KR20080105735A - A process for preparing an additionally glycosylated recombinant alpha-galactosidase and an additionally glycosylated recombinant alpha-galactosidase prepared therewith - Google Patents

Abstract

A method for preparing a recombination human alpha-galactosidase where a sugar chain is added, a recombination human alpha-galactosidase prepared by the method, and a composition for treating the Fabry disease containing the recombination human alpha-galactosidase are provided to improve stability and therapeutic effect. A method for preparing a recombination human alpha-galactosidase where a sugar chain is added comprises the steps of separating and purifying the recombination human alpha-galactosidase prepared by the gene recombination method; and adding a sugar chain to the purified recombination human alpha-galactosidase in vitro.

Description

Process for preparing an additionally glycosylated recombinant alpha-galactosidase and an additionally glycosylated recombinant alpha-galactosidase prepared therewith}

1 is a schematic diagram of a recombinant expression vector for expressing recombinant human alpha-galactosidase.

FIG. 2 shows the results of comparing peaks (pI) and molecular weights of Fabrazyme and ISU303. FIG. 2A is a result of comparing peaks through IEF (isoelectrofocusing), and FIG. 2B shows SDS-PAGE (SDS-polyacrylamide). Gel electrophoresis) was used to compare the molecular weight of pabrazyme with ISU303 (M; pI marker, SM; size marker, Fz; fabrazyme).

3 is a result of navigating the glycosylation reaction conditions, Figure 3 A is a SDS-PAGE result, FIG. 3B is a glycosylation level analysis via 2D gel electrophoresis experiment (two-dimensional gel electrophoresis).

Figure 4 shows the results of the sugar chain addition reaction conditions using IEF, Figure 4A shows the comparison of the two-fold sialic acid and two-fold combination reaction results, Figure 4B shows the degree of sialic acid addition by reaction time in the two-fold combination reaction conditions Seems to compare.

5 is a comparison result of the degree of sugar chain addition of Fabrazyme, ISU303, and ISU303-1, FIG. 5A is a result of SDS-PAGE, and FIG. 5B is a result of comparison of sugar chain addition using gel electrophoresis.

FIG. 6 shows sugar chain profile analysis results of ISU303 (FIG. 6A) and ISU303-1 (FIG. 6B) using Maldi-TOF (matrix-assisted laser desorption / ionization-time of flight).

Figure 7 shows the body activity according to the sugar chain addition of alpha-galactosidase.

Fabry disease, a sex chromosomal recessive genetic disease, is one of the representative Lysosomal Storage Diseases (LSD). Alpha-galactosidase serves to hydrolyze the alpha-galactoside linkage site at the terminal site of glycolipids. Deficiency of this enzyme causes the body to accumulate lipids known as globboriaosyceramide (Gb3) or ceramide trihexoside (CTH). Gb3 is mainly deposited on vascular endothelial cells such as kidney, heart, liver, spleen, or increased in the plasma of the patient. Gradual accumulation of glycolipids leads to acroparesthesia, hypohidrosis, angiokeratoma, corneal opacities, autonomic dysfunction, and gradually to the heart, brain, and kidneys. Developing vascular disease leads to death. The average life expectancy of patients is reported around 40 years of age, and current treatments include enzymatic replacement therapy (ERT), which involves recombination of a deficient alpha-galactosidase with symptomatic therapy for symptomatic relief. Two therapeutic enzyme products are available. The incidence rate is about 1 / 40,000, the number of patients in the US is estimated at 15,000, and the number of patients around the world is estimated at 15,000.

Alpha-galactosidase is an enzyme that cleaves alpha-D-galactose at the end of sugar chains attached to glycolipids. It is a glycoprotein with three N -linked sugar chains and acts as a homodimer. CDNA was first identified and cloned in 1986 (Bishop et al. (1986) Proc. Natl. Acad. Sci. USA , 83 , 4859).

Genzyme and TKT (TransKaryotic Therapeutics, Inc.) in Europe currently use recombinant alpha-galactosidase to treat Fabry disease under the trade names Fabrazyme and Replagal. It is developed as a medicine and sold in the United States and Europe. These products are proteins produced by expression in animal cells, CHO (Chinese Hamster Ovary) cells and human cells, respectively, using genetic recombination technology. Although the amino acid sequences of these proteins are identical, there are differences in the structure and composition of the attached sugar chains. Accordingly, differences in biodistribution and pharmacokinetics were observed (Lee K. et al. (2003) Glycobiol . 13 , 305).

Alpha-galactosidase, like other medicinal glycoproteins, depends greatly on the structure and components of the attached sugar chains, and the efficacy, body stability, and tissue distribution are greatly influenced. Among these, mannose-6-phosphate, which plays a very important role in the targeting of intracellular lysosomes, and sialic acid, which plays an important role in the half-life of the body, are found in alpha-galactosidase. It has a big impact on performance as a therapeutic agent. In particular, the extent to which the sugar chain ends attached are capped with sialic acid is known to have a great influence on the half-life of the body (Goochee et al. (1991) BioTechnology 9 , 1347). Normally, cells have the ability to secrete glycoproteins with sialic acid-capped sugar chains, but galactose or N-acetyl, which do not occur with sialic acid capping, depending on the type of glycoprotein secreted and the physiological state of the cells. Occurs with glucosamine-terminated sugar chains. When sugar-attached proteins that end with galactose or N-acetylglucosamine instead of sialic acid are injected into the blood, they are nonspecifically recognized and eliminated by the bisialic acid glycoprotein receptor in the liver (Weigel et al. 2002) Bioch.Biophy.Acta 1572 , 341). In this case, there is a problem that the half-life in the body is reduced compared to the glycoprotein having a sugar chain capped with sialic acid.

Recombinant protein products produced in animal cells are generally very expensive. In particular, enzyme treatment for Fabry's disease costs about 200 million won per patient per year, resulting in economic loss not only for patients and their families but also for the country. In order to reduce the production cost in animal cells, it is essential to overexpress the recombinant protein and mass produce it. However, overexpression of recombinant proteins above a certain level often results in cells secreting their sialic acid capping incompletely, since their ability to add sugar chains cannot keep up with the secretory expression of glycoproteins (Goochee et al. (1994). Curr. Opin. Biotechnol. 5, 546). Processes to add sialic acid have been developed to solve these problems (US6399336, Raju et al. (2001) Biochemistry 40, 8868), but succeeded by applying concretely to alpha-galactosidase, a treatment for Fabry disease. There was no report yet.

The inventors have established a reaction process for increasing sialic acid capping by using sialic acid and galactose addition enzymes to alpha-galactosidase produced in a highly productive cell line and in which sugar chain addition is incompletely reacted. The present invention was completed by confirming that the added recombinant alpha-galactosidase had an improved effect on the stability and therapeutic efficacy in the body compared to the existing alpha-galactosidase.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

It is an object of the present invention to provide a sugar chain-added alpha-galactosidase having improved stability and therapeutic efficacy by increasing sialic acid capping to a recombinant alpha-galactosidase in which a sugar chain addition reaction is incomplete.

Still another object of the present invention is to provide the sugar chain-added alpha-galactosidase as a composition for treating Fabry disease.

Still another object of the present invention is to provide a method for preparing the alpha-galactosidase and a therapeutic composition.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

As a first aspect to achieve the above object, the present invention is to modify the sugar chain structure of alpha-galactosidase used as a Fabry disease treatment agent to add a lot of sialic acid (sialic acid, N-acetylneuraminic acid) to the sugar chain terminal Thereby providing a method for preparing a sugar chain variant having increased body stability and therapeutic efficacy.

In the present invention, "alpha-galactosidase" (α-galactosidase) refers to alpha-galactoside including galactose oligosaccharides, galactomannans, galactolipids, and the like. It is an enzyme that catalyzes the hydrolysis of the ends of non-reducing alpha-d-galactose residues in -galactosides.

As used herein, the term "sugar chain" refers to a molecule in which various sugars such as glucose are linked like a ring. Sugar chains are key to the function of various proteins or bioactive substances in vivo, such as regulating the properties and functions of cells or recognizing, accepting and delivering substances, depending on which sugars are attached. Recently, the sugar chains attached to proteins and lipids are known to be linked to many diseases, and if it is possible to develop a drug that changes the combination of sugars that make up the function of sugar chains, it is expected that early detection and treatment of the diseases will be possible. Therefore, the basic and applied researches leading to sugar chain biology and sugar chain engineering following genomics are actively progressing in various countries around the world.

The alpha-galactosidase of the present invention, like other glycoproteins, is greatly influenced by the structure and components of the attached sugar chains, such as efficacy, stability and tissue distribution. Among these, mannose-6-phosphate, which plays a very important role in the targeting of intracellular lysosomes, has a great effect on the performance of alpha-galactosidase as a therapeutic agent. The results of the study have been reported (Sakuraba et al., 2006). In addition, as with other glycoproteins, in particular, the degree to which the sugar chain ends are capped with sialic acid is expected to have a great influence on the half-life of the body (Goochee et al. (1991) Biotechnology 9 , 1347).

In the present invention, "sialic acids" are also referred to as sialic acid, and are particularly contained in mucin of the submandibular gland. The chemical structure is n-acetyl or n-glycol, sometimes o-acetylated, by noiramic acid, the aldol condensate of pyruvic acid and mannosamine. Hemagglutination by influenza viruses has been found to be inhibited by the glycoprotein sialic acid. Sialic acid is present at the non-reducing end of these glycoproteins. Since sialic acid is involved in keto-glycosidic bonds, the carboxyl group is in a free state. At pH in vivo, this carboxyl group dissociates almost and has a negative charge. This fact is believed to bring about the important physiological significance of sialic acid, such as hemagglutination.

Sialic acid plays an important role in the half-life of alpha-galactosidase. In particular, the degree to which the sugar chain terminal attached to alpha-galactosidase is capped with sialic acid is known to have a great influence on the half-life of the body (Goochee et al. (1991) Biotechnology 9 , 1347). In the present invention, recombinant alpha-galacto is established by establishing a reaction process for increasing sialic acid capping to alpha-galactosidase (code name: ISU303), an overexpressed recombinant protein in which the sugar chain of the cell lacks the capacity of glycoprotein. Increased body stability and active sialic acid capping and therapeutic efficacy of sidase.

Specifically, the sugar chain addition process according to the present invention is at least 35 mU and at most 45 mU of alpha-2,3-sialyltransferase per mg of alpha-galactosidase produced by genetic recombination. (α-2,3-sialyltransferase), beta-1,4-galactosyltransferase (between 35 mU and 45 mU or less), beta-1,4-galactosyltransferase (UD-1,4-galactosyltransferase) and UDP-galactose (15 or more and 25 μmol or less) Alpha-galactosidase, characterized by the simultaneous treatment of UDP-Galactose), 15 μmol or more and 25 μmol or less CMP-NANA (N-acetylneuramic acid, sialic acid) and 15 μmol or more and 25 μmol or less of MnCl ₂ . Is a sugar chain addition method.

Preferably, the sugar chain addition process according to the present invention is characterized in that it proceeds by adding the treatment reaction once more. That is, (i) more than 35 mU or more and 45 mU or less alpha-2,3-sialyltransferase, 35 mU or more and 45 mU per mg of alpha-galactosidase produced by genetic recombination. Co-treatment with up to beta-1,4-galactosyltransferase, from 15 μmol to 25 μmol UDP-galactose, from 15 μmol to 25 μmol CMP-NANA, and from 15 μmol to 25 μmol MnCl ₂ After the reaction at 37 DEG C for at least 1 hour, (ii) alpha-2,3-sialyltransferase, beta-1,4-galactosyltransferase, UDP at the same concentration as described above in the reaction solution, respectively. It provides a method for adding a sugar chain of alpha-galactosidase, characterized by further adding galactose, CMP-NANA and MnCl ₂ for further 1 hour or more at 37 ° C.

More preferably, the sugar chain addition process according to the present invention is at least 38 mU to 42 mU alpha-2,3-sialyltransferase (α) per mg of alpha-galactosidase at each step. -2,3-sialyltransferase), 38 mU or more, 42 mU or less beta-1,4-galactosyltransferase (β-1,4-galactosyltransferase), 18 μmol or more and 22 μmol or less UDP-galactose (UDP- Galactose), CMP-NANA (N-acetylneuramic acid, sialic acid) of 18 μmol or more and 22 μmol or less and MnCl ₂ of 18 μmol or more and 22 μmol or less are simultaneously treated.

The reaction in each of the above steps is characterized in that for each reaction at 37 ℃ 1 hour or more, preferably 7-10 hours, more preferably 12 hours or more.

Recombinant human alpha-galactosidase, in which the sugar chain addition is incompletely applied for applying the sugar chain addition process according to the present invention, is obtained through any genetic recombination method well known to those skilled in the art. May be galactosidase. In a preferred embodiment of the invention, in order to prepare such recombinant human alpha-galactosidase, (i) polynucleotides encoding human alpha-galactosidase and dhfr (dihydrofolate reductase) ), The expression vector expressing the polynucleotides encoding each or together is transformed into CHO (Chinese hamster ovary) cells lacking the dhfr gene, and then (ii) human alpha-gal via gene amplification using MTX (methotrexate). Obtaining a cell line high expressing lactosidase, (iii) culturing the high expressing cell line to isolate and purify the recombinant human alpha-galactosidase from the culture, and code the recombinant human alpha-galactosidase thus separated. It was named ISU303. That is, in a preferred embodiment of the present invention, sugar chains were added to the recombinant human alpha-galactosidase ISU-303 thus prepared and separated according to the method of the present invention, and thus new recombinant human alpha- with sugar chains added thereto. Galactosidase (ISU-303-1) was obtained. However, the method of preparing recombinant human alpha-galactosidase for applying the in vitro sugar chain addition process according to the present invention due to incomplete sugar chain addition is not limited to the method of the preferred embodiment of the present invention described above. It will be understood by those skilled in the art that various methods known in the art can be applied to prepare recombinant human alpha-galactosidase in which such sugar chain addition has not occurred sufficiently. It is apparent to those skilled in the art that the scope of the present invention is included.

Accordingly, the present invention is another embodiment, alpha- which is prepared according to the above production method to add more sialic acid (N-acetylneuraminic acid) to the sugar chain terminal to increase the stability sialic acid capping and therapeutic efficacy in the body Provides galactosidase. Referring to FIG. 5 of the present specification, compared to recombinant human alpha-galactosidase (ISU303) (FIG. 5A) produced in an overexpressing cell line according to a genetic recombination method, it is prepared by further adding a sugar chain according to the method of the present invention. It can be seen that the sugar chain structure of one alpha-galactosidase (ISU303-1) (FIG. 5B) was altered and more sugar chain addition and sialic acid capping occurred. In addition, the sugar chain structure of the sugar chain-added ISU-303-1 can be confirmed to have a higher sialic acid content than Fabrazyme or Reflagal, a recombinant human alpha-galactosidase product currently on the market (Table 2).

In another aspect of the present invention, there is provided a composition for treating Fabry disease comprising alpha-galactosidase in which the sugar chain structure is modified as described above. As shown in Table 2, the sugar chain-added alpha-galactosidase according to the present invention, as shown in Figure 6, exhibits much improved therapeutic efficacy and commercial stability, compared to the existing Fabry disease, a commercially available Fabry disease treatment. .

The therapeutic compositions of the invention may be formulated in various formulations using conventional techniques in the art, by incorporating a compound of the invention or a pharmaceutically acceptable salt thereof with an inert pharmaceutical carrier or diluent suitable for oral, parenteral or topical administration. It can be formulated as.

Representative of parenteral formulations are injectable formulations, preferably aqueous isotonic solutions or suspensions. Formulations for oral administration include, for example, tablets, capsules, and the like, which include diluents (lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, etc.), lubricants (silica, talc) in addition to the active ingredients. , Stearic acid and its magnesium or calcium salt and / or polyethylene glycol). Tablets or may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally starch, agar, alginic acid or its sodium Disintegrating or boiling mixtures such as salts and / or absorbents, colorants, flavors and sweeteners.

The amount of the active compound to be administered may vary depending on various factors such as the subject to be treated, the severity of the disease, the mode of administration, the sex, the doctor's prescription, etc., and the effective amount can be easily determined by those skilled in the art. Can be. The compounds of the present invention can be administered orally in the form of injections on a daily basis, weekly basis or monthly basis in an amount of 0.1 to 10 mg / kg for the purpose of treating Fabry disease. May be administered.

As a result of the test on the stability and efficacy of the sugar chain-added alpha-galactosidase according to the present invention, the stability and efficacy was improved than the conventional recombinant alpha-galactosidase.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Preparation of Recombinant Human Alpha-Galactosidase (ISU303) Using Genetic Recombination Method

Example 1-1) Preparation of Alpha-galactosidase Expression Vector

CDNA library of fibroblast (WI38 cell line) was used to obtain human alpha-galactosidase gene cDNA. First, DNA was isolated and purified by amplifying the cDNA library and PCR was performed using the same. Pharge DNA was isolated and purified using Wizard Lambda Preps DNA Purification System (Promega) .In the process of constructing the expression plasmid, DNA manipulation and transformation of E. coli were performed by Sambrook et al. (Molecular Cloning, 1989, Cold Spring Harbor Laboratory Press). Was carried out according to the method of.

PCR primers were prepared such that the entirety of the protein encoding portion was synthesized based on the previously reported cDNA sequence of alpha-galactosidase (Gene Bank BC002689). First, amplify the alpha-galactosidase gene using the library DNA as a template by the first PCR, and recognize the recognition sequences of NheI and XhoI restriction enzyme for insertion into the expression vector by the second PCR at the 5 'and 3' ends. Each was introduced. In addition, to increase protein synthesis, the Kozac sequence (GCCACC) was included 5 'above the start codon at which protein synthesis begins.

1st PCR Primer 1: 5'-TCCGG TCACC GTGAC AATGC AGC-3 '(SEQ ID NO-1)

         Primer 2: 5'-GTCTT TTAAT GACAT CTGCA TAGTC C-3 '(SEQ ID NO-2)

2nd PCR Primer 3: 5'-CTA GCT AGC CAC CAT GCA GCT GAG GAA CCC AG-3 '(SEQ ID NO-3)

         Primer 4: 5'-CCG CTC GAG TTA AAG TAA GTC TTT TAA TG-3 '(SEQ ID NO-4)

The second PCR product was treated with NheI and XhoI restriction enzymes, purified by DNA fragments through agarose gel electrophoresis, and then pMSG vector (opened by NheI and XhoI restriction enzymes) (Korean Patent Application No. 10-2000-0043996, PCT application PCT / KR01 / 01285) to obtain a recombinant human alpha-galactosidase expression vector pMSG-α-gal (see FIG. 1).

After ligation for 2 hours at 25 ° C., E. coli JM109 was transformed. The transformants were plated in LB medium containing X-gal to select white colonies. Sequencing confirmed that human alpha-galactosidase cDNA was cloned correctly.

Example 1-2 Alpha-galactosidase High Expression Cell Line Construction

2ug recombinant plasmid (pMSG-α) using CHO cells (CHO / DHFR- (DG44), source Dr. Lawrence Chasin, columbia University) lacking the gene encoding the dihydrofolate reductase enzyme as a host cell -gal), 16 ng of pDCHIP (plasmid with DHFR gene) and 5.3 ul of Dosper (Roche) were added for transformation. The overall process for transformation was done according to the Dosper (Roche) protocol.

After 2 days the transformed cells were initially inoculated into 6 well plates (6-7 × 10 ⁵ cells / well) and incubated with α-MEM (w / o) + 10% dFBS medium to initially adapt cell lines. Got. In addition, adaptive cell lines were selected by increasing the concentration of MTX (methotrexate) in the medium from the initial 10 nM to 1 uM to amplify the expression level, and the expression level was measured by enzyme activity measurement. In order to select single cell lines with high expression efficiency, single clones were isolated by culturing at 0.5 cell / well in 96 well plates by limiting dilution, and high productivity clones were selected by enzyme activity assay. The productivity of the clones selected through this procedure was 150 mg / L / day or more, more than five times higher than that of the existing therapeutic drug, pabrazyme, and more than 30 times higher than the cell lines disclosed in the prior patent (EP01020528).

Example 1-3) Alpha-Galactosidase Purification

After centrifugation to remove cell debris and impurities in the culture medium with alpha-galactosidase, the insoluble particles were removed by filtration with a 0.45 um filter. The recovered solution was concentrated with buffer change using the UF system to prepare a sample to be purified using an anion exchange chromatography column.

Alpha-galactosidase is adsorbed on the column using anion exchange chromatography to remove components, impurities, pyrogen, etc. contained in the media, and NaCl gradient elution -Galactosidase was isolated. In the polishing step, HCP, HCD and other impurities are removed using a hydrophobic chromatography column to separate and purify high-purity alpha-galactosidase, and a flow through method is performed through a cation exchange chromatography column. Further purification was carried out to separate and purify high purity alpha-galactosidase suitable for the experiment. Finally, the final purification stock solution is separated and purified using a gel filtration chromatography column to remove the polymers and fragments, and then the formulation buffer is prepared using the UF-system to produce the desired concentration stock solution. ) And concentrated to form the final alpha-galactosidase stock solution.

Example 2 Comparison of Isometric and Molecular Weights of Fabrazyme and ISU303

Alpha-galactosidase (ISU303) and Fabra of Examples 1-3) obtained from the cell lines of Examples 1-2) which are five times more productive than alpha-galactosidase producing cell lines reported to date. Isoelectrofocusing (IEF), SDS-PAGE and 2D gel electrophoresis experiments were performed to compare the peaks and molecular weights of Zymeim (Zenzyme, USA). SDS-PAGE was performed according to the preceding method (Molecular Cloning, 1989, Cold Spring Harbor Laboratory Press).

After performing IEF for isotopic determination of ISU303, 5-10 ug of protein was loaded into an IEF gel (Invitrogen), followed by 100 V for the first hour, then 200 V for the first hour, and 500 V for the last 30 minutes. I put the voltage on. The IEF finished gel was fixed with protein using 12% TCA solution, stained using Coomassie Blue solution, and destained with methanol and acetic acid mixed solution to identify protein bands ( FIG. 2A ).

2D gel electrophoresis experiment for comparing the peak and the molecular weight was performed by the following method. After lyophilization of about 30 ug of protein, it is dissolved in 350 ul of rehydration buffer (8 M Urea, 2% CHAPS, 0.01% bromo phenol blue, 0.28% DTT), which is immobilized pH gradient (IPG) buffer. 5 ul of pH 3-10 (Amersham Biosciences) was added and placed in the holder. Place IPG immobiline DryStrip (pH 3-10 or 3-7) on top of it without bubbles, drop the oil and cover it. The prepared holder was placed on an Ettan IPGphor system (Amersham Biosciences) and rehydrated at 20 ° C. for 12 hours, and the voltage was increased from 200 V 1 hour, 500 V 1 hour, 100 V 1 hour, and 8,000 V to 80,000 V. In the end, it takes up to 50 uA per strip. Isoelectrofocusing strips were treated with first buffer (50 mM Tris-HCl pH 8.8, 6 M Urea, 30% glycerol, 2% SDS, bromophenol blue, 1% DTT) and second buffer (50 mM Tris-HCl pH8.8, Equilibration in 6 M Urea, 30% Glycerol, 2% SDS, Bromophenol Blue, 2.5% iodoacetamide for 15 minutes and then placed on 12% SDS-polyacrylamide gel (200, 200, 1.5 mm) . Then, electrophoresis was performed at 200 V for about 5 hours, and then stained with Coomassie Brilliant Blue or silver nitrate.

Alpha-galactosidase ISU303, which is highly expressed in CHO, had a slightly lower molecular weight when compared to conventional Fabrazyme ( FIG. 2B ). PNGase F (peptide: N-glycanse F) cleavage of attached N-linked sugar chains (PNGase F reaction conditions were carried out according to the manual provided by New England Biolabs) and compared to the same size. It was confirmed that the difference in molecular weight is due to the difference in attached sugar chains. In addition, pabrazyme was located in the acidic region from pi to 5.0 to 3.5, while ISU303 was located in the neutral region from pI to 5.5 to 4.0 in all three culture conditions. This difference is judged to be due to the inability of sugar chains such as sialic acid to be completely made due to overexpression of proteins in the case of ISU303 ( FIG. 2A ).

<Example 3> Searching for sugar chain addition reaction conditions

A sugar chain addition reaction was performed in vitro to control the sugar content of the alpha-galactosidase (ISU303) produced in Example 1.

First, the sialic acid and galactose addition reactions were performed under various conditions in order to search for the optimal sialic acid addition condition, and then the degree of sugar chain addition was confirmed by performing Isoelectrofocusing, SDS-PAGE and 2D gel electrophoresis as in Example 2. ( FIG. 3-4 ). 50 mM MES buffer (pH 6.4) was used as the sugar chain addition reaction solution. For this purpose, ISU303 was exchanged with 50 mM MES buffer (pH 6.4) using a YM-10 membrane manufactured by Millipore. 0.5 mg of protein was added to 1 ml of reaction solution, 10 mU / ul beta-1,4-galactosyltransferase (GT), 0.5 mU / ul alpha-2,3 The sugar chain portion of -sialylltransferase (ST) was added to the enzyme, 2 mM UDP-galactose, 2 mM CMP-NANA (N-acetylneuraminic acid, sialic acid), and a cofactor of 2 mM MnCl ₂ . The final amount described in Table 1 was added and reacted twice for 12 hours at 37 ° C. for a total of 24 hours.

Table 1 shows Sialic acid and galactose addition enzyme reaction conditions.

Reaction condition First reaction (12 hours) Second reaction (12 hours) GT (mU) UDP- Galactose (umol) ST (mU) CMP- NANA (umol) MnCl ₂ (umol) GT (mU) UDP- Galactose (umol)   ST (mU) CMP- NANA (umol) MnCl ₂ (umol) Sialylation - - 10 5 5 - - 10 5 5 Sequential 20 10 - - 5 - - 20 10 5 Combined 10 5 10 5 5 10 5 10 5 5 2x Sialylation - - 20 10 10 - - 20 10 10 2x Combined 20 10 20 10 10 20 10 20 10 10

Five reaction conditions were selected as in Table 1 to simulate various reactions. First, Sialylation reaction conditions were 12 hours with sialic acid addition enzyme alpha-2,3-sialylltransferase and sialic acid precursor CMP-NANA as substrate. In order to replenish the spent enzyme and substrate, additional alpha-2,3-sialyltransferase and CMP-NANA are added and reacted for 12 hours to react for a total of 24 hours.

Second, the sequential reaction conditions were first treated with N-acetyl using galactose addition enzymes beta-1,4-galactosyltransferase and UDP-galactose for 12 hours. After adding galactose to sugar chains terminated with glucosamine, reaction was performed for 12 hours with sialic acid addition enzymes alpha-2,3-sialyltransferase and CMP-sialylltransferase. This is a reaction condition for capping sialic acid to all sugar chains ending with galactose.

Third, the combined reaction condition is a condition for adding galactose and sialic acid at the same time, beta-1,4-galactosyltransferase, alpha-2,3-sialyltransferase and UDP-galactose. It is a condition that the galactose addition reaction and the sialic acid addition reaction occur at the same time by putting CMP-NANA at the same time, and reacting for 12 hours after adding the same enzyme and substrate after 12 hours.

Fourth and fifth, two-time sial oxidation reaction and two-time combination reaction conditions are the conditions under which the reaction is carried out in a state in which excess amounts of enzyme and substrate are added to the sial oxidation conditions and the combination reaction conditions, respectively. Under all the reaction conditions, the same amount was added again after 12 hours reaction to add a sufficient amount of cofactor MnCl ₂ ( FIGS. 4A and 4B ).

As a result of carrying out the reaction under various conditions as shown in Table 1 , the optimum condition under which the sialic acid addition occurs most frequently is a two-fold combination reaction condition in which galactose and sialic acid addition reactions are simultaneously performed in a state in which an enzyme and a substrate are added in excess. It was found that ( Fig. 3 and 4A ). Molecular weight was the highest in the two-fold combination reaction conditions and pI value was also the lowest (pI 5 or less), it was confirmed that the sialic acid added to the same degree as the conventional fabrazyme. As a result of the reactions performed at different times under the two-fold combination reaction conditions found to be optimal, after one hour of reaction, the same amount of enzyme and substrate were added, followed by an additional reaction for one hour, which was equivalent to Fabrazyme. It was found that sialic acid addition occurred to an extent ( FIG. 4B ). The sugar chain variant alpha-galactosidase with sufficient sialic acid addition was named ISU303-1. ISU303-1, similar to fabrazyme, has increased molecular weight and reduced pI value than ISU303 through SDS-PAGE and 2DE methods. It was confirmed that the molecular weight and pI value of the degree ( Fig. 5 ). As a result of comparing the amount of NANA and galactose by using Bio-LC, it was confirmed that the content of NANA increased 2.3 times compared to ISU303, and that the ratio of NANA and galactose was nearly 1 It could be confirmed ( Table 2 ).

Table 2 relates to NANA and galactose content analysis using Bio-LC.

Mol ratio per AGA molecule NANA / galactose ratio NANA Galactose Fabrazyme ^* 7.0 8.0 0.88 Repragal ^* 6.9 W0.6 12.2 0.56 ISU303 2.7 W0.5 5.3 0.51 ISU303-1 6.2 5.7 W1.0 1.09

* Literature data (Karen et al. (2003) Glycobiology 13, 305)

Example 4 Analysis of Sugar Chain Profiles of Sugar Chain Variants

In order to compare the sugar chains of ISU303-1 obtained in Example 3 with the sugar chains of ISU303, which is a protein before the competitor's fabrazyme and the sugar chain addition reaction, sugar chains were separated and a sugar chain profile structure analysis using MALDI-TOF was performed.

First, to obtain a sugar chain, the glycoprotein is mixed with denaturation buffer (5% SDS, 10% β-mercaptoethanol), boiled at 100 ° C. for 10 minutes, cooled to room temperature, and finally 0.05 M sodium phosphate (pH). 7.5) and 1% NP-40 were added and reacted with PNGase F for 16 hours at 37 ° C. This cleaved sugar chain was purified using 150 mg PGC column (Alltech Associates, Deerfield, IL). This column contains 1 M NaOH, deionized water, 80% (v / v) acetonitrile / 0.10% (v / v) trifluoroacetic acid (TFA), 25% (v / v) ) Acetonitrile / 0.05% (v / v) TFA, 25% (v / v) acetonitrile, and 3 ml of pre-washing 2 ml each in deionized water, 25% acetonitrile, 0.075% Glycans were isolated by TFA (Urs et. Al). Sugar chains purified using a PGC column were dried with a SpeedVac evaporator. 50 μl and 10 μl of anhydrous DMSO and methyl iodide were added to the dried sugar chains, and then mixed well. The mixture was left at room temperature for 2 hours, and then mixed with 100 μl of anhydrous DMSO. This sample was dried with a SpeedVac evaporator (takes about 6 hours) to perform MALDI-TOF analysis.

For profiling analysis of sugar chains to which sialic acid was added, methylesterfication or permethylation reaction was performed on the sugar chains prepared as described above. 50 μl and 10 μl of anhydrous DMSO and methyl iodide were added to the dried sugar chains for methylesterfication, and then mixed well and left at room temperature for 2 hours, followed by mixing 100 μl of anhydrous DMSO. This sample was dried on a SpeedVac evaporator (takes about 6 hours) to perform MALDI-TOF analysis. For polar methylation, first, NaOH 1g was added to 4 ml of anhydrous DMSO, and the mixture was prepared to be cloudy. 2-100 μl was added to the dried sugar chains, mixed well, and allowed to stand at room temperature for 30 minutes (at this time, votexing was performed intermittently). 1.5 times of CH ₃ I was added and left at room temperature for 1 hour in the same manner, and the same amount of DMSO / NaOH and CH ₃ I were further added and left at room temperature for 1 hour. And CHCl ₃ corresponding to 5 times the volume was added, and washed with 2 times the volume of distilled water. Distilled water washing was repeated to remove basicity, and finally, the organic solvent layer was dried to perform MALDI-TOF analysis.

For sugar chain profiling using MALDI-TOF, two matrices of DHB (2,5-dihydroxybenzoic acid) and 6ATT (6-aza-2-thiothymine) were saturated with 25% acetonitrile and 0.1% TFA, creating a 1: 1 solution. The mixture was prepared at the ratio of. The dried sugar chains were dissolved in the same solution as above, and then mixed with the prepared matrix solution in a ratio of 1 to 1 to prepare. At this time, TFA was added to maintain the acidic pH during the crystal formation of the sample and the matrix. The prepared samples were placed on a target and dried and analyzed using Microflex TOF (Bruker Daltonik GmbH. Bremen, Gemany).

According to the sugar chain profiling analysis using MALDI-TOF, ISU303 lacks biantenary and triantennary added sugar chains compared to its competitor, Pabrazyme, but has sufficient added sialic acid. It was confirmed that the variant ISU303-1 had a sugar chain structure of biantennary and triantennary to which sialic acid was added, similar to fabrazyme ( FIG. 6 ).

Example 5 Confirmation of Efficacy of ISU303-1

In order to confirm the treatment effect of Fabry disease of ISU303-1 obtained in Example 3, Dr. Alpha-galactosidase knockout mice developed by Roscoe O. Brandy team were used (Ohshima et al., PNAS. 18, 2540-4, 1997). After administration of recombinant alpha-galactosidase to Fabry mice, it was confirmed whether the residual amount of ceramide trihexoside (CTH) in vivo was reduced by administration of alpha-galactosidase enzyme, and alpha- Galactosidase enzyme activity was measured.

Example 5-1) Tissue Degradability Test of Ceramide Trihexoxide (CTH)

CTH is an intermediate that accumulates in the body due to a deficiency of alpha-galactosidase enzyme in Fabry disease patients, and CTH residual amount is decreased by alpha-galactosidase enzyme treatment. Therefore, by measuring the amount of CTH after alpha-galactosidase enzyme treatment it can be confirmed whether the alpha-galactosidase function properly in vivo. Tissues are homogenized in buffer A (28 mM citric acid, 44 mM indium phosphate, and 5 mg / ml sodium taurocholate, pH 4.4), Lipids were extracted with isopropanol. The extracted fat is saponified with chloroform and methanol and diluted with chloroform / methanol / water / ammonia (66: 30: 3.5: 0.5). Diluted glycolipids were analyzed using an evaporative light scattering detector. At this time, the temperature was 50 ℃, nitrogen pressure (nitrogen pressure) was 2.4 bar and CTH was obtained at 10 bar.

Example 5-2) Measurement of In vivo Distribution of Alpha-galactosidase

The distribution of recombinant alpha-galactosidase in the body was analyzed by measuring the activity of alpha-galactosidase by major organs. Tissue samples were homogenized in Buffer A (28 mM citric acid, 44 mM disodium phosphate, and 5 mg / ml sodium taurocholate, pH 4.4), followed by ultrasound Proteins were extracted by sonication and centrifugation at 20,000 g for 30 minutes. The measurement of alpha-galactosidase activity was determined by extracting the extracted protein from buffer B (28 mM citric acid, 44 mM disodium phosphate, 5 mM 4-methylumbelliferyl-aD-galactopyranoside (4-methylumbelliferyl-aD). -galactopyranoside), 4 mg / ml BSA, and 0.1 M N-acetylgalactosamine (N-acetylgalactosamine) for 1 hour, and then the fluorescence was measured using a fluorimeter to calculate the enzyme activity.

As a result of the efficacy evaluation using Fabry mouse, the activity of the enzyme targeted by tissue was higher in ISU303 and ISU303-1 than in the existing therapeutic drug, Fabrazyme, and especially in the kidney, the main target organ It was confirmed that the sugar structure was the highest in the modified ISU303-1 showing superior efficacy in the treatment of Fabry disease ( FIG. 7 ).

While specific embodiments of the present invention have been described in detail, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments of the present invention, and thus the scope of the present invention is not limited thereto. . In other words, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

As described above, alpha-galactosidase, an overexpressed recombinant protein produced in a high protein cell line of protein according to the present invention and lacking the sugar chain addition ability of the cell as compared to the glycoprotein expression ability, is used for sialic acid and galactose addition enzymes. By establishing a reaction process that increases sialic acid capping, it is possible to provide a sugar chain-added recombinant alpha-galactosidase with increased body stability and Fabry disease treatment efficacy compared to existing Fabry disease treatments.

<110> ISU ABXIS Inc. <120> A process for preparing an additionally glycosylated recombinant          alpha-galactosidase and an additionally glycosylated recombinant          alpha-galactosidase prepared therewith <130> PA9609-656 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for first PCR <400> 1 tccggtcacc gtgacaatgc agc 23 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for first PCR <400> 2 gtcttttaat gacatctgca tagtc 25 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> forward primer for second PCR <400> 3 ctagctagcc accatgcagc tgaggaaccc ag 32 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for second PCR <400> 4 ccgctcgagt taaagtaagt cttttaatg 29  

Claims (10)

(a) isolating and purifying recombinant human alpha-galactosidase produced by genetic recombination, and (b) adding sugar chains in vitro to the purified recombinant human alpha-galactosidase, wherein the method for producing sugar chain added recombinant human alpha-galactosidase. The method of claim 1, wherein the genetic recombination method of step (a) (i) Expression vectors expressing polynucleotides encoding human alpha-galactosidase and polynucleotides encoding dhfr (dihydrofolate reductase), respectively or together, are expressed as CHO (deficient in dhfr genes). Chinese hamster ovary) cells were transformed into (ii) obtaining a cell line highly expressing recombinant human alpha-galactosidase by gene amplification using MTX (methotrexate), and (iii) culturing the high-expressing cell line and isolating and purifying recombinant human alpha-galactosidase from the culture medium, the method for producing a sugar chain-added recombinant human alpha-galactosidase. The method of claim 1, wherein adding the sugar chain in step (b) comprises at least 35 mU and at most 45 mU of alpha-2,3-sialyltransferase per mg of alpha-galactosidase. α-2,3-sialyltransferase), 35 mU or more and 45 mU or less beta-1,4-galactosyltransferase (β-1,4-galactosyltransferase), 15 μmol or more and 25 μmol or less UDP-galactose (UDP) -Galactose), a sugar chain-added recombinant human, comprising the step of reacting simultaneously with 15 μmol to 25 μmol of CMP-NANA (N-acetylneuramic acid, sialic acid) and 15 μmol to 25 μmol of MnCl ₂ . Method for preparing alpha-galactosidase. The method for producing a sugar chain-added recombinant human alpha-galactosidase according to claim 1, wherein the step of adding the sugar chain in step (b) is repeated twice. The method of claim 4, wherein the reaction is performed at 37 ° C. for at least 1 hour. 6. The method of claim 5, wherein the reaction is performed for 12 hours each. The method according to any one of claims 3 to 6, wherein the alpha-2,3-sialyltransferase (α-2, not less than 38 mU and not more than 42 mU per mg of alpha-galactosidase) 3-sialyltransferase), beta-1,4-galactosyltransferase of 38 mU or more and 42 mU or less, β-, 4-galactosyltransferase, UDP-Galactose of 18 or more and 22 μmol or less, A sugar chain-added recombinant human alpha-galactosidase characterized by reacting at least 18 μmol and not more than 22 μmol of CMP-NANA (N-acetylneuramic acid, sialic acid) and at least 18 μmol and 22 μmol of MnCl ₂ . Method of preparation. Recombinant human alpha-galactosidase, wherein the sugar chain is added according to the method of any one of claims 1 to 7, wherein the content of sialic acid to galactose is 0.9 or more. Recombinant human alpha-galactosidase having a sugar chain profile as shown in FIG. 6B as determined by Maldi-TOF by sugar chain addition according to any one of claims 1 to 7. A composition for treating Fabry disease having improved stability and therapeutic efficacy in the body, comprising the recombinant human alpha-galactosidase of claim 8 or 9.

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