ES2525768A1 - Procedure for the production of glycosylated recombinant proteins. (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Procedure for the production of glycosylated recombinant proteins. The present invention belongs to the field of the production of recombinant proteins. In particular, it relates to a process for the production, in human cells, of recombinant proteins with a glycosylation pattern that approximates that obtained with the chinese hamster ovary cell line. It also refers to the recombinant protein obtainable by said method and its use for the preparation of a medicament. (Machine-translation by Google Translate, not legally binding)

Description

Procedure for the production of glycosylated recombinant proteins

Field of the Invention

The present invention belongs to the field of recombinant protein production, more specifically it relates to a process for the production of recombinant proteins in human cells with a glycosylation pattern that approximates that obtained with the cell line derived from hamster ovary cells Chinese (CHO cell line, Chinese Hamster Ovary).

Background of the invention

The growing demand for drugs for the treatment of diseases has driven the development of biotechnologies for the efficient and effective production of biopharmaceuticals, so that more and more therapeutic molecules or proteins produced in biological systems (biotechnological drugs) are being produced.

Similar biotechnological medicines, also called biosimilars or biosimilar medicines, are defined as those medicinal products of biotechnological origin similar, but not exactly identical, to other innovative medicinal products, the patent of which has expired. From the economic point of view, the development of biosimilars presents clear advantages over that of new products because a preclinical characterization similar to that of the reference product allows addressing only clinical phases I and III, avoiding the high economic cost and the time invested in the development of clinical phase II.

The expression systems used for the production of biotechnological drugs are based on vectors that allow the cloning of the gene that encodes the protein of interest and its expression in a host cell such as bacteria, yeasts or stable eukaryotic cell lines.

Initially, genetically modified microorganisms were used for the production of therapeutic proteins, however some of the proteins produced by them had certain structural deficiencies that made them unsuitable for human therapeutic use. One of the main drawbacks of these production systems is the lack of mechanisms that allow generating post-translational modifications that in many cases are fundamental to the functionality, life time and immunogenicity of the biomolecule.

These problems were solved with the use of stable eukaryotic cell lines, mainly mammalian cells, which are capable of producing therapeutic proteins.

minimally immunogenic, such as the CHO cell line. The CHO cell line is the expression system par excellence in the production of biotechnological drugs, so that they present a series of physical-chemical parameters characteristic of CHO cells. Within these parameters, the typical glycosylation pattern of CHO cells stands out (hereinafter referred to as the CHO glycosylation pattern or profile), characterized by being highly fucosylated (around 90%), and moderately galactosylated (approximately 50%), by present a fucosylation / galactosylation ratio between 1.3 and 1.8, and for presenting a ratio of major glycan species of approximately 1: 0.80: 0.26: 0.18 for G0F: G1Fa: G1Fb: G2F, taking as reference the species G0F (Xie et al. (Xie H., Chakraborty A., Ahn J., Yu YQ, Dakshinamoorthy DP, Gilar M., Chen W., Skilton SJ and Mazzeo JR (2010) mAbs 2: 4,379-394 , see page 388)).

The influence of the glycosylation pattern on the biological behavior of proteins is very high, reflecting this is the use of this glycosylation pattern by regulatory agencies as a critical parameter to evaluate whether or not to accept a biological product as a possible biosimilar. Thus, the use of cells other than those derived from Chinese hamsters, for example human cell lines, for the production of biosimilars of the reference products produced in the CHO line, is limited to the production of proteins that do not have post-translational modifications such as interferons, vaccines, insulins, etc. Human cells generate a series of post-translational modifications characteristic of the human cell line and not of hamster cells. The main consequence of these differences is that products obtained from human cells cannot be considered by the regulatory agencies as biosimilar medicines to those reference medicines that have been produced using the CHO cell line. These products will be qualified as improved products or "biobetters" (improved biotechnological products with respect to the reference product), being therefore considered as a new registration and having to pass all the relevant clinical phases.

On the other hand, it should be noted that the use of non-human eukaryotic cell lines, such as the CHO cell line, for the production of therapeutic recombinant proteins can cause problems, such as an incorrect folding of proteins that occurs during or after translation, which may be dependent on the presence of appropriate chaperone proteins. An aberrant folding can lead to a decrease or absence of biological activity of the recombinant protein, a decrease in the half-life in blood and even an increase in immunogenicity. In addition, some non-human eukaryotic cell lines, such as the CHO cell line, derive from tumors, which adds an additional risk to working with them and results in isolation procedures for very strict recombinant proteins, with the aim of avoiding transforming activity or the tumorigenic material in any protein or other preparations.

Taking into account all of the above, the authors of the present invention have developed a method for the production of recombinant proteins in human cells that,

Surprisingly, they have a glycosylation pattern that approximates that obtained with the CHO cell line, thus providing an optimal expression system for biosimilar production.

Object of the invention

One aspect of the present invention relates to a process (method of the invention) for the production of a recombinant protein characterized in that it comprises the following steps:

a) add a cereal protein hydrolyzate to a culture medium,

b) culturing a human cell transfected with an expression vector comprising a nucleic acid molecule encoding the recombinant protein, in the culture medium with cereal protein hydrolyzate of step a), where the human cell is not a cell human embryonic mother, and

c) isolate the recombinant protein produced by the human cell in step b), where the recombinant protein contains a N-glycan profile characterized by the fact that the majority glycan is G0F, which represents between 30% and 60% depending on of the total content of N-glycans, and the ratio of N-glycans G0F: G1Fa: G1Fb: G2F is 1: 0.400.95: 0.1-0.40: 0.02-0.40.

In another aspect, it refers to the use of a cereal protein hydrolyzate for the production of a recombinant protein that contains a N-glycan profile characterized in that the majority glycan is G0F, which represents between 30% and 60% depending on the total content of N-glycans, and the ratio of N-glycans G0F: G1Fa: G1Fb: G2F is 1: 0.40-0.95: 0.1-0.40: 0.02-0.40 , in a human cell transfected with an expression vector comprising a nucleic acid molecule encoding the recombinant protein, where the human cell is not a human embryonic stem cell.

Another aspect of the invention relates to a recombinant protein that contains an N-glycan profile characterized in that the majority glycan is G0F, which represents between 30% and 60%, depending on the total N-glycan content, and the ratio of N-glycans G0F: G1Fa: G1Fb: G2F is 1: 0.40-0.95: 0.1-0.40: 0.02-0.40, obtainable by the process of the invention. It also refers to a pharmaceutical composition comprising said recombinant protein and a pharmaceutically acceptable carrier, and the use of said recombinant protein for the preparation of a medicament.

Brief description of the figures

Figure 1 shows the N-glycan profile of a recombinant monoclonal antibody produced under different conditions (fluorescence (FLU) versus retention time in minutes). In AD panels the antibody has been produced in human cells grown for 12 days in culture medium without supplementation (panel A, Medium), supplemented with wheat hydrolyzate (panel B, Medium + H. wheat), supplemented with wheat hydrolyzate Y

albumin (panel C, Medium + H. wheat + Albumin), supplemented with albumin (panel D, Medium + Albumin). In panel E the antibody has been produced in the CHO cell line (CHO).

Figure 2 shows the difference of the relative percentages of G0F, G1Fa, G1Fb, G2F (panel A) and the difference of the relative percentages of fucosylation (F), galactosylation (G), fucosylation / galactosylation ratio (F / G) ( panel B) of a monoclonal antibody produced in human cells in different culture media: culture medium alone (A), medium supplemented with wheat hydrolyzate (B), medium supplemented with wheat hydrolyzate and recombinant human albumin (C) and medium supplemented with recombinant albumin (D), relative to the same antibody produced in the CHO cell line (E).

Figure 3 shows a graph depicting the production of monoclonal antibody (IgG) in g / L versus time in days, in human cells in different culture media: medium alone (A, rhombus), medium supplemented with albumin recombinant (D, cross), medium supplemented with wheat hydrolyzate (B, square), medium supplemented with wheat hydrolyzate and recombinant human albumin (C, triangle).

Detailed description of the invention

One aspect of the present invention relates to a process for producing a

Recombinant protein characterized in that it comprises the following steps:

a) add a cereal protein hydrolyzate to a culture medium,

b) culturing a human cell transfected with an expression vector comprising a nucleic acid molecule encoding the recombinant protein, in the culture medium with cereal protein hydrolyzate of step a), where the human cell is not a cell human embryonic mother, and

c) isolate the recombinant protein produced by the human cell in step b), where the recombinant protein contains a N-glycan profile characterized by the fact that the majority glycan is G0F, which represents between 30% and 60% depending on of the total content of N-glycans, and the ratio of N-glycans G0F: G1Fa: G1Fb: G2F is 1: 0.400.95: 0.1-0.40: 0.02-0.40.

In the context of the present invention, the term "protein hydrolyzate" refers to the product obtained after an enzymatic hydrolysis reaction. The cereal protein hydrolyzate may be of commercial origin or may be obtained by means known to the person skilled in the art, such as those described in Pasapuleti V.K. and Braun S. (Protein Hydrolysates in Biotechnology, Editorial Springer, 2010, 229 p, ISBN 987-1-40206673-3). As for the different species of N-glycans, in the context of the present invention, G0, G1 and G2 refer to complex biantene structures with 0, 1 and 2 terminal galactose residues, while G0F, G1F and G2F are corresponding fucosilated structures. G1Fa and G1Fb species are isoforms of the G1F species (Xie et al., 2010).

Throughout memory, reference to human cells excludes human embryonic stem cells. The culture medium to which the cereal protein hydrolyzate is added and in which human cells are grown depends on the type of cell used and is generally known to the person skilled in the art. The conditions under which stage b) of the process of the invention is carried out are also dependent on the type of cell used and are also known to the person skilled in the art.

In a particular embodiment of the process of the invention, in step a) a cereal protein hydrolyzate is added to the culture medium until a concentration of cereal protein hydrolyzate is reached between 1 g / L and 20 g / L (grams of cereal protein hydrolyzate per liter of culture medium). In another particular embodiment, in step a) a cereal protein hydrolyzate is added to the culture medium until a concentration of cereal protein hydrolyzate is reached between 3 g / L and 10 g / L, and more particular until reaching a concentration of cereal protein hydrolyzate in the medium of 6 g / L.

In a particular embodiment, the cereal protein hydrolyzate is a hydrolyzate of a cereal selected from the group consisting of spelled, barley, oats, millet, corn, rice, rye, wheat and mixtures thereof. In a more particular embodiment, the cereal protein hydrolyzate is a wheat protein hydrolyzate. Thus, in a particular embodiment of the process of the invention, in step a) a wheat protein hydrolyzate is added to the culture medium until a concentration of wheat protein hydrolyzate is reached between 1 g / L and 20 g / L (grams of wheat protein hydrolyzate per liter of culture medium). In another particular embodiment, in step a) a wheat hydrolyzate is added to the culture medium until a concentration of wheat protein hydrolyzate is reached between 3 g / L and 10 g / L, and more particularly up to reach a concentration of wheat protein hydrolyzate in the medium of 6 g / L. The wheat protein hydrolyzate may be of commercial origin or may be obtained by means known to the person skilled in the art, such as those described in Pasapuleti V.K. and Braun S. (Protein Hydrolysates in Biotechnology, Editorial Springer, 2010, 229 p, ISBN 987-1-40206673-3).

As indicated above, cereal protein hydrolyzate is the result of enzymatic treatment of cereal and subsequent purification of the resulting molecules, which are not only amino acids. In a particular embodiment, the cereal protein hydrolyzate is characterized in that it comprises, in mg per g of hydrolyzate:

-

free sugars (mg / g): 3-300 glucose, 0.001-20 fucose, 0.001-20 rhamnose, 0.00120 galactose, 1-100 fructose and 0.05-100 sucrose, the majority being glucose;

-

Total sugars (mg / g): 5-300 glucose, 0.1-100 fucose, 0.5-100 rhamnose, 0.05-100

galactose, 5-300 fructose

-

free amino acids (mg / g): 0.01-50 alanine, 1-100 arginine, 0.01-50 asparagine, 0.1-100 aspartic acid, 0.1-100 cysteine, 0.01-50 glutamic acid, 0.001-10 glutamine, 0.1001 glycine, 0.5-100 histidine, 0.5-100 isoleucine, 1-100 leucine, 0.05-100 lysine, 0.01-50

methionine, 0.5-100 phenylalanine, 0-10 proline, 0.5-100 serine, 0.5-100 threonine, 0.5-100 tyrosine and 0.5-100 valine;

-

Total amino acids (mg / g): 5-300 alanine, 5-300 arginine, 0-50 asparagine, 10350 aspartic acid, 5-300 cysteine, 50-600 glutamic acid, 0-10 glutamine, 5-300 glycine, 5 5 -300 histidine, 5-300 isoleucine, 10-300 leucine, 15-400 lysine, 1-100 methionine, 10-350

phenylalanine, 10-300 proline, 15-400 serine, 5-300 threonine, 5-300 tyrosine and 15-400 valine; -minerals (mg / g): 0.001-20 calcium, 0.0001-10 iron, 0.001-20 magnesium, 0.05-100 phosphorus, 0.01-50 potassium and 2-300 sodium; and -organic acids and other chemical compounds (mg / g): 3-300 oxalic acid, 0.5-100 10 succinic acid, 0.01-50 lactic acid, 3-300 formic acid and 0.1-100 gammaaminobutyric acid (GABA).

In another particular embodiment, the cereal protein hydrolyzate is characterized by comprising: 15 -free sugars (mg / g): 3-200 glucose, 0.001-10 fucose, 0.001-10 rhamnose, 0.00110 galactose, 1-50 fructose and 0.05-50 sucrose, the majority being glucose; Total sugars (mg / g): 5-200 glucose, 0.1-50 fucose, 0.5-50 rhamnose, 0.05-50 galactose, 5-200 fructose, free amino acids (mg / g): 0 , 01-30 alanine, 1-50 arginine, 0.01-30 asparagine, 0.1

20 50 aspartic acid, 0.1-50 cysteine, 0.01-30 glutamic acid, 0.001-5 glutamine, 0.1-50 glycine, 0.5-50 histidine, 0.5-50 isoleucine, 1-50 leucine , 0.05-50 lysine, 0.01-30 methionine, 0.5-50 phenylalanine, 0-5 proline, 0.5-50 serine, 0.5-50 threonine, 0.5-50 tyrosine and 0, 5-50 valine; Total amino acids (mg / g): 5-200 alanine, 5-200 arginine, 0-30 asparagine, 10

25 200 aspartic acid, 5-200 cysteine, 50-450 glutamic acid, 0-5 glutamine, 5-200 glycine, 5200 histidine, 5-200 isoleucine, 10-250 leucine, 10-200 lysine, 1-50 methionine, 10 -200 phenylalanine, 10-250 proline, 10-200 serine, 5-200 threonine, 5-200 tyrosine and 10-200 valine;

-

minerals (mg / g): 0.001-10 calcium, 0.0001-5 iron, 0.001-10 magnesium, 0.05-50 phosphorus, 0.01-30 potassium and 2-100 sodium; and 30 -organic acids and other chemical compounds (mg / g): 3-200 oxalic acid, 0.5-50 succinic acid, 0.01-30 lactic acid, 3-200 formic acid and 0.1-50 GABA.

In another particular embodiment, the cereal protein hydrolyzate is characterized in that it comprises, in mg per g of hydrolyzate; 35 -free sugars (mg / g): 8-30 glucose, 0.01-1.5 fucose, 0.1-2 rhamnose, 0.05-1.5 galactose, 3-18 fructose and 0.2-8 sucrose, the majority being glucose; -Total sugars (mg / g): 20-45 glucose, 0.2-5 fucose, 1-10 rhamnose, 0.9-5 galactose, 15-45 fructose, -free amino acids (mg / g): 0.5 -50 alanine, 3-15 arginine, 0.5-5 asparagine, 0.2-5

40 aspartic acid, 0.2-5 cysteine, 0.5-5 glutamic acid, 0.05-2 glutamine, 0.2-5 glycine, 0.5-10 histidine, 1-10 isoleucine, 3-20 leucine, 0.2-9 lysine, 0.5-8 methionine, 1-10 phenylalanine, 0-1 proline, 1-7 serine, 2-10 threonine, 0.5-10 tyrosine and 2-10 valine; Total amino acids (mg / g): 20-55 alanine, 20-50 arginine, 0-1 asparagine, 30-60

aspartic acid, 10-35 cysteine, 200-300 glutamic acid, 0-1 glutamine, 10-35 glycine, 10-35 histidine, 10-35 isoleucine, 40-130 leucine, 25-65 lysine, 3-15 methionine, 30 -70 phenylalanine, 70-130 proline, 25-60 serine, 15-50 threonine, 15-50 tyrosine and 25-65 valine;

-

minerals (mg / g): 0.05-2 calcium, 0.005-1 iron, 0.05-2 magnesium, 1-5 phosphorus, 0.5-3 potassium and 4-25 sodium; Y

-

Organic acids and other chemical compounds (mg / g): 5-20 oxalic acid, 1-10 succinic acid, 0.05-3.5 lactic acid, 10-25 formic acid and 0.2-5 GABA.

In another particular embodiment, in step a) the culture medium is a culture medium free of serum and proteins of animal origin. In another particular embodiment, said serum and protein-free culture medium of animal origin does not have lactate and comprises glutamine, glucose and glutamic acid. In a more particular embodiment the culture medium is the PerMab® medium (Thermofisher Scientific / 15 Hyclone, catalog number SH30871.01).

Following the procedure of the invention, recombinant proteins are produced with a glycosylation pattern that approximates that obtained with the CHO cell line (Figure 1, panel E). Surprisingly, this glycosylation pattern that approximates that obtained with the CHO cell line (hereinafter referred to as the "hamsterized" glycosylation pattern or profile) is achieved using human cells as an expression system. The "hamsterized" glycosylation pattern of the recombined proteins produced by the process of the invention is characterized in that the majority glycan is G0F, which represents between 30% and 60% depending on the total N-glycan content, and because the ratio of N-glycans G0F: G1Fa: G1Fb: G2F is 1: 0.40-0.95: 0.1-0.40: 0.02-0.40, taking as reference the species G0F, is that is, considering the maximum value of the content of N-glycan G0F.

In a particular embodiment, the recombinant protein obtained by the method of the present invention contains an N-glycan profile characterized in that the majority glycan is G0F, which represents between 40% and 50% depending on the total N content. -glycans, and the ratio of N-glycans G0F: G1Fa: G1Fb: G2F is 1: 0.40-0.95: 0.1-0.40: 0.02-0.40, taking as reference the species G0F .

In another particular embodiment, the recombinant proteins produced by the method of the invention have a fucosylation percentage of between 70% and 96% and a galactosylation percentage of between 40% and 85%, presenting a fucosylation / galactosylation ratio of between 1.13 and 1.8.

The glycosylation pattern of the recombinant protein can be analyzed using liquid chromatography, such as hydrophilic interaction liquid chromatography (HILIC) and / or other methods known to those skilled in the art, such as mass spectroscopy analysis.

Among the different human cells (excluding human embryonic stem cells) that can be used as a recombinant protein expression system, the PER.C6® cell line (Crucell NV (Leiden, The Netherlands), Percivia), deposited in the European Collection of Cell Cultures (ECAAC) under number 96022940, which is obtained from human embryonic retinal cells (EP 0833934 B1, also published as ES 2231813 T). One of the advantages offered by the use of PER.C6® cells is its ability to grow in cultures of extreme cell density which allows it to produce production titres 5 times higher than those traditionally obtained with the CHO cell line. Thus, in a particular embodiment of the process of the invention, in step b) the human cell is a human embryonic retina cell, preferably the cell the ECACC cell line with the number 96022940 (PER.C6®). The PER.C6® cell line is transfected with an expression vector comprising a nucleic acid molecule that encodes the recombinant protein to be produced (recombinant protein of interest). In a particular embodiment said nucleic acid molecule is a DNA molecule. In a particular embodiment, the transfected human cell is the ECACC cell line with the number 96022940 (PER.C6®) transfected with the vector v074 (SEQ ID NO 1) linearized by enzymatic digestion with ScaI, which has been deposited in the institution Health Protection Agency Culture Collections (HPACC, Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom) on March 7, 2012, with deposit number 12030701, that is, the transfected human cell is the cell deposited in the HPACC with deposit number 12030701. Vector v074 encodes the heavy and light chains of a monoclonal antibody against the Her2 membrane receptor.

The majority of biotechnological drugs currently produced are monoclonal antibodies whose production is carried out, in most cases, in CHO cells. Thus, in a particular embodiment of the process of the invention, the recombinant protein produced is an antibody and more particularly is a monoclonal antibody.

In a particular embodiment of the procedure described in the preceding paragraphs, step b) is carried out in Fed-Batch or by infusion, more particularly in Fed-Batch.

As indicated above, with the process of the invention, characterized by the addition of a cereal protein hydrolyzate to the culture medium, recombinant proteins are produced with a "hamsterized" glycosylation profile but using human cells as the expression system. Surprisingly, the addition of albumin to the culture medium of step a) causes an increase in the production of the recombinant protein without negatively affecting its glycosylation pattern, that is, the recombinant protein produced in culture medium with protein hydrolyzate of cereal and albumin still maintain a "hamsterized" glycosylation profile (Figures 1, panel C). In addition, unexpectedly, the combination of cereal protein hydrolyzate and albumin has a synergistic effect in terms of increasing recombinant protein production with a "hamsterized" glycosylation profile (Figure 3, triangle).

Another important advantage of the combination of cereal protein and albumin hydrolyzate is that the same protein production is achieved in a shorter time than in human cells grown in medium without supplementation, supplemented only with cereal protein hydrolyzate or with albumin only ( Figure 3). Therefore, with the combination of protein hydrolyzate of cereals and albumin, it is possible to increase and accelerate the production of recombinant protein. Thus, in a particular embodiment of the process of the invention, in step a) albumin is added to the culture medium. In a particular embodiment, in step a) albumin is added to the culture medium until a concentration of albumin in the medium is reached between 0.5 g / L and 20 g / L (grams of albumin per liter of culture medium). In another particular embodiment, albumin is added to the culture medium until reaching an albumin concentration in the medium between 1 g / L and 15 g / L and more particularly until reaching an albumin concentration in the medium between 2 g / L and 10 g / L, and preferably until reaching an albumin concentration in the medium of 4 g / L.

Albumin may be commercial or obtained by means known to the person skilled in the art. In another particular embodiment, the albumin is recombinant albumin, preferably recombinant human albumin. In another particular embodiment, the recombinant albumin is produced in a rice plant, free of animal proteins, thus avoiding the risks of using fetal bovine serum.

In a particular embodiment of the process of the invention, in step a) a cereal protein hydrolyzate, in particular a wheat protein hydrolyzate, is added to the culture medium, until a concentration of cereal protein hydrolyzate is reached, in particular of wheat protein hydrolyzate, in the medium between 1 g / L and 20 g / L and albumin is also added until reaching an albumin concentration in the medium between 0.5 g / L and 20 g / L. In another particular embodiment, a cereal protein hydrolyzate, in particular a wheat protein hydrolyzate, is added until reaching a concentration in the medium between 3 g / L and 10 g / L and albumin is also added until a concentration of albumin in the medium between 1 g / L and 15 g / L. In a preferred embodiment of the process of the invention, in step a) a cereal protein hydrolyzate, in particular a wheat protein hydrolyzate, is added to the culture medium, until a concentration of cereal protein hydrolyzate is reached, in particular a wheat protein hydrolyzate, in the medium between 3 g / L and 10 g / L and albumin is added until a concentration of albumin in the medium between 2 g / L and 10 g / L is reached, and more preferably the hydrolyzate of Cereal proteins, in particular a wheat protein hydrolyzate, are added until reaching a concentration of 6 g / L and albumin until reaching a concentration of 4 g / L.

Another aspect of the present invention relates to the use of a cereal protein hydrolyzate, in particular a wheat protein hydrolyzate, for the production of a recombinant protein containing an N-glycan profile characterized in that the majority glycan is G0F , which represents between 30% and 60% depending on the total content of N-glycans, and the ratio of N-glycans G0F: G1Fa: G1Fb: G2F is 1: 0.40-0.95: 0.1 -0.40:

0.02-0.40, in a human cell transfected with an expression vector comprising a nucleic acid molecule encoding the recombinant protein, where the human cell is not a human embryonic stem cell. In a particular embodiment, the invention relates to the use of a combination of a cereal protein hydrolyzate, in particular wheat, and albumin for the production of a recombinant protein with an N-glycan profile characterized in that the majority glycan is G0F, which represents between 30% and 60% depending on the total content of N-glycans, and the ratio of N-glycans G0F: G1Fa: G1Fb: G2F is 1: 0.40-0.95: 0, 1-0.40: 0.02-0.40, in a human cell transfected with an expression vector comprising a nucleic acid molecule encoding the recombinant protein, where the human cell is not a human embryonic stem cell. In another particular embodiment, the human cell is a human embryonic retinal cell, more particularly a PER.C6® cell. In an even more particular embodiment the transfected human cell is the cell line deposited in the HPACC institution with the deposit number 12030701.

As indicated above, the combination of cereal protein and albumin hydrolyzate increases and accelerates the production of recombinant protein. Thus, in a particular embodiment, the invention relates to the use of a cereal protein hydrolyzate, in particular a wheat protein hydrolyzate, and albumin to increase the production, in human cells, of a recombinant protein with an N profile. -glycans characterized by the fact that the majority glycan is G0F, which represents between 30% and 60% depending on the total content of N-glycans, and the ratio of N-glycans G0F: G1Fa: G1Fb: G2F is 1: 0 , 40-0.95: 0.1-0.40: 0.02-0.40. In another particular embodiment, the human cell, which is transfected with the expression vector comprising a nucleic acid molecule encoding the recombinant protein of interest, is a human embryonic retinal cell, more particularly a PER.C6® cell. . In an even more particular embodiment the transfected human cell is the cell line deposited in the HPACC institution with the deposit number 12030701.

The present invention relates in another aspect to a recombinant protein obtainable by the process of the invention as described in the preceding paragraphs (recombinant protein of the invention), that is, a recombinant protein with an N-glycan profile. characterized in that the majority glycan is G0F, which represents between 30% and 60% depending on the total content of N-glycans, and the ratio of N-glycans G0F: G1Fa: G1Fb: G2F is 1: 0.40 -0.95: 0.1-0.40: 0.02-0.40. Thus, in a particular embodiment the recombinant protein is obtainable by the process of the invention by adding in step a) a cereal protein hydrolyzate, in particular a wheat protein hydrolyzate, to the culture medium until a hydrolyzate concentration of cereal proteins, in particular of wheat protein hydrolyzate, in the medium between 1 g / L and 20 g / L, more particularly between 3 g / L and 10 g / L, and preferably up to a concentration in the middle of 6 g / L. In another particular embodiment the recombinant protein is obtainable by the method of the invention, wherein the human cell that is transfected with an expression vector comprising a nucleic acid molecule that

encoding the recombinant protein of interest is a human embryonic retinal cell, more particularly it is a PER.C6® cell. In a preferred embodiment the transfected human cell is the cell line deposited in the HPACC institution with the deposit number 12030701. In another particular embodiment, the recombinant protein obtainable by the process of the invention is an antibody, more particularly, an antibody. monoclonal In another particular embodiment, the recombinant protein is obtainable by the process of the invention by performing step b) in Fed-Batch or in perfusion. In another particular embodiment the recombinant protein is obtainable by the process of the invention where in stage a) albumin is added to the culture medium. In a particular embodiment, albumin is added to the culture medium until a concentration of albumin in the medium is reached between 0.5 g / L and 20 g / L. In another particular embodiment, said concentration of albumin in the medium is between 1 g / L and 15 g / L, more particularly it is between 2 g / L and 10 g / L, and preferably it is 4 g / L. In another particular embodiment the recombinant protein is obtainable by the process of the invention where in step a) a cereal protein hydrolyzate, in particular a wheat protein hydrolyzate, is added to the culture medium until a hydrolyzate concentration of Cereal proteins, in particular wheat protein hydrolyzate, in the medium between 1 g / L and 20 g / L and albumin is also added until a concentration of albumin in the medium between 0.5 g / L and 20 g is reached / L. In another particular embodiment, the cereal protein hydrolyzate, in particular a wheat protein hydrolyzate, is added to the culture medium until a concentration of between 3 g / L and 10 g / L and the albumin is reached until a concentration of between 1 g / L and 15 g / L, more particularly between 2 g / L and 10 g / L albumin, and more preferably the cereal protein hydrolyzate, in particular a wheat protein hydrolyzate, is added until reaching a concentration of 6 g / L and albumin until reaching a concentration of 4 g / L.

Finally, another aspect of the present invention relates to the use of the recombinant protein of the invention, that is, the recombinant protein obtainable by the process of the invention as described in the previous paragraph for the preparation of a medicament. , in particular a biosimilar medicament, and also refers to said recombinant protein for use as a medicament, in particular as a biosimilar medicament. It also refers to a pharmaceutical composition comprising the recombinant protein of the invention and a pharmaceutically acceptable carrier. It also refers to the use of said pharmaceutical composition for the preparation of a medicament, in particular a biosimilar medicament, and also refers to said pharmaceutical composition for use as a medicament, in particular as a biosimilar medicament.

BIOLOGICAL MATTER DEPOSIT

The ECACC cell line with the number 96022940 (PER.C6®) transfected with the vector v074 (SEQ ID NO 1) linearized by enzymatic digestion with ScaI has been deposited with the Health Protection Agency Culture Collections (HPACC, Porton Down,

Salisbury, Wiltshire, SP4 0JG, United Kingdom) on March 7, 2012, with deposit number 12030701 (Spanish patent application P201200465).

Examples

5 The following are concrete examples of embodiments of the invention that they serve to illustrate the invention.

In the present examples, reference is made to an anti-HER2 monoclonal antibody, to

10 which is called CRX01, encoded by the sequence expression vector SEQ ID NO 1, expressed in the cell line deposited with the HPACC institution with the deposit number 12030701.

EXAMPLE 1.-Production of a monoclonal antibody

15 Cultivation of HPACC cells No. 12030701 Throughout the culture process, PER.C6® cells were grown following the instructions indicated by the company that owns the PER.C6® cell line (Crucell-DSM). Briefly, PER.C6® cells were grown in PerMab medium (Thermo Fisher Scientific / HyClone, catalog number SH30871.01), which is specially designed

20 to allow the growth of cells in high density. The PerMab medium was completed with the addition of the 3mM glutamine (Ajinomoto) amino acid and the F-68 pluronic acid detergent (Sigma, catalog number P1300) to a final concentration of 1g / L and in no case was any type of serum added to the means, medium. Said PerMab medium supplemented with 3mM of glutamine and 1g / L of pluronic acid is referred to as "complete PerMab medium" hereinafter.

25 forward.

A vial of HPACC No. 12030701 cells was thawed and the cells were seeded at a density of 0.5x106 cells / ml in static T-flask for one week, at 36.5 ° C and 5% CO2, subculturing at 3 / 4 days. After this period of adaptation,

30 cells were grown under stirring at 75 rpm and after 4 days passed at 125 rpm stirring.

After this time, the cells were centrifuged at 300g, the supernatant was removed and the cells were resuspended in:

35 A. Half PerMab complete.

B. Complete PerMab medium and hydrolyzed wheat at a concentration of 6 g / L.

C. Complete PerMab medium, hydrolyzed wheat at a concentration of 6 g / L and recombinant human albumin at a concentration of 4 g / L.

D. Complete PerMab medium and recombinant human albumin at a concentration of 4 g / L.

Wheat hydrolyzate and albumin are from Kerry Bioscience, reference U1-5X00792 and 5X00163, respectively.

45 C cultivation strategy, that is, in which whole wheat hydrolyzate and albumin is added to the complete PerMab medium is called CURAMAB.

HPACC No. 12030701 cells were grown in Fed-Batch for 12 days following the instructions given by the company that owns the PER.C6® cell line (Crucell-DSM).

EXAMPLE 2.-Purification of the monoclonal antibody

The process of purification of the antibody produced in control medium (A) and supplemented (B-D) begins with centrifugation to remove cells and cell debris from the culture. Once the clarified solution of the protein of interest is obtained, the separation of the other proteins produced by the cell is carried out using a combination of different chromatographic techniques. These techniques separate mixtures of proteins based on their load, degree of hydrophobicity, affinity and size. Several different chromatography resins are commercially available for each of these techniques, allowing a precise adaptation of the purification scheme to the protein involved.

The purification process performed consists of three chromatographic stages, which begin with a purification process by affinity chromatography using a column with immobilized Protein A. Most of the contaminants (host cell proteins) pass through the column while the protein of interest is adsorbed to immobilized protein A in addition to other contaminants that unspecifically bind to the solid phase. Elution of IgG1 was performed by pH changes.

The next stage of purification consists of a cation exchange chromatography. The column was loaded with the Protein A pool conditioned in conductivity and pH, after several washes, the elution of the protein of interest was performed by a change in conductivity.

Finally, a final step of polishing chromatography or "polishing" of the eluted protein preparation was performed to reduce the contaminating proteins to ppm levels and decrease the amount of aggregates. For this stage a bimodal chromatography was used whose ligand bound to the solid phase is an N-benzyl-N-methyl-ethanol amine that exhibits several functional groups with different interaction behavior. The most pronounced is the ionic interaction, behaving as a strong anion exchanger, and the hydrophobic interaction.

The pool of the recovered protein was formulated by diafiltration with a tangential filtration system in a series of pharmaceutically suitable excipients to maintain its stability. In particular, L-histidine was used as a buffer to maintain a pH = 6, Trehalose dihydrate as a toning agent and polysorbate 20 as a stabilizer, which reduces the potential aggregation that the protein can undergo in solution.

EXAMPLE 3.-Glycosylation Profile

Monoclonal antibodies produced under different conditions (culture medium alone (A), supplemented with wheat hydrolyzate (B), supplemented with wheat and albumin hydrolyzate (C, CURAMAB) and supplemented with albumin (D) were deglycosylated by a first Denaturation stage with RapidGest SF (Water, Water, Milford, MA, USA, 5 ref. 186001861), followed by two stages of reduction and alkylation, with dithiothreitol and iodoacetamide, respectively. The N-glycans of the deglycosylated antibodies were digested by addition of PNGase F and incubation at 37 ° C. overnight The digested samples were purified by solid phase extraction in 96-well plates HILIC microELUTION SPE (Water, Milford, MA, USA, ref. 186002780) according to the protocol described by Xie et al. (2010). Purified glycans were labeled with 2 amino-benzamide (2-AB) by the Sigma GlycoProfile 2-AB Kit (St. Louis, MO, USA), then a second puri Solid phase fication with HILIC microELUTION SPE was carried out to remove excess 2-AB. The glycans labeled with 2-AB were separated on a HILIC column (Water, Milford, MA, USA, ref. 186006095) at 60 ° C and

15 detected by an L-2485 LaChrom ELITE. Separations of the different glycan species were made using a 40-minute gradient (28% -38% B) with 100 mM ammonium formate pH 2.7 (mobile phase B) and 100% acetonitrile (mobile phase A) a 0.6 ml / min linear flow.

The analysis showed a glycan profile of the reference antibody produced in the CHO cell line, highly fucosylated (around 90%) and moderately galactosylated (approximately 50%), presenting a characteristic fucosylation / galactosylation ratio of 1.68 ± 0.002, characteristic of this cell culture (Table 1). This value is typical of CHO cells where the majority species of this culture presented for G0F, G1Fa, G1Fb and G2F a

25 G0F: G1Fa: G1Fb: G2F ratio of 1: 0.86: 0.30: 0.24, considering as a maximum value the percentage of the G0F species (Table 1; Figure 1, panel E).

Table 1.-Relative percentages of intensity of major glycans of the monoclonal antibody produced in HPACC cells No. 12030701.

TO

B C D AND

G0F

24.03 ± 0.117 38.43 ± 0.014 32.75 ± 0.075 22.50 ± 0.149 36.99 ± 0.226

G1Fa

31.30 ± 0.262 30.15 ± 0.049 30.30 ± 0.066 33.15 ± 0.080 31.89 ± 0.148

G1Fb

10.82 ± 0.225 11.73 ± 0.014 11.21 ± 0.037 11.93 ± 0.036 11.15 ± 0.02

G2F

14.58 ± 0.273 9.96 ± 0.001 12.01 ± 0.077 16.91 ± 0.115 8.80 ± 0.09

F

87.07 ± 0.465 94.44 ± 0.014 92.68 ± 0.104 92.25 ± 0.358 92.49 ± 0.494

G

94.63 ± 0.485 70.47 ± 0.001 79.53 ± 0.263 97.41 ± 0.319 54.93 ± 0.212

F / G

1.09 ± 0.011 1.34 ± 0.002 1.16 ± 0.002 0.94 ± 0.006 1.68 ± 0.002

Table 2.-Difference of the relative percentages of Table 1 with respect to the same antibody produced in the CHO cell line.

B-E

EC A-E FROM

G0F

1.44 -4.24 -12.96 -14.49

G1Fa

-1.74 -1.59 -0.59 1.26

G1Fb

0.58 0.06 -0.33 0.78

G2F

1.16 3.21 5.78 8.11

F

1.95 0.19 -5.42 -0.24

G

15.54 24.6 39.7 42.48

F / G

-0.34 -0.52 -0.59 -0.74

In Tables 1 and 2 the letters from A to E refer to the antibody being produced in human cells in different culture media: PerMab medium (A), PerMab medium supplemented with wheat hydrolyzate (B), supplemented with Wheat hydrolyzate with recombinant human albumin (C) and supplemented with recombinant albumin (D), or produced in the CHO cell line (E).

The oligosaccharide profile of the antibody produced in human cell culture without supplementation (A) showed higher levels of galactosylation (approximately 94.63%) than the profile obtained in CHO (E) (Table 1). This profile of highly galactosylated glycans, typical of human proteins, showed values of majority species G0F, G1Fa, G1Fb and G2F with a G0F: G1Fa: G1Fb: G2F ratio of 0.76: 1: 0.34: 0.46 , considering as a maximum value the percentage of the species G1Fa (Table 1 and Figure 1, panel A). In this case, the fucosylation / galactosylation ratio was lower than in CHO, of 1.09 ± 0.011 (Table 1), typical of human cell cultures.

Surprisingly, the glycan profile of the proteins produced in HPACC human cell culture No. 12030701 supplemented only with wheat hydrolyzate (B) or with wheat hydrolyzate plus albumin (C), was more similar to that obtained with CHO cells (E) than in conditions without supplementing (Figure 1, panels B, C and E; Figure 2). This oligosaccharide profile obtained with culture medium supplemented with wheat hydrolyzate and albumin or supplemented only with wheat hydrolyzate showed fucosylation and galactosylation values very close to that obtained with CHO cells. More in detail, the profile obtained in the medium supplemented with wheat hydrolyzate plus albumin (C), presented values of majority species G0F, G1Fa, G1Fb and G2F with a G0F: G1Fa: G1Fb: G2F ratio of 1: 0, 92: 0.34: 0.36, considering as a maximum value the percentage of the G0F species and presented a fucosylation / galactosylation ratio of 1.16 ± 0.002 (Table 1).

The results obtained show that wheat hydrolyzate allows modifying the glycosylation profile of PER.C6® cells, making it similar to that produced by the CHO cell line. In addition, this profile is obtained at standard cultivation times of 12 days, which implies a

important advantage in terms of product quality. Long-term cultures of PER.C6® cells (more than 12 days) have the disadvantage of producing modifications in some physical-chemical parameters that affect the quality of the product, for example in the load heterogeneity profile. At a qualitative level the profile of glycans analyzed, of a total of

5 23 (data not shown), of the recombinant protein produced in the Per.C6® cell line in culture medium without supplementation, supplemented with wheat hydrolyzate and supplemented with wheat and albumin hydrolyzate showed no significant differences, the percentage of Major glycans G0F, G1Fa, G1Fb and G2F (they represent between 80% and 91% of the glycan species) which characterize the profile.

EXAMPLE 4.-Yield of monoclonal antibody production

Figure 3 shows that in PER.C6® cells grown in PerMab medium without supplementation (A, rhombus) the maximum production of IgG1 is 3.7 g / L on day 12 of culture. Addition to the culture medium of recombinant albumin (D, cross) or wheat hydrolyzate (B,

15 square) generated production levels of IgG1 at day 12 of 4.3 g / L and 3.3 g / L, respectively. Surprisingly, when a mixture of wheat hydrolyzate and recombinant human albumin (C, triangle) was added to the culture medium, a synergistic effect in production was observed, producing 4.9 g / L of IgG1.

20 The combination of wheat hydrolyzate with recombinant human albumin (C, CURAMAB) produces a significant increase in protein production reaching levels very similar to the absolute maximum of 5.1 g / L obtained by the culture not supplemented on day 18. This Improvement in the efficiency of the process, from the industrial point of view, allows a reduction of up to 6 days in the time necessary to achieve a production of

25 recombinant proteins maximum, considerably increasing the profitability of the process since: -It results in a reduction of costs associated with culture media, energy, personnel etc. -Allows an increase in the annual production capacity of a biopharmaceutical plant to

30 can make a larger number of lots.

Claims (12)

1-Procedure for the production of a recombinant glycosylated protein characterized because it includes the following stages: a) add a cereal protein hydrolyzate to a culture medium, b) culturing a human cell transfected with an expression vector comprising a nucleic acid molecule encoding the recombinant protein in the culture medium with cereal protein hydrolyzate of step a), where the human cell is not a stem cell human embryonic, and c) isolate the recombinant protein produced by the human cell in step b), where the recombinant protein contains a N-glycan profile characterized by the fact that the majority glycan is G0F, which represents between 30% and 60% depending on of the total content of N-glycans, and the ratio of N-glycans G0F: G1Fa: G1Fb: G2F is 1: 0.400.95: 0.1-0.40: 0.02-0.40. 2. Method according to the preceding claim, wherein in step a) a cereal protein hydrolyzate is added to the culture medium until a concentration of cereal protein hydrolyzate is reached between 1 g / L and 20 g / L . 3. Method according to any one of the preceding claims, wherein the cereal protein hydrolyzate is a wheat protein hydrolyzate. 4. Method according to any one of the preceding claims, wherein in step b) the human cell is a human embryonic retinal cell. 5. Method according to the preceding claim, wherein the human embryonic retinal cell is ECAAC cell No. 96022940. 6. Method according to any one of the preceding claims, wherein the recombinant protein is a monoclonal antibody. 7. Method according to any one of claims 1 to 3, wherein in step b) the transfected human cell is the cell deposited in the HPACC with the deposit number 12030701. 8. Method according to any one of the preceding claims, wherein step b) is carried out in Fed-Batch or perfusion. 9. Method according to any one of the preceding claims, wherein in step a) albumin is added to the culture medium. 10. Method according to the preceding claim, wherein the albumin is added until reaching a concentration between 0.5 g / L and 20 g / L in the culture medium. 11. Method according to any one of claims 9 and 10, wherein the albumin is recombinant human albumin. 12. 12. Use of a cereal protein hydrolyzate for the production of a glycosylated recombinant protein according to the method of any one of claims 1 to 8. 13. Use of a combination of a cereal protein and albumin hydrolyzate for Production of a glycosylated recombinant protein according to the method of any one of claims 9 to 11. TO B t (min)  FIG. one   t (min)  FIG. one   TO 10.00 5.00 % relative B-E 0.00 EC

-

5.00

A-E

-

10.00 -15.00

B 50 40 % relative B-E EC A-E 10 FROM 0 -10  FIG. 2 3 4 5 6 7 8 910 11 12 Days  FIG. 3   SEQUENCE LISTING <110> CURAXYS S.L. <120> Glycosylated recombinant proteins and production method <130> 011/13 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 8841 <212> DNA <213> Artificial Sequence <220> <223> vector v074 <400> 1 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 60 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 120 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag 180 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga 240 tagtgcgcga ggtcgctgag agctacaaca gcaaaattta tgaccgacaa aggcaaggct 300 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgctagg tggtcaatat 360 tggccattag ccatattatt cattggttat atagcataaa tcaatattgg ctattggcca 420 ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg tccaacatta 480 ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta 540 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc 600 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg 660 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg 720 aagtgtatca gcagtacatc tatgccaagt acgcccccta ttgacgtc aa tgacggtaaa 780 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac 840 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg 900 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg 960 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat ctccgcccca gtcgtaacaa 1020 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta 1080 gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac 1140 cgggaccgat ccagcctccg cggccgggaa cggtgcattg gaagcttggt accgagctcg 1200 gatccgccac catggagtgg tccggcgtgt tcatgttcct gctgtccgtg accgctggcg 1260 tgcactccga ggtgcagctg gtcgagtctg gcggcggact ggtgcagcct ggcggctccc 1320 tgcggctgtc ctgcgccgcc tccggcttca acatcaagga cacctacatc cactgggtgc 1380 ggcaggcccc tggcaagggc ctggagtggg tggcccggat ctaccctacc aacggctaca 1440 ccagatacgc cgactccgtg aagggccggt tcaccatctc cgccgacacc tccaagaaca 1500 ccgcctacct gcagatgaac tccctgcggg ccgaggacac cgccgtgtac tactgctcca 1560 gatggggcgg agatggcttc tacgccatgg actactgggg ccagggcacc ctggtgaccg 1620 tgtcctccgc tagcaccaag ggcccttccg tgttccctct ggccccttcc tccaagtcca 1680 cctccggcgg caccgccgct ctgggctgcc tggtgaagga ctacttccct gagcctgtga 1740 gaactctggc ccgtgagctg gccctgacca gcggcgtgca caccttccct gccgtgctgc 1800 agtcctccgg cctgtactcc ctgtcctccg tggtgacagt gccttcctcc tccctgggca 1860 cccagaccta catctgcaac gtgaaccaca agccttccaa caccaaggtg gacaagaagg 1920 tggagcctaa gtcctgcgac aagacccaca cctgccctcc ctgccctgcc cctgagctgc 1980 tgggcggacc ctccgtgttc ctgttccctc ctaagcctaa ggacaccctg atgatctccc 2040 ggacccctga ggtgacctgc gtggtggtgg cgaggaccca acgtgtccca gaggtgaagt 2100 tcaattggta cgtggacggc gtggaggtgc acaacgccaa gaccaagcct cgggaggaac 2160 agtacaactc cacctaccgg gtggtgtccg tgctgaccgt gctg caccag gactggctga 2220 acggcaagga atacaagtgc aaggtgtcca acaaggccct gcctgccccc atcgaaaaga 2280 ccatctccaa ggccaagggc cagcctcgcg agcctcaggt gtacaccctg cctccctccc 2340 gggacgagct gaccaagaac caggtgtccc tgacctgtct ggtgaagggc ttctaccctt 2400 ccgatatcgc cgtggagtgg gagtccaacg gccagcctga gaacaactac aagaccaccc 2460 ctcctgtgct ggactccgac ggctccttct tcctgtactc caagctgacc gtggacaagt 2520 cccggtggca gcagggcaac gtgttctcct gctccgtgat gcacgaggcc ctgcacaacc 2580 actacaccca gaagtccctg tccctgagcc ctggcaagtg ataatctagc gaattcaccg 2640 taagtttaaa gtaccaagct ccgctgatca gcctcgactg tgccttctag ttgccagcca 2700 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 2760 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 2820 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct 2880 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg 2940 tatccccacg cgccctgtag c ggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 3000 gtgaccgcta cacttgccag cgcctagcgc ccgctccttt cgctttcttc ccttcctttc 3060 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 3120 tttacggcac gatttagtgc aaaaacttga ctcgacccca ggttcacgta ttagggtgat 3180 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 3240 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg 3300 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 3360 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg 3420 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg 3480 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc 3540 ccgcccctaa aaccatagtc cccgccccta ctccgcccat gttccgccca actccgccca 3600 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc 3660 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa 3720 gctcccggga gcttggatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt 3780 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct 3840 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct 3900 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga 3960 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc 4020 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg 4080 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc 4140 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca 4200 cgagcacgta tcgcatcgag ctcggatgga agccggtctt gtcg atcagg atgatctgga 4260 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc 4320 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc tcatggtgga ttgccgaata 4380 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca 4440 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg 4500 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct 4560 tcttgacgag ttcttctgag cgggactctg gggttcggtg ctacgagatt tcgattccac 4620 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat 4680 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc 4740 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 4800 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc 4860 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg 4920 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg 4980 tgcctaatga gtgagctaac t cacattaat tgcgttgcgc tcactgcccg ctttccagtc 5040 gggaaacctg tcgtgccaga attgcatgaa gaatctgctt agggttaggc gttttgcgct 5100 gcttcgctag gtggtcaata ttggccatta gccatattat tcattggtta tatagcataa 5160 atcaatattg gctattggcc attgcatacg ttgtatccat atcataatat gtacatttat 5220 attggctcat gtccaacatt accgccatgt tgacattgat tattgactag ttattaatag 5280 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt 5340 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 5400 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 5460 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 5520 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 5580 cttggcagta gactttccta catctacgta ttagtcatcg ctattaccat ggtgatgcgg 5640 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc 5700 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa 5760 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 5820 tatataagca gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt 5880 atagaagaca tttgacctcc ccgggaccga tccagcctcc gcggccggga acggtgcatt 5940 ggaagcttgg taccggtgaa ttcggcgcgc caccatggag tggtccggcg tgttcatgtt 6000 cctgctgtcc gtgaccgctg gcgtgcactc cgacatccag atgacccagt ccccctcctc 6060 cctgtccgcc tccgtgggcg accgggtgac catcacctgc cgggcctccc aggacgtgaa 6120 caccgccgtg gcctggtatc agcagaagcc tggcaaggcc cctaagctgc tgatctactc 6180 cgcctccttc ctgtactccg gcgtgccttc ccggttctcc ggctcccggt ccggcaccga 6240 cttcaccctg accatctcct ccctgcagcc tgaggacttc GCCA cctact actgccagca 6300 gcactacacc acccctccta ccttcggcca gggcaccaag gtggagatca agcggaccgt 6360 ggccgctcct tccgtgttca tcttccctcc ctccgacgag cagctgaaga gcggcaccgc 6420 tgcctgctga cagcgtggtg acaacttcta ccctcgggag gccaaggtgc agtggaaggt 6480 ggacaacgcc ctgcagtccg gcaactccca ggaaagcgtc accgagcagg actccaagga 6540 cagcacctac tccctgtcct ccaccctgac cctgtccaag gccgactacg agaagcacaa 6600 ggtgtacgcc tgcgaggtga cccaccaggg cctgtccagc cctgtgacca agtccttcaa 6660 ccggggcgag tgctgatgag ttaacggatc gatccgagct cggtaccaag cttaagttta 6720 aaccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc 6780 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag 6840 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag 6900 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct 6960 atggcttctg aggcggaaag aaccagctgc attaatgaat cggccaacgc gcggggagag 7020 gcggtttgcg tattgggcgc t cttccgctt cctcgctcac tgactcgctg cgctcggtcg 7080 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 7140 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 7200 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 7260 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 7320 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 7380 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 7440 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 7500 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 7560 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 7620 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 7680 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 7740 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 7800 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 7860 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 7920 atgaagtttt taaattaaaa aaagtatata aaatcaatct tggtctgaca tgagtaaact 7980 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 8040 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 8100 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 8160 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 8220 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 8280 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcg tttggt atggcttcat 8340 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 8400 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 8460 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 8520 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 8580 ggcgtcaata gctcttgccc cgggataata ccgcgccaca tagcagaact ttaaaagtgc 8640 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 8700 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 8760 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 8820 cacggaaatg ttgaatactc a 8841