoclonal antibody in sample solution in prepare liquid
Under, it can faster judge the Dynamic binding capacities size of each affinity chromatography filler, filter out suitable affinity chromatography
Filler.
(2) time is saved.Traditional screening technique needs primary multiple circular flows that could carry out to single chromatographic stuffing
Evaluation.At the initial stage of chromatographic stuffing screening, the evaluation to a variety of chromatographic stuffing retention times, the work of entire screening process can be related to
It measures quite huge.For example at least implement 3 circulations to every kind of chromatographic stuffing using 1mL system, totally five kinds of chromatographic stuffings wait sieving
Choosing, each loading are subject to 50mg/mL carrying capacity, then need 5*3 time=15 times circulations, need the completion of 3 days or so time, whole process
It is not only cumbersome, also quite expend the time.Compared to traditional screening technique, the screening technique knot of the filler of above-mentioned affinity chromatography
High-throughput techniques are closed, while running a variety of multiple experiment conditions of affinity chromatography filler, realize retention time under more filler many conditions
With evaluation process while combining carrying capacity, evaluation process is greatly simplified, and is made entirely to assess efficiency and is greatly improved, and operation is also very big
Simplify.
(3) sample and consumptive material are saved.Traditional screening technique recycles sample detections since every kind of chromatography media all needs to take turns more,
The consumption of sample is very huge.It is by above-mentioned each loading by taking 50mg/mL carrying capacity as an example, then estimated to consume 50mg/ * of sample
15 times=750mg, or sample stability more precious for sample is undoubtedly increased there are for the early development of certain risk
The time cost and material cost of entire process exploitation.And each experiment condition of screening technique of the filler of above-mentioned affinity chromatography
Loading sample volume minimum can reduce to 200 μ L, then only need 0.8mg albumen for 80mg/mL designs carrying capacity, entire 96
Orifice plate applied sample amount is less than 50mg, and sample consumption reduces by 10 times or more, and buffer consumption is also less, is a kind of extremely economic exploitation
Method.
(4) using traditional evaluation method, convection current during loading is needed to pierce into row repeatedly sampling, then institute in the process
Generate flow through collect sample number will up to hundred grades or more, then to each collections sample implement detect respectively, opened in purifying process
Hair initial stage, loading sample are often fermented feed liquid, this allows for that A280 method cannot be directlyed adopt when carrying out protein content detection,
And must first pass through and carry out A280 or HPLC detection process again after purification, for sample of the sample size at hundred grades or more,
Brought detection pressure and challenge as one can imagine.The screening technique of the filler of above-mentioned affinity chromatography is collected into evaluation process
Multiple samples that flow through can disposably carry out antibody content evaluation, can by high throughput detect, no longer exist detection pressure, it is right
It is a kind of process development method of fast, economical for the antibody process exploitation remained high in time and economic cost.
Specific embodiment
Using 96 hole filter plates (Pall) and corresponding sealing plate film, concussion shaking table (its woods Bell), Epoch (BioTeK, band
Take3 microwell plate), 96 hole UV without baffle (Corning), vacuumizing assembly (Pall), be concentrated by ultrafiltration pipe (Millipore),
Stopwatch etc. carries out high-throughput antibody content detection, when being directed to chromatography with 1*PBS (10mM PB, 150mM NaCl,
It pH7.4) is equilibration buffer (Buffer A) that with 20mM NaAc, pH3.4 is elution buffer (Buffer B1), with 0.1M
NaOH (Buffer B2) is cleaning buffer solution to carry out.
Embodiment 1
(1) sample pretreatment and the dilution of gradient concentration sample: 80mg PD1 (programmed cell death is taken
Protein 1) monoclonal antibody (self-produced, Fei Peng bio-pharmaceuticals limited liability company of Shenzhen), it is managed through being concentrated by ultrafiltration
(Millipore,Ultra 15mL Centrifugal Filters, MWCO:30KDa) buffering displacement is carried out,
Isometric batch operation 5 times or more (it completes, changes liquid into 1*PBS, A280 method is corrected using Take3& 96-well light path, it is quasi-
Sample concentration after liquid is changed in really detection concentration, by Take3A280 result by its accurate dilutions to 4mg/mL.
(2) gradient concentration sample dilutes: being that 4mg/mL is denoted as SPL1, as carrying capacity 80mg/ by the concentration of monoclonal antibody
ML sample, the volume of SPL1 are 11mL.2.625mL SPL1 is taken, 375 μ L PBS are added, mixes, is denoted as SPL2, as carrying capacity
70mg/mL sample.2.25mL SPL1 is taken, 75 μ L PBS are added, mixes, is denoted as SPL3, as carrying capacity 60mg/mL sample.It takes
1125 μ L PBS are added in 1.875mL SPL1, mix, are denoted as SPL4, as carrying capacity 50mg/mL sample.3mL SPL1 is taken, is added
3mL PBS mixes, is denoted as SPL5, as carrying capacity 40mg/mL sample.1.125mL SPL1 is taken, 1875 μ L PBS are added, is mixed,
It is denoted as SPL6, as carrying capacity 30mg/mL sample.3mL SPL5 is taken, 3mL PBS is added, mixes, is denoted as SPL7, as carrying capacity
20mg/mL sample.3mL SPL7 is taken, 3mL PBS is added, mixes, is denoted as SPL8, as carrying capacity 10mg/mL sample.
(3) prepared by glue suspension: 5 15mL centrifuge tubes are taken, 5 kinds of affinity chromatography fillers are respectively charged into, the affinity chromatography filler
It is respectively derived from GE MabSelect SuRe (being hereafter denoted as " Resin A "), MabSelect SuRe LX (is hereafter denoted as
" Resin B "), Merck Eshmuno A (being hereafter denoted as " Resin C "), Tosoh TOYAPEARL AF-rProtein A
HC-650F (being hereafter denoted as " Resin D "), BestChrom AT Protein A Diamond) (hereafter it is denoted as " Resin
E "), the volume of every kind of affinity chromatography filler is 2mL, is centrifuged (175g*5min) after trim.3min~5min is stood, according to centrifugation
Scale on pipe is sucked out or continuously adds the 20%EtOH of certain volume, makes the volume of affine chromatographic stuffing in final each centrifuge tube
Concentration is 50%, is resuspended uniformly, obtains glue suspension.
(4) bed board: after the glue suspension of 50% gum concentration of step (3) is resuspended with the volley of rifle fire, according to position shown in table 1, with
(20 hole μ L/ glue suspensions correspond to the affinity chromatography filler in 10 holes μ L/ in 20 holes μ L/ addition corresponding 96 hole filter plate (Pall)
Volume).
Table 1
(5) clean and balance: first ultrapure water uses 1*PBS (10mM PB, 150mM NaCl, pH7.4) equilibration buffer again
All holes for the 96 hole filter plates that (Buffer A) completes the bed board that step (4) obtains are clear every time with the hole 0.2mL/ cleaning 3 times
Removal clear liquid is directly filtered after washing, clear liquid is removed in centrifugation after last time balances, and obtains 96 hole filter plates.
(6) it is right without baffle (Corning) that SPL1~SPL8 loading: is transferred to 96 hole UV of cleaning according to 230 holes μ L/
It answers in hole, 96well light path correction method A280 detection obtains initial loading sample concentration.Then by the SPL1 in 96 orifice plates~
SPL8 sample is transferred in the 96 hole filter plates that step (5) obtains with the volley of rifle fire with 200 holes μ L/.
(7) it is incubated for and flows through antibody content detection: being shaken under 675rpm revolving speed, so that sample and filler can mix well,
Suspend after being incubated for 30min, 150g*5min centrifugation, the supernatant after collecting centrifugation, with 96-well light path correction method &
Take3A280 method measures the concentration that albumen is not associated in supernatant, checks and is not associated with albumen situation in supernatant.Then, continue
The supernatant loading that will be collected into, same to be incubated for, concussion (concussion shaking table, woods Bell, QB-9001) under 675rpm revolving speed,
Suspend after 30min, 150g 5min centrifugation, supernatant is collected by centrifugation in collection, with 96-well light path Jiao Zhengfa &Take3A280 method
Concentration mensuration is carried out, checks and is not associated with albumen situation in supernatant.It repeats above-mentioned incubation-loading-Concentration Testing process totally five times,
Gained supernatant is denoted as FT1~FT5 (penetrating number number) respectively.It is drawn according to albumen situation is not associated in each supernatant
MAb concentration accounts for the concentration ratio of monoclonal antibody in loading sample in each chromatographic stuffing, as a result such as FIG. 1 to FIG. 8 institute
Show.
Through detecting, the initial sample concentration of SPL8~SPL1 is respectively 0.749mg/mL, 1.455mg/mL, 2.188mg/
ML, 2.863mg/mL, 3.496mg/mL, 3.963mg/mL, 4.238mg/mL and 4.423mg/mL, sample load are equal to initial
Divided by affinity chromatography packing volume, the corresponding practical carrying capacity of SPL8~SPL1 is respectively the product of sample concentration and loading volume
14.9mg/mL、29.1mg/mL、43.7mg/mL、 57.2mg/mL、69.9mg/mL、79.2mg/mL、84.7mg/mL、
88.4mg/mL。
By FIG. 1 to FIG. 8 it is found that Resin E and Resin D are shown stronger under the conditions of multiple sample concentrations
Adsorption capacity, indication Resin E and Resin D have high adsorption efficiency.Resin B is to inhale in < 60mg/mL carrying capacity
Attached most slow filler, Resin B have change in high carrying capacity.Resin C generally joint efficiency is below other fillers.Resin
B, Resin D has higher static binding capacity, followed by Resin E, the static binding capacity phase of Resin A and Resin C
To more lower.
Embodiment 2
To the 1mL column of Resin B affinity chromatography filler, following step is carried out respectively:
(1) sample pump is pre-filled with sample solution: Buffer, loading mouth S, A1 being cleaned with Buffer A in advance, and low
Flow velocity, chromatographic column, which are set, is carefully pumped into sample under By-pass state sample pump S, until ultraviolet appearance, no longer pumps loading and carry out
Wash, while wash again is pumped to A, make in sample pump full of to loading feed liquid.(2) retention time=3min is set, with
The continuous loading of 0.33mL/min flow velocity uses 96 orifice plates to be collected since the loading with 1.5mL/ pipe, loading wherein flowing through liquid
Stop loading to setting volume (33.13mL), pumps the rinse of Buffer pipeline and Sample pump wash without sample.Its
In, using it is above-mentioned be collected into flow through and be pushed forward 30 samples from after end of the sample as prepare liquid, number is respectively corresponded after going to certainly
For Resin B FT1~FT30.Then conventional method elutes affinity column.
(3) step (1)~(2) are also carried out respectively to the 1mL column of Resin D and Resin E both affinity chromatography fillers
Corresponding operation, the liquid that flows through being collected into are pushed forward 30 samples from after end of the sample as corresponding prepare liquid, divide after going to certainly
Other reference numeral is Resin D FT1~FT30, Resin E FT1~FT30.
(4) antibody content high throughput assay in prepare liquid: by (4)~(5) the step of 96 empty hole filter plates reference embodiment 1
It carries out bed board, cleaning and balance and obtains the 96 hole filter plates for measuring monoclonal antibody in prepare liquid.Wherein, 96 hole filter plates
Position is arranged as shown in table 2 (not setting multiple holes).Then 20 μ L steps (2) are obtained respectively flowing through liquid Resin B FT1~FT30
Corresponding be added of liquid is flowed through with Resin D FT1~FT30, the Resin E FT1~FT30 that step (3) obtains to flow through for measuring
96 hole filter plates of antibody in liquid.Wherein, Protein A affinity elution sample (oneself of 1.2mg/mL, 0.6mg/mL, 0.1mg/mL
Produce, Fei Peng bio-pharmaceuticals limited liability company of Shenzhen) it is positive control.Then it is shaken using 675rpm revolving speed, every 10min
Suspend 15s, be incubated for 2h, 150g*5min is centrifuged later, penetrates the list being not associated in liquid using 96-well light path correction method measurement
The concentration of clonal antibody.If penetrate in liquid be not associated with monoclonal antibody concentration in loading sample the monoclonal antibody it is dense
The ratio of degree be greater than 5%, then repeatedly loading and continue be incubated for 1h in the same way.Then 96 holes of monoclonal antibody will be combined with
Filter plate carries out subsequent cleaning, elution, CIP, detection, obtains the concentration of monoclonal antibody in each prepare liquid.
Table 2
According to content, volume and the correspondence of the monoclonal antibody in each prepare liquid of Resin B, Resin D and Resin E
Loading total volume, calculate the ratio of the concentration of monoclonal antibody in the concentration of monoclonal antibody and sample solution in each prepare liquid
Value, and draw the content change diagram for flowing through the monoclonal antibody in liquid of every kind of affinity chromatography filler.
As a result: the chromatography map of Resin D, Resin E and Resin B are respectively as shown in Fig. 9~Figure 11.Fig. 9~Figure 11
In, UV1_280 indicates the UV absorption (mAU) under 280nm, and Cond indicates conductivity (mS/cm), and pH indicates pH value.Resin
E, the changes of contents for flowing through the monoclonal antibody in liquid of Resin D and Resin B is as shown in figure 12.
As shown in Figure 12, when retention time is 3min, the DBC of ResinD, ResinE10%It is higher.About 70mg monoclonal
Antibody/mL filler, and the DBC of Resin B10%It is lower, about 31mg monoclonal antibody/mL filler.
Therefore, ResinD, ResinE be more suitable under the retention time desired monoclonal antibodies affinity chromatography it is pure
Change filler.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of screening technique of affinity chromatography filler, which comprises the following steps:
Under identical flow velocity, retention time, the sample solution containing monoclonal antibody is continuously loaded to multiple affine layers respectively
It analyses in column, makes each affinity chromatography column overload, contain a kind of affinity chromatography filler in each affinity column, and extremely
It is few that there are two the affinity chromatography filler in the affinity column is different；
The liquid that flows through of each affinity column is packed as multiple prepare liquids according to the sequencing penetrated；
Detect the concentration of monoclonal antibody described in each prepare liquid；
According to the concentration of monoclonal antibody described in the prepare liquid, determine various affinity chromatography fillers to the monoclonal antibody
Dynamic binding capacities；And
The affinity chromatography filler is screened according to the Dynamic binding capacities.
2. screening technique according to claim 1, which is characterized in that be added on described in each affine layer column
The volume of sample liquid and the product of the mass concentration of monoclonal antibody described in the sample solution are greater than the intrinsic of the affinity chromatography filler
The product of carrying capacity and the volume of the affine filler.
3. screening technique according to claim 1, which is characterized in that Dan Ke described in each prepare liquid of detection
The step of concentration of grand antibody includes:
Each affinity chromatography filler is loaded on porous filter plate respectively, each affinity chromatography is balanced with equilibrium liquid and fills out
Material, obtains micropore chromatoplate；
Multiple prepare liquids of each affinity column are added in equal volume in the corresponding hole of the micropore chromatoplate respectively,
It obtains multiple to Incubating Solution；
Will be each described to Incubating Solution incubation, centrifugation elution, obtain multiple eluents；And
The concentration for detecting monoclonal antibody described in each eluent obtains monoclonal antibody described in each prepare liquid
Concentration.
4. screening technique according to claim 3, which is characterized in that it is described by it is each it is described to Incubating Solution be incubated for, centrifugation wash
De-, the step of obtaining multiple eluents, includes:
Will be each described to Incubating Solution incubation, centrifugation, obtain multiple centrifugates；
The concentration of the monoclonal antibody in each centrifugate is detected, if the monoclonal antibody in the centrifugate
Concentration and the sample solution described in monoclonal antibody concentration ratio be greater than 5%, then the centrifugate is added again
To being incubated in the corresponding hole of the micropore chromatoplate, it is then centrifuged for, detects the concentration of the monoclonal antibody, until each
The ratio of the concentration of the grand antibody of monomer described in the concentration of the monoclonal antibody in the centrifugate and the sample solution is not
Greater than 5%, the micropore chromatoplate for being adsorbed with the monoclonal antibody is obtained；And
With elution buffer clean described in be adsorbed with the micropore chromatoplate of the monoclonal antibody, obtain multiple eluents.
5. screening technique according to claim 1, which is characterized in that the sample solution contains the Dan Ke of same concentrations
Grand antibody, the concentration of the monoclonal antibody according to the prepare liquid determine every kind of affinity chromatography filler to institute
The step of stating the Dynamic binding capacities of monoclonal antibody include:
Calculate the corresponding loading total volume of each prepare liquid；And
Calculate the dense of the concentration of monoclonal antibody described in each prepare liquid and monoclonal antibody described in the sample solution
The ratio of degree, under the identical ratio, the Dynamic binding capacities of the big affinity chromatography filler of the loading total volume are big.
6. screening technique according to claim 1, which is characterized in that each affinity column it is multiple described to be measured
In liquid, the concentration of the monoclonal antibody of at least one prepare liquid and the concentration of the monoclonal antibody in the sample solution
Ratio be in 0.05~0.15 between.
7. screening technique according to claim 1, which is characterized in that the sample solution is to contain the monoclonal antibody
Fermented liquid supernatant.
8. screening technique according to any one of claims 1 to 7, which is characterized in that described in identical flow velocity, guarantor
It stays under the time, the sample solution containing monoclonal antibody is continuously loaded in multiple affinity columns respectively, makes each parent
It is further comprising the steps of before the step of chromatography column overload:
Confirm every kind of affinity chromatography filler to the static binding capacity of the monoclonal antibody.
9. screening technique according to claim 8, which is characterized in that every kind of affinity chromatography filler of the confirmation is to institute
The step of stating the static binding capacity of monoclonal antibody include:
The monoclonal antibody is configured to the loading sample of various concentration；
Each affinity chromatography filler is loaded on porous filter plate in equal volume respectively, balances every kind with affinity chromatography equilibrium liquid
The affinity chromatography filler, obtains micropore chromatoplate；
The loading sample of various concentration is added in equal volume on the micropore chromatoplate respectively, and is incubated for, be centrifuged, received
Collect supernatant, detects the concentration of the monoclonal antibody in the supernatant；And
According to MAb concentration described in the supernatant of every kind of affinity chromatography filler, every kind of affinity chromatography is confirmed
Static binding capacity of the filler to the monoclonal antibody.
10. screening technique according to claim 9, which is characterized in that use gradient dilution method by the monoclonal antibody
It is configured to the loading sample of various concentration.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111521813A (en) * | 2020-03-20 | 2020-08-11 | 天德瑞(北京)生物科技有限公司 | Preparation method of green fluorescent protein fusion protein immunoaffinity column, immunoaffinity column and application thereof |
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| CN114832439A (en) * | 2022-06-07 | 2022-08-02 | 杭州奕安济世生物药业有限公司 | Method for automatically controlling sample loading capacity of continuous chromatography and chromatography method |
| CN115819608A (en) * | 2022-10-08 | 2023-03-21 | 盛禾(中国)生物制药有限公司 | Method for purifying fusion protein |
| CN114280208B (en) * | 2021-11-03 | 2024-05-24 | 鼎康(武汉)生物医药有限公司 | Method for measuring dynamic load of affinity chromatography packing |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111521813A (en) * | 2020-03-20 | 2020-08-11 | 天德瑞(北京)生物科技有限公司 | Preparation method of green fluorescent protein fusion protein immunoaffinity column, immunoaffinity column and application thereof |
| CN111529563A (en) * | 2020-05-26 | 2020-08-14 | 浙江府衡生物科技有限公司 | Method for preparing product component for treating and preventing gastric ulcer, gastritis and other gastropathy and product |
| CN114280208A (en) * | 2021-11-03 | 2022-04-05 | 鼎康(武汉)生物医药有限公司 | Method for measuring dynamic loading capacity of affinity chromatography filler |
| CN114280208B (en) * | 2021-11-03 | 2024-05-24 | 鼎康(武汉)生物医药有限公司 | Method for measuring dynamic load of affinity chromatography packing |
| CN114832439A (en) * | 2022-06-07 | 2022-08-02 | 杭州奕安济世生物药业有限公司 | Method for automatically controlling sample loading capacity of continuous chromatography and chromatography method |
| CN115819608A (en) * | 2022-10-08 | 2023-03-21 | 盛禾(中国)生物制药有限公司 | Method for purifying fusion protein |
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Address after: 523808 Room 301, building 10, No.1 Taoyuan Road, Songshanhu Park, Dongguan City, Guangdong Province Patentee after: Guangdong Feipeng Pharmaceutical Co.,Ltd. Address before: 518000 Room 201, building A, No. 1, Qian Wan Road, Qianhai Shenzhen Hong Kong cooperation zone, Shenzhen, Guangdong (Shenzhen Qianhai business secretary Co., Ltd.) Patentee before: FAPON BIOTECH Inc. |