CN101358225B - Method for preparing high sialic acid content immune globulin antibody and application - Google Patents
Method for preparing high sialic acid content immune globulin antibody and application Download PDFInfo
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- CN101358225B CN101358225B CN2007100093044A CN200710009304A CN101358225B CN 101358225 B CN101358225 B CN 101358225B CN 2007100093044 A CN2007100093044 A CN 2007100093044A CN 200710009304 A CN200710009304 A CN 200710009304A CN 101358225 B CN101358225 B CN 101358225B
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Abstract
A preparation method for an immunoglobulin antibody with high content of N-acetylneuraminic acid and an application thereof, which relate to an immunoglobulin antibody, in particular to a method for increasing the content of N-acetylneuraminic acid on the immunoglobulin antibody by adopting the catalysis of glycosyl transferase and an application thereof. The invention provides a preparation method for an immunoglobulin antibody with high content of N-acetylneuraminic acid and an application thereof. N-acetylneuraminic acid is catalyzed by cytidine-5'-phosphate-N-acetylneuraminic acid synthetase to react with cytidine-5'-triphosphate to produce cytidine-5'-phosphate-N-acetylneuraminic acid; galactose is catalyzed by Beta 1, 4 galactosyltransferase to be linked to N-acetylglucosamine which is the terminal of immunoglobulin glycosyl with uridinediphosphate-galactose as the substsrate; N-acetylneuraminic acid is catalyzed by sialyl transferase with cytidine-5'-phosphate-N-acetylneuraminic acid as the substrate to be linked to galactose which is the terminal of the immunoglobulin glycosyl. The prepared immunoglobulin antibody with high content of N-acetylneuraminic acid can be used for preparing the medicines for treating the autoimmune diseases, especially the rheumatoild diseases.
Description
Technical field
The present invention relates to a kind of immune globulin antibody, especially relate to a kind of method and application thereof of adopting glycosyltransferase catalysis to increase the sialic acid content on the immune globulin antibody.
Background technology
Immune globulin antibody like IGIV G, is widely used in treating (1.Imbach such as chronic self-Immunological diseases; P., Barandun, S.; D ' Apuzzo, V., et al; High-dose intravenous gammaglobulin foridiopathic thrombocytopenic purpura in childhood, Lancet i, 1228 (1981); 2.Blanchette, V, Imbach; P., Andrew, M.; Et.al.Randomised trial of intravenous immunoglobulin G, intravenous anti-D, andoral prednisone in childhood acute immune thrombocytopenic purpura; Lancet 344,703 (1994); 3.Y.Sultan; M.D.Kazatchkine; P.Maisonneuve; U.E.Nydegger, Anti-idiotypic suppression ofautoantibodies to factor VIII (antihaemophilic factor) by high-dose intravenous gammaglobulinLancet ii, 765 (1984); 4.D.R.Jayne, M.J.Davies, C.J.Fox, C.M.Black, C.M.Lockwood, Treatment of systemic vasculitis with pooled intravenous immunoglobulin Lancet 337,1137 (1991); 5.P.LeHoang, N.Cassoux, F.George; N.et.al.D.Kazatchkine; Intravenousimmunoglobulin (IVIg) for the treatment of birdshot retinochoroidopathy, Ocul.Immunol.In β amm.8,49 (2000)).The structure of the glycosyl on the immune globulin antibody is non-homogeneous, and the immune globulin antibody that contains the glycosyl of different structure has different physiologically actives.Such as, IGIV G contains two N-and connects glycosyl, and its N-connects glycosyl has different end structures; End structure is that the ratio of sialic IGIV G in total IGIV G is lower than 5% (Routier; F.H., Hounsell, E.F.; Rudd; P.M., et.al.Quantitation of theoligosaccharides of human serum IgG from patients with rheumatoid arthritis:a critical evaluationof different methods J.Immunol.Methods 213,113 (1998)).And this contains quantity not sufficient 5%, contains effective constituent (the Yoshikatsu Kaneko that sialic IGIV G is the treatment anti-inflammatory exactly; FalkNimmerjahn; Jeffrey V.Ravetch, Anti-Inflammatory Activity of Immunoglobulin G Resulting fromFc Sialylation, Science 313; 670 (2006)), heavy dose of IGIV G can bring out serious side reaction in human body.Therefore improve IGIV G and go up the terminal sialic content of N-connection glycosyl to improving its drug effect, reducing dosage has important medical value to alleviate side reaction.
Summary of the invention
The object of the present invention is to provide a kind of preparation method and application thereof of immune globulin antibody of high sialic acid content.
Technical scheme of the present invention is to adopt the glycosyltransferase catalysis process that the N-that sialyl is connected to immune globulin antibody is connected on the glycosyl; Be used for effectively improving the sialic acid content of Tegeline; Through changing into and contain sialic effective constituent, and improve the ratio of its active pharmaceutical ingredient not containing sialic composition.
The preparing method's of the immune globulin antibody of a kind of high sialic acid content of the present invention concrete steps are:
1) use Cytidine-5 '-phosphoric acid-sialyl synthetic enzyme catalysis sialyl and Cytidine-5 '-triphosphoric acid (CTP) reaction generation Cytidine-5 '-phosphoric acid-sialyl;
2) utilizing with uridine diphosphate(UDP) (UDP)-semi-lactosi is β 1,4 galactosyltransferase of substrate, and catalysis connects semi-lactosi to the terminal N-acetylglucosamine of Tegeline glycosyl;
3) utilize with Cytidine-5 '-phosphoric acid-sialyl is the sialyl based transferase of substrate, catalysis connects sialyl to the semi-lactosi of Tegeline glycosyl end.
Tegeline can be PIg antibody or monoclonal antibody.PIg antibody is selected from IGIV G (Intravenous IgG) etc.Described substrate is uridine diphosphate(UDP)-semi-lactosi or uridine diphosphate(UDP)-semi-lactosi-glycogenetic uridine diphosphate(UDP)-semi-lactosi of 4-epimerase enzyme catalysis uridine diphosphate(UDP)-grape etc.
The sialyl based transferase is α 2,6 sialyl based transferases, α 2,3 sialyl based transferases, α 2,8 sialyl based transferases, α 2,9 sialyl based transferases or α 2,8/9 polysialic acids based transferases etc.
The immune globulin antibody of prepared high sialic acid content can be applicable to preparation treatment autoimmune disorder medicine, like the treatment rheumatoid arthritis.
The invention has the beneficial effects as follows: connect the N-that sialyl is connected to IGIV on the glycosyl through glycosyltransferase catalysis, sialicly do not have the composition of anti-inflammatory activity to change the ratio that contains sialic effective constituent and improve its active pharmaceutical ingredient into not containing.Because the IGIV G that handles through the present invention has high-content sialyl (70%), therefore reduced IGIV G and be used to treat the pharmaceutical quantities of autoimmune disorder, and then reduced heavy dose of spinoff that is brought.
Description of drawings
Fig. 1 is the different structure synoptic diagram that the N-of the IGIV of the embodiment of the invention connects glycosyl.
Embodiment
Following examples will combine accompanying drawing that the present invention is further described.
In the buffered soln (100mmol phosphoric acid solution, pH 7.5, contain 2mmol divalent manganesetion and 5mmol divalence mg ion) of 100ml, add IGIV G (0.5g); Uridine diphosphate(UDP)-glucose (0.2g); Cytidine-5 '-triphosphoric acid (0.2g), sialyl (0.1g), uridine diphosphate(UDP)-semi-lactosi-4-epimerase enzyme (20 units activity); β 1; 4 galactosyltransferases ((20 units activity), Cytidine-5 '-phosphoric acid-sialyl synthetic enzyme (20 units activity) and α 2,6 sialyl based transferases (20 units activity).Reaction solution slowly stirs 12h at 4 ℃.Reaction solution is removed all glycosyltransferases through His-Tag protein purification post.Reaction solution is removed small molecules such as responseless uridine diphosphate(UDP)-glucose through dialysis then, buffered soln and metals ion and obtain the sialic IGIV G of high-content.Through detection sialic content, to compare with responseless IGIV G, the sialic content of the catalytic IGIV G of process glycosyltransferase has improved 10～15 times.
The N-that Fig. 1 provides the IGIV of the embodiment of the invention connects the different structure synoptic diagram of glycosyl, the N-that is connected to IGIV G to sialyl through glycosyltransferase catalysis connect on the glycosyl end and there not being the composition of anti-inflammatory activity to be converted into effective constituent (A).Immune globulin antibody contains two N-and connects sugared side chain, for illustrated simple, only draws one.β 1,4 galactosyltransferase is a substrate with uridine diphosphate(UDP)-semi-lactosi, and catalysis connects semi-lactosi (Fig. 1, circle) makes C be converted into D to the terminal N-acetylglucosamine of Tegeline glycosyl; The sialyl based transferase is with Cytidine-5 '-phosphoric acid-sialyl is that substrate catalysis connection sialyl (Fig. 1, rhombus) makes composition B and D be converted into A to the semi-lactosi of Tegeline glycosyl end.
Through experiment; The IGIV G of high sialic acid content and the IGIV G that handles without the present invention are expelled to respectively, and (0.1g/Kg) compares with IGIV G in the mouselet body of rheumatoid arthritis, and the IGIV G of high sialic acid content can protect the joint inflammation degree (drug effect improves 4～5 times) of mouselet effectively.
Claims (7)
1. the preparation method of the immune globulin antibody of high sialic acid content is characterized in that the steps include:
1) Cytidine-5 '-phosphoric acid sialyl synthetic enzyme catalysis sialyl and Cytidine-5 '-triphosphoric acid reaction generation Cytidine-5 '-phosphoric acid-sialyl;
2) β 1,4 galactosyltransferase is a substrate with uridine diphosphate(UDP)-semi-lactosi, and catalysis connects semi-lactosi to the terminal N-acetylglucosamine of Tegeline glycosyl;
3) α 2,6 sialyl based transferases are with Cytidine-5 '-phosphoric acid-sialyl is that substrate catalysis connects sialyl to the semi-lactosi of Tegeline glycosyl end.
2. the preparation method of the immune globulin antibody of high sialic acid content according to claim 1 is characterized in that Tegeline is PIg antibody or monoclonal antibody.
3. the preparation method of the immune globulin antibody of high sialic acid content according to claim 2 is characterized in that PIg antibody is selected from IGIV G.
4. the preparation method of the immune globulin antibody of high sialic acid content according to claim 2 is characterized in that monoclonal antibody is selected from immunoglobulin M or immunoglobulin A.
5. the preparation method of the immune globulin antibody of high sialic acid content according to claim 1; It is characterized in that substrate is uridine diphosphate(UDP)-semi-lactosi, or uridine diphosphate(UDP)-semi-lactosi-glycogenetic uridine diphosphate(UDP)-semi-lactosi of 4-epimerase enzyme catalysis uridine diphosphate(UDP)-grape.
6. treat the application in the autoimmune disorder medicine according to the immune globulin antibody of the prepared high sialic acid content of claim 1 in preparation.
7. application according to claim 6 is characterized in that described treatment autoimmune disorder medicine is a medicine for treating rheumatoid arthritis.
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| CN2007100093044A CN101358225B (en) | 2007-07-30 | 2007-07-30 | Method for preparing high sialic acid content immune globulin antibody and application |
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| CN2007100093044A CN101358225B (en) | 2007-07-30 | 2007-07-30 | Method for preparing high sialic acid content immune globulin antibody and application |
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| CN101358225B true CN101358225B (en) | 2012-02-01 |
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Non-Patent Citations (4)
| Title |
|---|
| Ramesh Jassal et.al.Sialalation of human IgG-Fc Carbohydrate by transfected rat a2,6-sialyltransferase.《Biochemical and Biophysical Research Communication》.2001,第286卷243-249. * |
| RameshJassalet.al.SialalationofhumanIgG-FcCarbohydratebytransfectedrata2 6-sialyltransferase.《Biochemical and Biophysical Research Communication》.2001 |
| T. Shantha Raju et.al.Glycoengineering of Therapeutic Glycoproteins: In Vitro Galactosylation and Sialylation of Glycoproteins with Terminal N-Acetylglucosamine and Galactose Residues.《Biochemistry》.2001,第40卷参见第8871页图解1－2，第8869页左栏倒数第1段. * |
| Yoshikatsu Kaneko, et al..Anti-Inflammatory Activity of Immunoglobulin G Resulting from Fc Sialylation.《Science》.2006,第313卷(第670期),参见第670页摘要,第672页最右栏倒数第2段倒数1-8行,第673页最左栏第2段. * |
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