CN107746424A - A kind of Bioconjugation method of IgG4 antibody - Google Patents
A kind of Bioconjugation method of IgG4 antibody Download PDFInfo
- Publication number
- CN107746424A CN107746424A CN201711032283.8A CN201711032283A CN107746424A CN 107746424 A CN107746424 A CN 107746424A CN 201711032283 A CN201711032283 A CN 201711032283A CN 107746424 A CN107746424 A CN 107746424A
- Authority
- CN
- China
- Prior art keywords
- antibody
- igg4
- product
- igg4 antibody
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
Abstract
The present invention discloses a kind of Bioconjugation method of IgG4 antibody, including：Step 1, reduction：IgG4 is reduced, makes 4 pairs of interchain disulfide bonds in gG4 antibody fully open；Step 2, partial oxidation：By, by 4 pairs of fully open interchain disulfide bond partial oxidations, the free sulfhydryl groups of obtained gG4 antibody reach the average coupling number 4 ± 1 of target in IgG4 antibody；Step 3, coupling reaction：Bioconjugation is carried out using the free sulfhydryl groups and connexon conjugate compound of lgG4 antibody；The purifying of step 4, Bioconjugation product：The Bioconjugation product that step 3 obtains obtains end-product by removing unnecessary coupling compound.There is coupled product mixture made from the method for the present invention more preferable product to be distributed, connect the coupled product content that number is 0 and be less than 5%, connect the coupled product content that number is 8 and be less than 15%, obtained coupled product mixture may be directly applied to animal or clinical test after general purification.
Description
Technical field
The present invention relates to the Bioconjugation technical field of bio-pharmaceuticals industry, more particularly, to one kind optionally by
Interchain disulfide bond in IgG4 antibody, and the technology being coupled with medicine is allowed to, and its at antibody-drug conjugates (ADC)
Application in production.
Background technology
Bioconjugate such as ADC (antibody-drug conjugates), which is that one kind is extremely potential, is applied especially to tumor target
To the biological agent for the treatment of.From a structural point, ADC can be divided into three parts：Antibody, connexon and lps molecule.Antibody
It is responsible for targets identification tumor cells, lps molecule is responsible for killing tumour cell, and connexon is then the bridge that medicine is connected with antibody
Beam, the stabilizations of ADC in vivo were both necessary to ensure that, and had needed to make lps molecule effectively to play work after tumour cell is reached again
With.Different from traditional chemotherapy, ADC utilizes the interaction of antibody-antigene, efficiently by the antineoplastic with high activity
It is delivered in tumor tissues to targeting, the effect for killing tumour cell is reached by directionally inside tumor cells release medicine.
Meanwhile the drug concentration that ADC can also make tumor tissues local is much higher than normal tissue site.Thus, ADC can be with specific killing
Tumour cell is without to normal affecting cells, the treatment for improving medicine is specific.With the high speed development of molecular biology,
ADC is applied not only to the clinical treatment of tumour, also begins to be applied to other fields.
ADC coupling can be divided into two kinds of site-directed coupling and non-site-directed coupling at present, and ADC site-directed couplings are to develop in recent years
That gets up is a kind of by way of special reaction group is introduced in antibody so as to realize coupling.Site-directed coupling can be compared
Single product, but more complicated technique is usually needed due to needing to transform antibody, and in coupling, thus
Final production cost also can be higher.
The non-site-directed couplings of traditional ADC are then that existing active reaction functional group is coupled in itself using antibody, generally
There is two ways.First way is using the amino on lysine residue, with active function groups (such as succinyl on connexon
Imines ester) reaction, so as to which lps molecule is coupled into antibody up.Because multiple lysines, at least 20- be present in antibody surface
The lysine residue of 30 antibody surfaces possesses the possibility of reaction, thus its connection number of the ADC obtained by this method is different
Matter is very strong, generally averagely connects number (DAR) control in 3.5 ADC, the connection number of antibody is at 0 to 10.Second
Kind mode is then to obtain free cysteine by the interchain disulfide bond gone back in original antibody, then recycles and has on cysteine
The sulfydryl for having reactivity is coupled, and the ADC products that this mode obtains are still a mixing with a variety of connection numbers
Thing.
For the second way using the coupling for reducing interchain disulfide bond progress, due to the interchain disulfide bond number in antibody
Limited (such as IgG1 and IgG4 only have four pairs of cystine linkages), its maximum number of connections is controllable.But in actual production, pass through this
The ADC with maximum number of connections that kind coupling mode obtains generally is unstable in vivo, it is difficult to plays ADC effect；Separately
On the one hand, ADC effect can be weakened by being not connected with the antibody of medicine.Therefore, in order to ensure ADC validity and security, in reality
Often required that in the production of border and ADC mixtures are obtained by this coupling mode, and the antibody of medicine is not connected with ADC mixtures
And the ADC components that connection number is higher, it should all be maintained at as far as possible than relatively low level.
For IgG1 antibody, prior art is directly to carry out partial reduction to interchain disulfide bond using reducing agent, then
Directly it is coupled, it is possible to obtain more satisfactory ADC.But for IgG4 antibody, due to its structure and IgG1
Difference, it is coupled according to identical partial reduction mode, resulting average connection number identical ADC is unsatisfactory, its
In the ADC contents of the antibody that is not connected with and high connection number it is all very high, it is impossible to directly application, resulting IgG4-ADC are necessary
By being further separated off the ADC of not connected antibody and high connection number, animal or clinic just can apply to.Increase
Isolate and purify operation, not only substantially reduce antibody starting material utilization rate, also add processing step, production technology is become more
Add complexity, add cost.
Therefore, need to develop a kind of new method being coupled using IgG4 antibody interchain disulfide bonds for IgG4 antibody
To obtain preferable ADC, make to solve the antibody being not connected with product during coupling and the high ADC contents for being connected number are inclined
The problem of high, be not required to further to be separated off not connected antibody and high connection number ADC cans be applied to animal or
Clinic, so as to reach the utilization rate for improving antibody starting material, production process is become more simple, be saved greatly the skills such as cost
Art effect.
The content of the invention
The technical problem to be solved by the invention is to provide it is a kind of be coupled using IgG4 antibody interchain disulfide bonds it is new
Method, the antibody being not connected with the ADC obtained after being coupled by this method is low with the high ADC contents levels for being connected number, be not required into
One step is separated off not connected antibody and the ADC cans of high connection number are applied to animal or clinic.
A kind of in order to solve the above technical problems, side being coupled using IgG4 antibody interchain disulfide bonds provided by the invention
Method, including：
Step 1, reduction：4 pairs of interchain disulfide bonds in IgG4 antibody are reduced, open more than 80% interchain disulfide bond.
There are 4 pairs of interchain disulfide bonds in IgG4 antibody, it is general to reduce IgG4 with excessive reducing agent, make in reaction raw materials IgG4 antibody
More than 80% interchain disulfide bond is able to fully open.
The terminal of reaction can be by monitoring, to disconnect gained light chain after interchain disulfide bond the methods of high performance liquid chromatography
And the content of heavy chain accounts for IgG4 antibody total amounts more than 80% to reach reaction end.For example the reversed phase high performance liquid of PLRP posts can be used
Phase chromatogram detects, testing result：Light chain be added with heavy chain peak area shared by antibody total peak area more than 80%.Reach reaction eventually
During point, reaction product is 4 pairs of interchain disulfide bonds by Restore All or the IgG4 antibody of partial reduction, typically be can't detect in chromatogram
Complete unreduced IgG4 antibody is in itself (as shown in Figure 4).
When carrying out reduction reaction, IgG4 antibody is mixed and in suitable temperature in buffer solution with reducing agent with proper ratio
Lower culture so that the interchain disulfide bond in IgG4 antibody is opened, and retains the intrachain disufide bond in antibody.The source of IgG4 antibody
Including people source, mouse source and other.
Used reducing agent can be that the reducing power commonly used in any biochemical test is suitably enough to make IgG4 antibody
In 4 pairs of interchain disulfide bonds be able to the reducing agent opened.It is different according to the degree of reduction reaction, it should at least make more than 80%
IgG4 antibody interchain disulfide bond is opened, and more excellent reaction effect is that the IgG4 antibody interchain disulfide bond for making 90%-100% is beaten
Open.
Reducing agent includes but is not limited to commonly used in the prior art three (2- carboxyethyls) phosphines (TCEP), dithiothreitol (DTT)
(DTT) etc..
Reducing agent can determine that generally 10-100 equivalents, preferably 10-50 work as using equivalent with real reaction effect
Amount, such as the TCEP using 10,20,30,40 or 50 equivalents.
Also can open the interchain disulfide bond of antibody, still reservation intrachain disufide bond is advisable the temperature of reduction reaction, typically
Reaction temperature is 4-37 DEG C, reaction time 5-24h, for example is reduced 18 hours in 37 DEG C of water-bath.One in reduction reaction
As should not influence intrachain disufide bond in antibody light and heavy chain.
Step 2, partial oxidation：The interchain disulfide bond being opened in IgG4 antibody is subjected to partial oxidation with oxidant, obtained
To IgG4 antibody have suitable quantity and be available for coupling free sulfhydryl groups.
Using suitable oxidant, appropriate time, portion are incubated in proper proportions in buffer solution with the antibody being reduced
Divide the interchain disulfide bond that has been opened closed in antibody, free sulfhydryl groups number is reached target and be averagely coupled number.The target is averagely even
The value range for joining number is 4.0 ± 1.0, and more preferable value range is 4.0 ± 0.5.
The terminal of reaction can be by monitoring the methods of high performance liquid chromatography, and the interchain being opened with 45%-55% is double
Sulfide linkage re-forms as reaction end.For example the RPLC of HIC posts can be used to detect, to the antibody after oxidation
With conjugate detect after quick coupling, testing result：It is 4.0 ± 1.0 to be averagely coupled number.
Used oxidant can be that the oxidability commonly used in any biochemical test can suitably make in IgG4 antibody
The oxidant that the interchain disulfide bond being opened is oxidized and closes, such as oxidant can be only done the weak of partial oxidation for excessive
Oxidant, or middle strong oxidizer of enough completion partial oxidations of limitation etc..Oxidant includes but is not limited to dehydrogenation Vitamin C
Sour (DHAA), 5,5'- bis- thiobis (2- nitrobenzoic acids) (DTNB), NADH (NAD), nicotinoyl amine gland
Purine dinucleotides phosphoric acid (NADP) etc..
Oxidation reaction is averagely coupled number to form appropriate interchain disulfide bond, so as to reaching target to be advisable.It is for this purpose, anti-
Cosolvent in buffer solution system, the ratio of oxidant and antibody, oxidizing temperature, oxidization time and reaction system used in answering
Addition can adjust.
The ratio of oxidant and antibody is generally (2-100)：1, including but not limited to 2:1、4:1、5:1、6:1、7:1、
8:1、10:1、20:1、40:1、60:1、70:1、100:1 etc..
The temperature of oxidation reaction is generally 4-37 DEG C, is advisable with 4-10 DEG C, can be incited somebody to action for some antibody for being difficult to aoxidize
Reaction temperature is improved to room temperature or 37 DEG C.
The time of oxidation reaction should be advisable with closing suitable number of free sulfhydryl groups, and oxidization time is generally at least 3 hours,
For example can be even to stay overnight for 3 hours, 4 hours, 5 hours.
The organic solvent of proper proportion can be also added in oxidation system to help the generation of oxidation reaction.
Between step 1 and step 2, it is also an option that the buffer exchange step of increase by one of property, possible unnecessary to remove
Reducing agent.Reducing agent unnecessary in reaction system is removed by the way of buffer exchange, while completes buffer exchange, is made
Reaction system is applied to carry out the oxidation reaction of step 2 and the coupling reaction of subsequent step.The mode of buffer exchange includes
But it is not limited to carry out the displacement of buffer solution using super filter tube, tangential flow apparatus, bag filter, or desalting column.Replace to aoxidize
Include but is not limited to PBS (pH6.5, pH7.0, pH8.0) etc. with the buffer solution of coupling.
Step 3, coupling reaction：Biology is carried out using the free sulfhydryl groups and connexon-conjugate compound of lgG4 antibody occasionally
Connection.
By the IgG4 antibody that the part interchain disulfide bond that connexon-conjugate compound and step 2 obtain is opened suitable
It is coupled under the conditions of conjunction, obtains antibody coupling matter product (ADC).
The coupled product formula of IgG4 antibody and conjugate is IgG4- (C-L-D) n, wherein, IgG4 represents the hypotype of antibody
For IgG4 antibody；C represents the active function groups of the sulfydryl reaction with dissociating in IgG4 antibody；L represents connection antibody and coupling
The connexon of thing；D expressions and the attachment with biological function, the including but not limited to medicine with antitumor activity, resist
Medicine, fluorescein, polypeptide, nucleotide fragments or protein of inflammatory reaction etc.；N is the average coupling number of coupled product, this
N span should be 4.0 ± 1.0, more preferably 4.0 ± 0.5 in invention.
Coupling reaction betides an active reaction functional group of the free sulfhydryl groups and connexon on lgG4 antibody.The function
Roll into a ball with the stronger ability with sulfydryl reaction, including but not limited to maleimide, α-halogenatedacetamide, α-halogenatedketone,
Alpha-halogen ester, halogeno-benzyl compound, alkenyl sulfone etc..
It is connected between conjugate and antibody typically by a connexon, usual this kind of connexon can be divided into and can be broken
Type and can not breaking type.
The connexon that can not be broken directly can be made up of carbochain, or the middle PEG etc. that adds improves water miscible function
Group, or some structures are added to increase the steric hindrance of connexon.
The connexon that can be broken can then be disconnected by various cut-out modes, two such as disconnected using intracellular reproducibility environment
Sulfide linkage connexon, the connexon for including hydrazone key disconnected using intracellular lysosomal acid environment, can be by the enzyme in lysosome
Dipeptides connexon of cut-out etc..Conjugate includes but is not limited to the micromolecular compound of belt lacing, polypeptide or protein.
Conjugate can be with compared with strong cytotoxicity small molecule, such as tubulin polymerization inhibitor such as maytansine or
MMAE, DNA synthetic inhibitor such as SN-38 or multi-kanamycin, or the compound such as calicheamicin or PBD bis- for causing DNA to damage
Aggressiveness etc..Other conjugates then include but is not limited to cytotostatic agent, immunodepressant or antibiotic etc..
The detailed process of coupling reaction is, it would be desirable to the conjugate of progress mixes with by the IgG4 antibody of partial oxidation,
A certain proportion of organic solvent can be added to help to dissolve.The organic solvent of addition includes but is not limited to DMA, DMF, DMSO etc.
There is the solvent of relatively good compatibility with water, the additional proportion of organic solvent is not premised on destroying antibody structure, Ke Yiwei
5%, 10%, 15% and 20% etc..
The carry out temperature and time of coupling reaction can adjust according to reaction complexity.Temperature is generally 4 DEG C -37 DEG C,
Can be 4 DEG C, room temperature or 37 DEG C etc.；Reaction time is generally at least 0.5 hour, can be 0.5 hour, 1 hour, 2 hours
Etc..Excessive conjugate should be added during coupling reaction, free sulfydryl is all coupled, makes to be not present in reaction product and dissociates
Sulfydryl.Coupling reaction terminal can be detected by HIC-HPLC, should be substantially without odd number coupling peak in HIC-HPLC.
It is usually mixture to be coupled obtained product, on the premise of average connection number is 4, the idol that is obtained by step 3
Co-product, most of article all concentrate on the product that connection number is 4, and the ADC contents that not connected antibody and height connect number are low,
Thus product has preferably distribution.
After the oxidation reaction of step 2 is completed, it can directly carry out the Bioconjugation of step 3 or be removed by purifying
Bioconjugation is carried out again after oxidant.
The purifying of step 4, Bioconjugation product：The Bioconjugation product that step 3 obtains is by removing unnecessary coupling
Compound, that is, obtain end-product.
When carrying out the purifying unnecessary coupling compound of removal, the exchange of buffer solution can also be realized simultaneously.Bag can be used
Include but be not limited to following means：Buffer-exchanged is carried out using desalting column, buffer-exchanged is carried out using rotation super filter tube, thoroughly
Analysis or cross-flow ultrafiltration system etc..
The present invention obtains free sulfhydryl groups to carry out Bioconjugation using IgG4 antibody middle part disjunction interchain disulfide bond of turning up the soil.Pass
The antibody and the ADC of high connection number being not connected with the product that the method being directly coupled after the partial reduction IgG4 antibody of system obtains
Contents level is too high, will be unable to directly apply to animal or clinical test if not being further purified.And increase to remove and be not connected with
Antibody and high connection number ADC the step of, the utilization rate for making antibody starting material is greatly reduced, on the other hand makes purge process more
Add cumbersome, also increase process costs.
Partial oxidation in the present invention is for reduction, will pass through the interchain disulfide bond that reduction reaction is opened
The free sulfhydryl groups formed, then make the free sulfhydryl groups of part close to form interchain disulfide bond through peroxidization.Described opening
Or close the fracture and generation for referring to interchain disulfide bond.To reach the effect that target is averagely coupled 4.0 ± 1.0, to reduction step
Control be to make more than 80% IgG4 antibody interchain disulfide bonds be opened to be formed, the control to oxidation step is to make about half
The interchain disulfide bond that (45%-55%) is opened regenerates.
The method provided by the invention for first reducing again partial oxidation, its advantage are：
1. there is obtained coupled product mixture more preferable product to be distributed.It is 4 that most of article, which all concentrates on connection number,
Product, connection number be 4 coupled product content can be more than 50%, in a preferred embodiment can be more than 60%；Mixture
In the ADC contents levels of the antibody that is not connected with and high connection number substantially reduce, the coupled product content that connection number is 0 is less than
5%, the coupled product content that connection number is 8 is less than 15%, and obtained coupled product mixture can be direct after general purification
Applied to animal or clinical test.
2. improve the utilization rate of antibody starting material.Due to reducing the content ratio of not connected antibody and high connection number conjugate
Example, the reaction later stage need not remove the conjugate of unreacted antibody and high connection number, thus substantially increase antibody starting material
Utilization rate.Meanwhile the requirement when producing to reaction volume also becomes smaller.
3. the method for the present invention is more easy because product post-processes, production technology becomes simpler so that conjugate
Production cost is greatly lowered.
Brief description of the drawings
Fig. 1 is the reaction principle schematic diagram of the present invention.
Fig. 2A be in embodiment 1 using classical partial reduction method IgG4 antibody is coupled obtained by coupled product
Product distribution map and ratio.
Fig. 2 B be embodiment 1 in using the present invention method IgG4 antibody is coupled gained coupled product product divide
Butut and ratio.
Fig. 3 A be in embodiment 2 using classical partial reduction method IgG4 antibody is coupled obtained by coupled product
Product distribution map and ratio.
Fig. 3 B be embodiment 2 in using the present invention method IgG4 antibody is coupled gained coupled product product divide
Butut and ratio.
It is overlapping with the PLRP-HPLC without going back original antibody through going back original antibody after Fig. 4 is step 1 reduction through the present invention
Spectrum, there is no completely unreduced antibody presence after reduction for antibody.
Fig. 5 is the endpoint monitoring figure of step 1 reduction reaction of embodiment 1.
Fig. 6 is the endpoint monitoring figure of step 1 reduction reaction of embodiment 2.
Embodiment
Clear, complete description is carried out to technical scheme below in conjunction with accompanying drawing, it is clear that described implementation
Example is the part of the embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, the common skill in this area
The every other embodiment that art personnel are obtained on the premise of creative work is not made, belong to the model that the present invention protects
Enclose.
A kind of method that Bioconjugation is carried out using cystine linkage inside IgG4 antibody that the present invention develops, as shown in figure 1, bag
Include the reduction of IgG4 antibody, the partial oxidation of IgG4 antibody, and the coupled reaction of IgG4 antibody and conjugate, and last production
Product purify.Its basic method steps is described as follows：
Step 1, reduction：IgG4 is reduced with reducing agent, enables 4 pairs of most of openings of interchain disulfide bond in gG4 antibody.
IgG4 antibody is mixed in buffer solution with reducing agent with proper ratio and cultivated at appropriate temperatures so that 80%
Interchain disulfide bond in above IgG4 antibody is opened, and retains the intrachain disufide bond in antibody.Between step 1 and step 2, also
Can selectivity the buffer exchange step of increase by one, so that remove may unnecessary reducing agent.
Step 2, partial oxidation：The 4 pairs of interchain disulfide bond partial oxidations that will be opened with oxidant in IgG4 antibody, are obtained
GG4 antibody have suitable quantity and be available for coupling free sulfhydryl groups.
Using suitable oxidant, appropriate time, portion are incubated in proper proportions in buffer solution with the antibody being reduced
Divide the cystine linkage that has been opened closed in antibody, free sulfhydryl groups number is reached the average coupling number of target.The target is averagely coupled
Several ideal values is 4.0 ± 0.5.Step 2 oxidation reaction complete after, can directly carry out step 3 Bioconjugation or
Person carries out Bioconjugation again after removing oxidant by purifying.
Step 3, coupling reaction：Biology is carried out using the free sulfhydryl groups and connexon-conjugate compound of lgG4 antibody occasionally
Connection.
The IgG4 antibody that connexon-conjugate compound of excess is opened with part interchain disulfide bond is in suitable condition
Under be coupled, obtain antibody coupling matter product (ADC).
The purifying of step 4, Bioconjugation product：The Bioconjugation product that step 3 obtains is by removing unnecessary coupling
Compound, that is, obtain end-product.
By above-mentioned basic method steps, the present invention also provides the following specific implementation for preparing antibody coupling matter product (ADC)
Example.Do not do specified otherwise, the reagent used in following embodiment is customary commercial reagent or tried using customary commercial
Agent prepares to obtain by the conventional methods of this area.
Embodiment 1
(1) reduction of IgG4 antibody.Antibody is dissolved in 20mM Histidine, 5% (w/v) with 8mg/ml concentration
In sucrose, PH 5.5 buffer solution, the TCEP of 20 equivalents is added, places reaction liquid into 37 DEG C of water-bath and places 21 hours,
PLRP-HPLC monitorings reach reaction end (as shown in Figure 5).
(2) unnecessary reducing agent is removed.Molecular weight retention is used to delay for 40KD desalting column to reduction reaction system
Fliud flushing exchanges.By antibody protein buffer-exchanged into 50mM NaPi, 50mM NaCl, 2mM EDTA, PH 6.5, while remove anti-
Unnecessary TCEP in answering.
(3) antibody of reduction is subjected to partial oxidation.Add buffer solution into the antibody by desalting column purifying, DMSO with
And DHAA DMSO liquid storages, it is 5mg/ml to make antibody concentration, and DMSO contents are 5%, DHAA and the equivalent proportion of antibody is 6, will be anti-
Answer liquid to be placed in 4 DEG C to place 3 hours, PLRP-HPLC monitorings reach reaction end, make free sulfhydryl groups reach the average coupling of target
Number 4 ± 1.
(4) coupling of antibody and medicine.DMA and MC-vc-PAB-MMAE is added into the solution of oxidation reaction, and (commodity try
Agent, supplied by Levena Biopharma) DMA liquid storages, make the final DMA content be for the equivalent proportion of 5%, MMAE and antibody
9.Reaction solution continues to be placed in react 1 hour at 4 DEG C.
(5) purifying products.Buffer-exchanged is carried out to reaction system for 40KD desalting column using molecular weight retention, simultaneously
Remove organic solvent and unreacted MC-vc-PAB-MMAE.Final coupled product is stored in 20mM Histidine
In Acetate, PH5.0 buffer solution.
Test control group is being used directly with TCEP partial reductions and the result that is coupled using identical antibody.With examination
ADC products obtained by testing the method for ADC products and the present invention obtained by control group detect the distribution of its product under the same conditions, such as scheme
Shown in 2A, 2B.
From experimental data, obtain average coupling number (DAR) it is similar in the case of, the inventive method is used in Fig. 2 B
In obtained ADC products, be not connected with medicine antibody (D0) content be 4.51% (＜ 5%), compared to test control group (i.e. according to
Partial reduction directly is carried out to interchain disulfide bond using reducing agent to IgG4 antibody in the prior art, is then directly coupled
Method) it have dropped 61%；ADC (D8) content of height connection number is 13.09% (＜ 15%), be have dropped compared to test control group
38%；It is 61.5% to connect ADC (D4) content that number is 4, and 20% is improved compared to test control group.It is prepared by the method for the present invention
Coupled product mixture there is more preferable product to be distributed, most of article all concentrates on the product that connection number is 4, be suitable for through
Zoopery is directly applied to after general purification.
Embodiment 2
(1) reduction of IgG4 antibody.Antibody is dissolved in PBS with 10mg/ml concentration, in PH 6.0 buffer solution, added
Enter the DTT of 40 equivalents, place reaction liquid into 37 DEG C of water-bath and place 21 hours, PLRP-HPLC monitorings reach reaction end (such as
Shown in Fig. 6).
(2) unnecessary reducing agent is removed.Molecular weight retention is used to delay for 40KD desalting column to reduction reaction system
Fliud flushing exchanges.By antibody protein buffer-exchanged into 50mM NaPi, 50mM NaCl, 2mM EDTA, PH 6.5, while remove anti-
Unnecessary DTT in answering.
(3) antibody of reduction is subjected to partial oxidation.Add buffer solution into the antibody by desalting column purifying, DMSO with
And NAD DMSO liquid storages, it is 5mg/ml to make antibody concentration, and DMSO contents are 23%, NAD and the equivalent proportion of antibody is 65, will be anti-
Answer liquid to be placed in 15 degree and place 23 hours to complete partial oxidation, free sulfhydryl groups is reached the average coupling number 4 ± 1 of target.
NAD is added at twice, adds 32.5 for the first time at that time, reaction adds other 32.5 equivalent after 6.5 hours, and continues reaction 16.5
Hour.
(4) coupling of antibody and medicine.DMA and MC-vc-PAB-DM is added into the solution of oxidation reaction (by WuXi
Apptec is provided) DMA liquid storages, the content for making final DMA is 5%, and the equivalent proportion of maytansine and antibody is 9.Reaction solution continues
It is placed at 4 DEG C and reacts 1.5 hours.
(5) purifying products.Buffer-exchanged is carried out to reaction system for 40KD desalting column using molecular weight retention, simultaneously
Remove organic solvent and unreacted MC-vc-PAB-DM.Final coupled product is stored in 20mM Histidine Acetate,
In PH5.0 buffer solution.
Test control group is being used directly with TCEP partial reductions and entered with MC-vc-PAB-MMAE using identical antibody
The result of row coupling.Detected under the same conditions with ADC products obtained by the method for ADC products obtained by test control group and the present invention
Its product is distributed, as shown in Fig. 3 A, 3B.
From experimental data, obtain average coupling number (DAR) it is similar in the case of, the inventive method is used in Fig. 3 B
In obtained ADC products, antibody (D0) content for being not connected with medicine is 4.63% (＜ 5%), be have dropped compared to test control group
68%；ADC (D8) content of height connection number is 10.97% (＜ 15%), compared to test control group (i.e. according to right in the prior art
IgG4 antibody directly carries out partial reduction, the method being then directly coupled using reducing agent to interchain disulfide bond) it have dropped
41%；It is 51.71% to connect ADC (D4) content that number is 4, and 12% is improved compared to test control group.The method system of the present invention
There is standby coupled product mixture more preferable product to be distributed, and most of article all concentrates on the product that connection number is 4, is suitable for
Zoopery is directly applied to after purification through conventional.
In summary, the various embodiments described above and accompanying drawing are only presently preferred embodiments of the present invention, not limiting this
The protection domain of invention, within the spirit and principles of the invention, any modification, equivalent substitution and improvements done etc., all should
Comprising within the scope of the present invention.
Claims (15)
- A kind of 1. Bioconjugation method of IgG4 antibody, it is characterised in that including：Step 1, reduction：4 pairs of interchain disulfide bonds in IgG4 antibody are reduced, open interchain disulfide bond；Step 2, partial oxidation：By, by 4 pairs of fully open interchain disulfide bond partial oxidations, obtained IgG4 resists in IgG4 antibody The free sulfhydryl groups of body reach the average coupling number 4 ± 1 of target；Step 3, coupling reaction：Bioconjugation is carried out using the free sulfhydryl groups and connexon-conjugate compound of IgG4 antibody；The purifying of step 4, Bioconjugation product：The Bioconjugation product that step 3 obtains produces by removing unnecessary conjugate To end-product.
- 2. the method as described in claim 1, it is characterised in that in the step 1, by IgG4 antibody in buffer solution with reduction Agent is mixed with proper ratio and cultivated at appropriate temperatures so that the interchain disulfide bond in more than 80% IgG4 antibody is opened.
- 3. method as claimed in claim 2, it is characterised in that in the step 1, the interchain disulfide bond in IgG4 antibody is at least 90% is opened.
- 4. method as claimed in claim 2, it is characterised in that in the step 1, three (2- carboxyethyls) phosphines that reducing agent includes (TCEP), dithiothreitol (DTT) (DTT).
- 5. method as claimed in claim 2, it is characterised in that in the step 1, reducing agent dosage is 10-100 equivalents, instead It is 4-37 DEG C to answer temperature, reaction time 5-24h.
- 6. the method as described in claim 1, it is characterised in that in the step 2, using in suitable oxidant and step 1 Appropriate time, the quilt that part is closed in IgG4 antibody are incubated in proper proportions in buffer solution through reducing obtained IgG4 antibody Cystine linkage is opened, free sulfhydryl groups number is reached the average coupling number of target, the average coupling number of the target is 4.0 ± 0.5.
- 7. method as claimed in claim 6, it is characterised in that in the step 2, the oxidant includes hydroascorbic acid (DHAA), the thiobis of 5,5'- bis- (2- nitrobenzoic acids) (DTNB), NADH (NAD), nicotinoyl amine gland are fast Nicotinamide adenine dinucleotide phosphoric acid (NADP).
- 8. method as claimed in claim 6, it is characterised in that in the step 2, the ratio of the oxidant and IgG4 antibody For (2-100)：1, the temperature of oxidation reaction is 4-37 DEG C, and oxidization time is at least 3 hours.
- 9. the method as described in claim 1, it is characterised in that in the step 3, by connexon-conjugate compound and portion The IgG4 antibody for dividing interchain disulfide bond to be opened is coupled under the proper conditions, obtains antibody coupling matter product；The antibody The formula of coupled product is IgG4- (C-L-D) n, wherein, IgG4 represents that the hypotype of antibody is IgG4 antibody；C is represented and IgG4 The active function groups of the sulfydryl reaction to dissociate in antibody；L represents the connexon of connection antibody and conjugate；D represents there is biology Learn function attachment, including but not limited to the medicine with antitumor activity, the medicine of anti-inflammatory response, fluorescein, polypeptide, Nucleotide fragments or protein etc.；N is the average coupling number of antibody coupling matter product, and n span should be 4.0 ± 1.0.
- 10. method as claimed in claim 9, it is characterised in that in the step 3, the temperature of coupling reaction is 4 DEG C -37 DEG C, Reaction time is at least 0.5 hour.
- 11. the method as described in claim 1, it is characterised in that between step 1 and step 2, in addition to a buffer exchange Step, unnecessary reducing agent is removed from reaction system in the form of buffer-exchanged, while antibodies buffer is replaced as It is suitable for the buffer solution for aoxidizing and being coupled.
- 12. the method as described in claim 1, it is characterised in that after the oxidation reaction of step 2 is completed, can directly enter The Bioconjugation of row step 3 carries out step 3 again after purifying removing oxidant.
- 13. the coupled product of IgG4 antibody made from the method according to claim any one of 1-12.
- 14. the coupled product of IgG4 antibody as claimed in claim 13, it is characterised in that in the coupled product, connect number It is less than 5% for 0 coupled product content, the coupled product content that connection number is 8 is less than 15%.
- 15. the coupled product of IgG4 antibody as claimed in claim 13, it is characterised in that in the coupled product, connect number It is more than 50% for 4 coupled product content.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711032283.8A CN107746424A (en) | 2017-10-30 | 2017-10-30 | A kind of Bioconjugation method of IgG4 antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711032283.8A CN107746424A (en) | 2017-10-30 | 2017-10-30 | A kind of Bioconjugation method of IgG4 antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107746424A true CN107746424A (en) | 2018-03-02 |
Family
ID=61254204
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711032283.8A Pending CN107746424A (en) | 2017-10-30 | 2017-10-30 | A kind of Bioconjugation method of IgG4 antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107746424A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113289587A (en) * | 2021-05-10 | 2021-08-24 | 苏州君盟生物医药科技有限公司 | Sulfydryl modified magnetic nano-microsphere and preparation method and application thereof |
| CN114216971A (en) * | 2021-10-13 | 2022-03-22 | 上海药明生物技术有限公司 | Ultra-high performance liquid chromatography detection of dithiothreitol residue in protein product |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005084390A2 (en) * | 2004-03-02 | 2005-09-15 | Seattle Genetics, Inc. | Partially loaded antibodies and methods of their conjugation |
| CN106456725A (en) * | 2014-02-11 | 2017-02-22 | 西雅图基因公司 | Selective Reduction Of Proteins |
| CN106687472A (en) * | 2014-09-12 | 2017-05-17 | 麦迪穆有限责任公司 | Pyrrolobenzodiazepines and conjugates thereof |
-
2017
- 2017-10-30 CN CN201711032283.8A patent/CN107746424A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005084390A2 (en) * | 2004-03-02 | 2005-09-15 | Seattle Genetics, Inc. | Partially loaded antibodies and methods of their conjugation |
| CN106456725A (en) * | 2014-02-11 | 2017-02-22 | 西雅图基因公司 | Selective Reduction Of Proteins |
| CN106687472A (en) * | 2014-09-12 | 2017-05-17 | 麦迪穆有限责任公司 | Pyrrolobenzodiazepines and conjugates thereof |
Non-Patent Citations (1)
| Title |
|---|
| SUN M M C ET AL.: "Reduction-alkylation strategies for the modification of specific monoclonal antibody disulfides", 《BIOCONJUGATE CHEMISTRY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113289587A (en) * | 2021-05-10 | 2021-08-24 | 苏州君盟生物医药科技有限公司 | Sulfydryl modified magnetic nano-microsphere and preparation method and application thereof |
| CN113289587B (en) * | 2021-05-10 | 2023-11-28 | 苏州君盟生物医药科技有限公司 | Sulfhydryl modified magnetic nano microsphere and preparation method and application thereof |
| CN114216971A (en) * | 2021-10-13 | 2022-03-22 | 上海药明生物技术有限公司 | Ultra-high performance liquid chromatography detection of dithiothreitol residue in protein product |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0408546B1 (en) | Improvements relating to drug delivery systems | |
| Paddock et al. | Proton transfer pathways and mechanism in bacterial reaction centers | |
| Lemberg | Cytochrome oxidase. | |
| CN1997661B (en) | Polymer conjugates with decreased antigenicity, methods of preparation and uses thereof | |
| NO166618B (en) | PROCEDURE FOR THE PREPARATION OF IMMUNOENZYMATIC CONJUGATES WHICH, BY COVALENT BINDING, BINDES AN ENZYM AND ANTIBODY. | |
| WO2000076550A2 (en) | Carrier-drug conjugate | |
| JPH04501260A (en) | Fractionation method | |
| JP2008500961A5 (en) | ||
| NO20064676L (en) | Pharmaceutical salts of 3-O- (3'3'-dimethylsuccinyl) betulinic acid | |
| JPH05502880A (en) | Hybrid molecule with a translocation region and a cell binding region | |
| CN102575244A (en) | Dissociation method and dissociation agent for avidin and biotin | |
| DE10012120A1 (en) | New ligand, comprising therapeutic or diagnostic agent bonded non-covalently with substance having high affinity to transport molecule | |
| CN107746424A (en) | A kind of Bioconjugation method of IgG4 antibody | |
| Oke et al. | Advances in the application of deep eutectic solvents based aqueous biphasic systems: An up-to-date review | |
| US20140275509A1 (en) | Multifunctionalized materials | |
| CN109265512A (en) | The preparation method of protein conjugate based on pyridine dicarbaldehyde | |
| CN107849121A (en) | End-blocking and uncapped antibody cysteine, and its purposes in antibody drug is conjugated | |
| Wan et al. | Robust Strategy for Antibody–Polymer–Drug Conjugation: Significance of Conjugating Orientation and Linker Charge on Targeting Ability | |
| CN106880848A (en) | Biodegradable poly HPMA Gd magnetic resonance imaging probes and preparation method thereof | |
| Federico et al. | Histaminase PEGylation: preparation and characterization of a new bioconjugate for therapeutic application | |
| Willmore | Silver nanoparticles for cancer research | |
| Sakurai et al. | Design and synthesis of small molecule-conjugated photoaffinity nanoprobes for a streamlined analysis of binding proteins | |
| Fottrell | Dehydrogenase isoenzymes from legume root nodules | |
| Houba et al. | Improved Characteristics of a Human β-Glucuronidase− Antibody Conjugate after Deglycosylation for Use in Antibody-Directed Enzyme Prodrug Therapy | |
| CN107261137A (en) | Two kinds of Anti-HER 2 chaetocin conjugates and preparation method thereof and antitumor application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 200131 701, 7 floor, 2 Hua Jing Road, Pudong New Area, Shanghai. Applicant after: SHANGHAI YAOMING BIOTECHNOLOGY CO., LTD. Applicant after: Wuxi Yaoming Biotechnology Co., Ltd. Applicant after: SUZHOU YAOMING KANGDE INSPECTION TESTING CO., LTD. Address before: 200131 701, 7 floor, 2 Hua Jing Road, Pudong New Area, Shanghai. Applicant before: SHANGHAI YAOMING BIOTECHNOLOGY CO., LTD. Applicant before: WUXI APPTEC BIOPHARMACEUTICALS CO., LTD. Applicant before: SUZHOU YAOMING KANGDE INSPECTION TESTING CO., LTD. |
|
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 200131 701, 7 floor, 2 Hua Jing Road, Pudong New Area, Shanghai. Applicant after: SHANGHAI YAOMING BIOTECHNOLOGY CO., LTD. Applicant after: Wuxi Yaoming Biotechnology Co., Ltd. Applicant after: Suzhou Pharmacopoeia Testing and Inspection Co., Ltd. Address before: 200131 701, 7 floor, 2 Hua Jing Road, Pudong New Area, Shanghai. Applicant before: SHANGHAI YAOMING BIOTECHNOLOGY CO., LTD. Applicant before: Wuxi Yaoming Biotechnology Co., Ltd. Applicant before: SUZHOU YAOMING KANGDE INSPECTION TESTING CO., LTD. |
|
| CB02 | Change of applicant information | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180302 |
|
| RJ01 | Rejection of invention patent application after publication |