CN101624614A - Culture method for anti-CD25 antibody - Google Patents
Culture method for anti-CD25 antibody Download PDFInfo
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- CN101624614A CN101624614A CN200910057759A CN200910057759A CN101624614A CN 101624614 A CN101624614 A CN 101624614A CN 200910057759 A CN200910057759 A CN 200910057759A CN 200910057759 A CN200910057759 A CN 200910057759A CN 101624614 A CN101624614 A CN 101624614A
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Abstract
The invention discloses a culture method for an anti-CD25 antibody, on the basis of three stages of fed-back culture, basic medium is added with galactose in concentration of 20-100mg/L, fed-back medium is added with glucose in concentration of 3-12mmol/L and glutamine in concentration of 5.0-7.8mmol/L, and glucose concentration is maintained to be 0.5-2.0g/L in the culture process. By applying the culture method disclosed by the invention, difference between protein expressed by cell and protein naturally existed in human body can be reduced, and safety of culture product on human body is improved.
Description
Technical field
The present invention relates to the animal cell culture field, more specifically, the present invention relates to the method that a kind of improved large scale culturing technology efficiently expresses antibody.
Background technology
Organ transplantation is one of the significant achievement of 20th century medical science, is the whole end property disease of multiple internal organs unique treatment means at present.Only annual newly-increased liver, kidney and the cardiac failure patient of China just reaches millions ofly, and wherein major part can prolong life by organ transplantation.With the renal transplantation is example, ten thousand renal transplantations surplus the whole world adds up to have implemented 50 at present altogether, and this numeral has surpassed 30,000 in China.The renal transplantation number of China in 2000 is broken through 5000 examples, is only second to the U.S., occupies the second in the world.In the face of annual newly-increased 200,000 uremic patient, renal homotransplantation has become a kind of very important means of treatment chronic renal failure.China recent years is except that traditional renal transplantation, and heart transplantation, liver transplantation are accelerated growth situation, and the liver transplantation in 2003 1 year example number surpasses 2000 examples, and is also more than the summation of each year in the past.The subject matter that influences the organ transplantation curative effect is the problem of immunological rejection except that the donor shortage; And immunological rejection has increased the weight of this organ origin of shortage.
Divide from the donor organ source, organ transplantation can be divided into syngraft (as the transplanting between autotransplantation, the twin children), allogeneic transplantation and xenotransplantation.Present most organ transplantation is allogeneic transplantation.Because the height diversity of the main histocompatibility antigen of human body (MHC antigen) causes donor internal organs antigen incompatible with the acceptor immunity system inevitably, the operation back produces serious rejection.For keeping the survival of graft, reach postoperative before the transplantation and must conventionally use immunosuppressor, suppress the attacking ability of immunity system to graft.Traditional immunosuppressor comprises small molecules chemicalses such as glucocorticosteroid (prednisone class), cyclosporin A, azathioprine.The invention of these medicines, obvious facilitation has been played in development to organ transplantation, yet, because its specificity is lower, bigger to liver, kidney and neural toxicity, have a strong impact on the life quality of transplanting the receptor, caused the chronic forfeiture of long term complication such as organ dysfunction, easy infection and the neoplastic disease etc. sent out.
Along with the continuous research of people for the immune response process, the mechanism of immunological rejection is further illustrated, therefore, many in recent years investigators attempt at the key link in the immunological rejection process, take intervening measure, attempt to eliminate specifically of the immune response of receptor's immunity system, keep the immunologic function such as anti-infective, antitumor of body as much as possible, reduce the generation of other toxic side effects simultaneously at graft.
Acute rejection is modal a kind of graft-rejection, can occur in this reaction of untreated person and transplant the back within a couple of days; And through the immunosuppressant therapy person, can after several months or several years, take place suddenly, showing as heating, general malaise clinically, graft enlargement and pain go down suddenly with graft function simultaneously.
Acute rejection belongs to the cellular immunization phenomenon of delayed allergy, the HLA antigenic stimulation receptor T lymphocyte that is graft makes it differentiation and proliferation, produce and have specific primed lymphocyte in a large number, but direct killing or kill and wound target cell by discharging various lymphokines is repelled graft.Think that now humoral immunization also participates in acute rejection.Tissue pathologies change is tunica intima damage and fibrinoid necrosis companion monocyte infiltration.Acute rejection is through correct processing in time, and 80-90% can be reversed.
Leukocyte differentiation antigen CD25 claims Tac antigen again, is the alpha subunit (P55 α, R alpha subunit) of interleukin II (IL-2) acceptor.T lymphocytic cell surface IL-2 acceptor is made up of α (p55), β (p75), three subunits of γ (p64), and alpha subunit (claiming Tac or CD25 again) only is expressed in and activates the T lymphocytic cell surface.
Normal IL-2 acceptor is to be combined with a large amount of covalent linkage by three α of subunit, β, γ three chains, and is very low in tranquillization T cell surface expression rate, and the activated T cell surface then has more fully expresses.Its alpha subunit is derivable, can not be accounted for by other acceptor branches, can only constitute low-affinity receptor; β and γ key then can constitute medium avidity acceptor, can be by other receptors bind.α, β, γ three's polymer could constitute complete high-affinity receptor.As the acceptor of IL-2, CD25 is with after IL-2 combines, though can not conducts information, can start the T cell and enter m period.In the renal transplantation acceptor, because the heteroplastic transplantation kidney stimulates body immune system, the cytokine IL-2 that is produced by immunocyte such as T cell, NK cell, LAK cell can combine with the acceptor (CD25) through antigen activated T lymphocytic cell surface, the fast breeding of inducing T cell produces rejection.
Because CD25 mainly is expressed in activated T lymphocyte, therefore, if can be at the CD25 molecule, design specific medicine, only suppress this group T cell activity, or remove this group T cell, then can when the allogeneic organ transplantation, suppress immune response specifically at graft.Monoclonal antibody technique is because the specificity and the target of its height are listed in preferred option very naturally.
Anti-CD25 monoclonal antibody combines with the specificity of Tac has blocked combining of IL-2 and IL-2 receptor complex, thereby has suppressed IL-2 by high-affinity IL-2 receptor-inducible T cell activation, has also just suppressed the critical passage of cellular immunization in the rejection process.It brings into play immunosuppressant main mechanism: suppress the T hyperplasia that IL-2 relies on by the binding competition ground with IL-2 acceptor Tac subunit; In the external T cytolysis (this may be the immunosuppressant another kind of mechanism of this medicine) that can cause anti-Tac monoclonal antibody mediation by the cytotoxicity (ADCC) of antibody-dependant cell mediation.Indirect immunity conditioning mechanism is reduced as the IL-2 expression level; By Fc section and Fc acceptor interaction, optionally deactivation and destruction Tac male lymphocyte cause the reaction-ive T cell colony to be eliminated, thereby strengthen or amplify optionally immunosuppressive effect.
Yet mouse source antibody can cause the intensive immune response when being applied to human body, has seriously hindered the curative effect of the anti-CD25 monoclonal antibody in mouse source as neotype immunosuppressant.Maturation along with the humanized antibody technology, the foreign study person has carried out humanization modified to mouse source antibody, make it keep original specific while, immunogenicity to human body reduces greatly, thereby be developed as the anti-immunological rejection medicine of ideal (Immunosuppression for neural xenografts:a comparison of cyclosporin andanti-CD25 monoclonal antibody.Honey CR, Shen H.J Neurosurg 1999 Jul; 91 (1): 109-13.Daclizumab.Linda R.W, Diana F.Drugs 1999,58 (6): 1029-42.Placebo-controlled study of a humanized anti-TAC monoclonal antibody in dualtherapy for prevention of acute rejection after renal transplantation.CharpentierB, Thervet E.Transplant Proc 1998 Jun; 30 (4): 1331-2).At present, the monoclonal antibody of FDA and 2 anti-CD25 of European Union's approved, a kind of is humanization monoclonal antibody (Daclizumab); Another kind is the anti-CD25 monoclonal antibody of people-mouse mosaic type (Basiliximab).These two antibody all have been successfully used to prevent the acute rejection after the renal transplantation.Its therapeutic index is wide, untoward reaction is little, long half time, the Basiliximab transformation period is about 6 days, the Daclizumab transformation period surpasses 20 days (Daclizumab:outcome of phase III trials and mechanism of action.Double Therapy and theTriple Therapy Study Groups.Vincenti F, Nashan B, Light S.Transplant Proc1998Aug; 30 (5): 2155-8 Reduction of acute renal allograft rejection bydaclizumab.Daclizumab Double Therapy Study Group.Nashan B, Light S, Hardie IR, Lin A, Johnson JR.Transplantation 1999 Jan 15; 67 (1): 110-5 Thesafety and efficacy of a two-dose daclizumab (zenapax) induction therapy in livertransplant recipients.Eckhoff DE, McGuire B, Sellers M, Contreras J, Frenette L, Young C, Hudson S, Bynon JS.Transplantation 2000 May 15; 69 (9): 1867-72Results of 3-year phase III clinical trials with daclizumab prophylaxis forprevention of acute rejection after renal transplantation.Bumgardner GL, HardieI, etc.Transplantation 2001 Sep 15; 72 (5): 839-45.Daclizumab:a review ofits use in the management of organ transplantation.Carswell CI, Plosker GL, Wagstaff AJ.BioDrugs2001; 15 (11): 745-73).These two kinds of antibody all are to utilize genetic engineering technique, the recombinant protein that adopts the mammalian cell fermentation technique to express.
Daclizumab (trade(brand)name Zenapax) is by U.S. Protein Design Labs company exploitation, after it licensed to Hoffmann-La Roche (Luo Shi) company produce, obtained the approval of FDA in 1997, be used to prevent the acute rejection of renal transplantation.This antibody comprises 90% human IgG sequence and 10% mouse sequence, and wherein the human sequence is made up of human IgG1's constant region and the variable framework region of Eu myeloma antibody, and the mouse sequence is made of the CDR of mouse-anti Tac antibody, molecular weight 144KDa.
In view of Declizumab shows definite effect before clinical and in the clinical study,
Obtained the drugs approved by FDA listing on December 10th, 1997, indication is " prevention allograft renal transplantation patient's acute organ rejection reaction can be share with the immunosuppressant scheme that comprises ciclosporin and corticosteroid hormone ".
Protein Design Labs has selected the NSO cell as host cell at the beginning of exploitation, has adopted serum-free suspension batch best cultivation.NSO is as host cell, has following geneogenous defective: the α of NSO host cell-1,3 galactoside transferases can produce the Gal α 1 with high immunogenicity, 3-Gal β 1,4-GlcNac, this non-existent glycosylation modified when being applied to human body in human body, having stimulates body to produce the immune response of this class heterologous antigen of diagnosis, thereby reduce the curative effect of medicine, even severe anaphylactic reaction takes place.
In the NSO cell, the sialic acid form of glycosylation end is mainly N-glyculyl-neuraminicacid (NGNA) (N-n acetylneuraminic acid n), different with the N-acetyl-neuraminic acid (NANA) (N-n acetylneuraminic acid n) of human body, therefore utilize the NSO cell to cultivate the anti-CD25 monoclonal antibody that obtains and be used for also having the potential immunogenicity behind the human body, may stimulate body to produce immune response and affect the treatment even anaphylaxis takes place as host cell.
Has retroviral particle in the NSO cell, in with the process of this cell as the anti-CD25 monoclonal antibody preparation of industrialization of host cell, a large amount of retroviral particles is secreted in the culture supernatant, if the residual virus that has not removing to obtain deactivation can cause uncertain risk in the clinical treatment in the finished product.Therefore the purge process in downstream need be passed through very complicated step removal or the virus in the deactivation supernatant, and these steps must be by effectively verifying.
Summary of the invention
In order to overcome the shortcoming of utilizing the NSO cell to cultivate anti-CD25 monoclonal antibody, thereby need utilize proper host cell and optimize culture condition and reduce that the albumen of cell expressing and human body are natural to exist proteic difference, improve its security human body as host cell.
The present inventor utilizes Chinese hamster ovary celI as host cell, and be that 200610147535.7 denominations of invention contain the Chinese hamster ovary celI of anti-CD 25 antibody sequence for disclosed three step stream addition culture bag in " adopting the method for zooblast feeding culture mode highly effective expressing recombinant protein " according to the number of patent application of on October 20th, 2006 application, with utilize the NSO cell to cultivate to compare as host cell, the result shows, the antibody glycosylation form consistent with the Natively glycosylated form of human body that obtains increases greatly, cause immunogenic possibility thereby reduced, and the ratio that semi-lactosi is not exclusively modified reduces significantly also.Confirm after testing, no endogenic retroviral particle in the engineering cell of structure, the antibody that utilizes this project cell cultures to obtain does not have the pollution of virus.
The present inventor further improves cultural method, thereby has finished the present invention.
The invention discloses a kind of method of utilizing Chinese hamster ovary celI feeding culture mode to efficiently express anti-CD 25 antibody, be three step feeding culture methods, i.e. a) 36 ℃-38 ℃ of cell cultures temperature, pH value 6.5-6.9, cultivated 24-48 hour, when zooblast grows to 2.0-3.5 * 10
6During cells/ml, add fed-batch medium in basic medium, carry out feeding culture, it is 0.1-0.3/ days that stream adds thinning ratio, 72-96 hour feeding culture time; B) the cell cultures temperature is reduced to 32.5 ℃-35 ℃, and pH value 7.0-7.3 carries out feeding culture, and it is 0.1-0.2/ days that stream adds thinning ratio, 120-144 hour feeding culture time; C) stopping stream adding, the cell cultures temperature rises to 36 ℃-38 ℃, pH value 7.0-7.3, cultivate, incubation time is 24-48 hour, it is characterized in that step a) adds semi-lactosi 20～100mg/L in basic medium, add glucose 3-12mmol/L and glutamine 5.0-7.8mmol/L in the fed-batch medium, glucose concn maintains 0.5-2.0g/L in the control culturing process.More preferably, add semi-lactosi 50mg/L in basic medium, add glucose 7mmol/L and glutamine 6.0mmol/L in the fed-batch medium, glucose concn maintains 0.8-1.0g/L in the control culturing process.
By the glycosylated ratio of cultured products in the culturing process is detected, the result shows in the serum free medium that adding semi-lactosi can significantly increase the glycosylated ratio of G2 type, thereby reduces the immunogenicity brought by glycosylation difference and the function difference problem of antibody.The virus of culturing process and product detects is undertaken by a series of experiments, comprise that cellular form observation, hemadsorption test, passage cell cultivation, transmission electron microscope detect retroviral particle, animal and the test of chicken embryo, animal's antibody and produce test, reverse transcriptase activity determination experiment, the result shows that Chinese hamster ovary celI is all negative, has reached purpose of the present invention.
Description of drawings:
Fig. 1: Chinese hamster ovary celI retrovirus detected result.
Fig. 2: the NSO cell reversal is recorded viral detected result.
Embodiment
Following examples, experimental example are described the present invention in further detail.Yet should be appreciated that and enumerate these embodiment, experimental example, and be not to be used for limiting the present invention just for an illustration.
Embodiment 1 Construction of eukaryotic
Antibody sequence is referring to, Chinese invention patent application number 89109618.3, and denomination of invention is a disclosed sequence in " new interleukin 2 receptor specific immunoglobulin ".
The construction process of expression vector referring to: Chinese invention patent application number 02112493.0, denomination of invention are disclosed method in " anti-CD25 monoclonal antibody, its encoding sequence and the application of reorganization ".
The stably express and the purifying of embodiment 2 humanized antibodies
Referring to: Chinese invention patent application number 02112493.0, denomination of invention are disclosed method in " anti-CD25 monoclonal antibody, its encoding sequence and the application of reorganization ".
The extensive serum-free culture that embodiment 3 optimizes
Three sections stream additions carry out in the employing patent 200610147535.7 " a kind of method of highly effective expressing recombinant protein ", and in the culturing process, fermentation parameter has carried out following optimization:
In step 1), in basic medium, add semi-lactosi 50mg/L, add glucose 7mmol/L and glutamine 6.0mmol/L in the fed-batch medium, glucose concn maintains 0.8-1.0g/L in the control culturing process.
The extensive serum-free culture that embodiment 4 optimizes
Three sections stream additions carry out in the employing patent 200610147535.7 " a kind of method of highly effective expressing recombinant protein ", and in the culturing process, fermentation parameter has carried out following optimization:
In the step 1), add semi-lactosi 100mg/L in basic medium, add glucose 3mmol/L and glutamine 5.0mmol/L in the fed-batch medium, glucose concn maintains 0.5-0.6g/L in the control culturing process.
The extensive serum-free culture that embodiment 5 optimizes
Three sections stream additions carry out in the employing patent 200610147535.7 " a kind of method of highly effective expressing recombinant protein ", and in the culturing process, fermentation parameter has carried out following optimization:
Add semi-lactosi 20mg/L mg/L in the step 1) in basic medium, add glucose 12mmol/L and glutamine 7.8mmol/L in the fed-batch medium, glucose concn maintains 2.0g/L in the control culturing process.
The product that embodiment 3～5 is obtained detects, the result shows, glucose concn in the control culturing process: pass through on-line monitoring, PID control glucose concn maintains 0.5-2.0g/L, glucose concn helps utilizing fully of glucose and less lactic acid accumulation in this scope, makes culturing process pH maintain state preferably.
Experimental example 1 cultured products glycosylation relatively
Cultivate by embodiment 3, embodiment 4, embodiment 5 steps, cultivate and finish after product glycosylation relatively (utilizing the BECKMAN capillary electrophoresis to operate) in conjunction with the method in BECKMAN glycosylation inspecting reagent box (Beckman Coulter, the 477600) by specification.
| ??G0 | ??G1，G1’ | ??G2 | Other | |
| Embodiment 3 | ??8.6 | ??12.6 | ??78.3 | ??0.5 |
| Embodiment 4 | ??6.1 | ??11.3 | ??82.1 | ??0.5 |
| Embodiment 5 | ??9.8 | ??19.2 | ??70.6 | ??0.4 |
| Three sections feeding culture | ??38.6 | ??31.2 | ??29.6 | ??0.6 |
| ??Zenapax (NSO cell) | ??31.2 | ??52.7 | ??11.0 | ??5.1 |
Numerical value is the shared percentage compositions of various glycosylation forms in the form, and wherein the G2 type is most complete glycosylation form.The structure of various glycosylation forms is as follows:
Add semi-lactosi in the serum free medium, can significantly increase the glycosylated ratio of G2 type, glycosylation form natural in this complete glycosyl galactose form and the human body is consistent, thereby reduces the immunogenicity brought by glycosylation difference and the function difference problem of antibody.
The virus of experimental example 2 culturing process and product detects
NSO and CHO (cultivate by embodiment 3～5 steps, cultivate the product that obtains after finishing) cell virus detected result is as shown in the table.
| ?CHO | ??NSO | |
| Cellular form is observed | ?- | ??+ |
| Hemadsorption test | - | ??+ |
| Passage cell is cultivated | - | ??+ |
| Transmission electron microscope detects retroviral particle (transmission electron microscope observation) | - | ??+ |
| Animal and the test of chicken embryo | - | ??+ |
| Animal's antibody produces test | - | ??+ |
| Reverse transcriptase activity is measured | - | ??+ |
Related experimental methods is undertaken by State Food and Drug Administration's " the virus safe evaluation technique of biological tissue extracted goods and eukaryotic cell expression goods is evaluated rule ", " human monoclonal antibody Quality Control Technology governing principle ", and method is with reference to three appendix of the Pharmacopoeia of the People's Republic of China.
Three appendix IX of Pharmacopoeia of the People's Republic of China M reverse transcriptase activity test procedure.
Three appendix XII of Pharmacopoeia of the People's Republic of China C virus exogenous factor test procedure.
Three appendix XII of Pharmacopoeia of the People's Republic of China H mouse borne virus test procedure.
The retrovirus detected result is seen Fig. 1, Fig. 2, and the result shows, does not see retroviral particle in the Chinese hamster ovary celI, in the NSO cell, and visible a large amount of retroviral particle (point of black among the figure).
Claims (4)
1. the cultural method of an anti-CD 25 antibody may further comprise the steps:
A) the cell cultures temperature is 36 ℃-38 ℃, and pH value 6.5-6.9 cultivated 24-48 hour, when zooblast grows to 2.0-3.5 * 10
6During cells/ml, add fed-batch medium in basic culture solution, carry out feeding culture, it is 0.1-0.3/ days that stream adds thinning ratio, 72-96 hour feeding culture time;
B) the cell cultures temperature is reduced to 32.5 ℃-35 ℃, and pH value 7.0-7.3 carries out feeding culture, and it is 0.1-0.2/ days that stream adds thinning ratio, 120-144 hour feeding culture time;
C) stop stream and add, the cell cultures temperature rises to 36 ℃-38 ℃, and pH value 7.0-7.3 cultivates, and incubation time is 24-48 hour;
It is characterized in that, add semi-lactosi 20～100mg/L in the step a) basic medium, add glucose 3-12mmol/L and glutamine 5.0-7.8mmol/L in the fed-batch medium, glucose concn maintains 0.5-2.0g/L in the control culturing process.
2. the cultural method of the described anti-CD 25 antibody of claim 1, it is characterized in that in the step a) basic medium, adding semi-lactosi 50mg/L, add glucose 7mmol/L and glutamine 6.0mmol/L in the fed-batch medium, glucose concn maintains 0.8-1.0g/L in the control culturing process.
3. the cultural method of the described anti-CD 25 antibody of claim 1, it is characterized in that in the step a) basic medium, adding semi-lactosi 100mg/L, add glucose 3mmol/L and glutamine 5.0mmol/L in the fed-batch medium, glucose concn maintains 0.5-0.6g/L in the control culturing process.
4. the cultural method of the described anti-CD 25 antibody of claim 1, it is characterized in that in the step a) basic medium, adding semi-lactosi 20mg/L, add glucose 12mmol/L and glutamine 7.8mmol/L in the fed-batch medium, glucose concn maintains 2.0g/L in the control culturing process.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320388A (en) * | 2012-03-20 | 2013-09-25 | 无锡药明康德生物技术有限公司 | Cell culture method capable of improving antibody expression levels and improving glycosylation levels |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320388A (en) * | 2012-03-20 | 2013-09-25 | 无锡药明康德生物技术有限公司 | Cell culture method capable of improving antibody expression levels and improving glycosylation levels |
| CN103320388B (en) * | 2012-03-20 | 2015-10-28 | 无锡药明康德生物技术股份有限公司 | Improve the cell culture processes of antibody expression amount and improvement level of glycosylation |
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