JP6920280B2 - Method for Producing Purified Cat-Derived Erythropoietin - Google Patents

Description

The present invention relates to a method for producing purified cat-derived erythropoietin, which can be used as an active ingredient of veterinary medicine.

Cats have long been attached to humans as pets, but recently they are establishing themselves as members of human society as "animals as companions, companions, and companions." On the other hand, it has been used as an experimental animal in medicine, pharmacy, veterinary medicine, psychology, etc., and has recently been used for drug safety tests and efficacy tests. In such a situation where the social importance of cats is increasing, there is a great deal of interest in cat diseases and infectious diseases, and effective treatment methods for them are desired. In recent years, medicinal proteins have been attracting attention in the treatment of cat diseases, and mainly human medicinal proteins have been diverted. However, since the medicinal protein for humans has a different amino acid sequence from the in-vivo protein originally possessed by cats, the effect in the living body may be different. In addition, differences in amino acid sequences may cause allergies, or worse, cause anaphylaxis-like symptoms. Therefore, since it cannot withstand high-frequency administration, it is required to develop a medicinal protein originally possessed by cats.

Therefore, the applicant has developed a technique for producing cat-derived erythropoietin, which can be used for the treatment of diseases such as renal anemia, as a cat-derived pharmaceutical protein using transgenic birds (Patent Document 1, Patent Document 1, 2).

Patent Documents 1 and 2 disclose a method for obtaining cat-derived erythropoietin from the egg white of a transgenic bird that has been genetically modified to express cat-derived erythropoietin. In Patent Documents 1 and 2, in order to purify and recover cat-derived erythropoetin from the egg white of transgenic birds, a salting out method, an adsorption column chromatography method (blue sepharose chromatography, heparin chromatography, etc.), an ion exchange column chromatography, etc. It is disclosed that the graph method, the gel filtration column chromatograph method, and the antibody column method are used alone or in combination for purification.

In Example 9 of Patent Document 1, the egg white is diluted with water, the pH is adjusted to 5.0, the mixture is stirred for 15 minutes or more, centrifuged, and the supernatant is pretreated to adjust the pH to 7.0. In addition, blue sepharose chromatography, heparin chromatography, desalting column, anion exchange column chromatography, and gel filtration chromatography can be performed on the pretreated egg white liquor in this order to purify the cat-derived erythropoetin. It is disclosed.

In paragraph 0026 of Patent Document 2, egg white is diluted with a buffer as a pretreatment to adjust the pH, and then subjected to a blue Sepharose column. After dialysis, the target product is fractionated by a cation exchange column (SP column), which is not limited. It is disclosed that cat erythropoetin having a purity of 99% or more is obtained by purification of an anion exchange column (DEAE-sepharose column) after concentration and then desalting column treatment.

Patent Document 3 discloses a method of expressing a variant of erythropoietin derived from a cat having a modified amino acid sequence using cells such as CHO (Chinese hamster ovary) cells and purifying the cells from the medium or cell lysate. ing. Affinity chromatography, anion exchange chromatography, cation exchange chromatography and the like are exemplified as methods for purification. In Example 8 of Patent Document 3, the culture supernatant of cells expressing the variant of erythropoietin derived from cat was adjusted to pH 8.5, and phenylboronate chromatography, anion exchange chromatography and hydrophobic interaction chromatography were performed in this order. It is disclosed to perform and purify a variant of erythropoietin derived from a cat.

On the other hand, in Example 24 of Patent Document 4, the egg white of a transgenic chicken egg producing human-derived erythropoietin is diluted with 3 times the amount of 50 mM sodium acetate having a pH of 4.6, mixed, filtered, and cepharose. It is disclosed to purify human-derived erythropoietin using a cation exchange column. In Patent Document 5, as a method for culturing human-derived erythropoetin-producing mammalian cells and purifying and recovering human-derived erythropoetin from the culture supernatant, a first step of purification by dye affinity chromatography and a second purification by a hydroxyapatite column are performed. A method of performing a step and a third step of purification by cation exchange chromatography is disclosed.

In Patent Document 6, as a method for pretreating egg white for chromatography treatment, about 0.5% by weight to about 5% by weight of an acidic buffer solution is added to the egg white pool, and the pH is about about 0.5% by weight. A method of mixing the egg white and the acidic buffer so as to form a mixed egg white of 5 to 6.5 is disclosed. Patent Document 6 discloses a buffer solution containing about 5 M to about 6 M sodium acetate as the acidic buffer solution.

JP-A-2007-89578 Japanese Unexamined Patent Publication No. 2010-11195 Japanese Patent Publication No. 2012-504136 Special Table 2010-509327 Special Table 2010-511378 Gazette International release WO2015 / 164320

However, the purification method specifically disclosed in Patent Documents 1 and 2 uses blue sepharose chromatography. The columns used in blue sepharose chromatography have dye molecules immobilized on a carrier, but there is a risk that these molecules will be mixed into the purified product of erythropoietin derived from cats.

Further, Patent Documents 4 and 5 do not describe the production and purification of cat-derived erythropoietin. Further, Patent Document 4 discloses that the pH of the protein solution is adjusted in the acidic region before the treatment of cation exchange chromatography and that a buffer solution having a buffering action in the acidic region is used when loading the column. Not. Further, in the method of Patent Document 5, since purification is performed using dye affinity chromatography, there is a risk that dye molecules are mixed in the purified product of erythropoietin, as in Patent Documents 1 and 2.

Further, Patent Document 6 does not describe the production and purification of cat-derived erythropoietin. Of course, Patent Document 6 does not disclose suitable means for purifying and recovering erythropoietin derived from cats.

From the above, as a method for purifying and recovering cat-derived erythropoietin from a protein solution, a conventionally known method using dye affinity chromatography (Patent Documents 1, 2, etc.) is not always satisfactory. Further, Patent Document 3 describes not only a protein purification method using affinity chromatography but also a protein purification method not using it, but when affinity chromatography is not used, it is generally increased in order to increase the purification ratio. There is a problem that stepwise chromatography is required, and there is room for improvement.

Therefore, it is an object of the present invention to provide an optimized means for producing purified cat-derived erythropoietin.

The following inventions are disclosed in the present specification as means for solving the above problems.
(1) A method for producing purified cat-derived erythropoietin.
A pH adjustment step for adjusting the pH of a protein solution containing cat-derived erythropoietin to a range of 4 or more and 7 or less, and
In the pH adjusting step, the pH-adjusted protein solution is brought into contact with a carrier equipped with a cation exchange group to adsorb cat-derived erythropoetin, and then the adsorbed cat-derived erythropoetin is eluted from the carrier. How to include.
(2) The method according to (1), further comprising a precipitate removing step of removing the precipitate from the pH-adjusted protein solution in the pH adjusting step before the purification step.
(3) The method according to (1) or (2), wherein the pH of the protein solution is adjusted to a range of 4.7 or more in the pH adjusting step.
(4) In the pH adjusting step, the protein solution and a buffer solution having a buffering action in an acidic region containing a component having a buffering action in an acidic region at a concentration of 1 M or less are mixed (1) to (3). The method described in any of.
(5) In the pH adjusting step, the protein solution and a buffer solution having a buffering action in an acidic region, which is 1.5 parts by volume or more with respect to 1 part by volume of the protein solution, are mixed (1) to (4). ).
(6) The protein solution used in the pH adjustment step is a protein solution prepared using eggs of a transgenic bird having an exogenous gene encoding erythropoietin derived from a cat, according to any one of (1) to (5). The method described.
(7) A step of producing purified cat-derived erythropoietin by the method according to any one of (1) to (6), and
Water-soluble long-chain molecule-added cat-derived erythropoetin, which comprises a step of chemically modifying the purified cat-derived erythropoetin with a water-soluble long-chain molecule to obtain water-soluble long-chain molecule-added cat-derived erythropoetin. Production method.
(8) The method according to (7), further comprising a virus removal step of removing a virus by passing erythropoietin derived from a water-soluble long-chain molecule-added cat obtained in the water-soluble long-chain molecule addition step through a filter.
(9) A preparation containing erythropoetin derived from a water-soluble long-chain molecule-added cat obtained in the water-soluble long-chain molecule addition step and having the same osmotic pressure and pH as the body fluid of a mammal. The method according to (7) or (8), further comprising a chemical conversion step.

This specification includes the disclosure content of Japanese Patent Application No. 2016-072815, which is the basis of the priority of the present application.

According to the present invention, an improved means for producing purified cat-derived erythropoietin is provided.

In the present invention, cat-derived erythropoetin can be efficiently purified by using the pH adjustment step and the purification step using a carrier provided with a cation exchange group in combination.

Further, according to the embodiment of the present invention in which an insoluble component is precipitated as a precipitate from a protein solution by a pH adjusting step and then a purification step is performed, cat-derived erythropoietin can be efficiently purified.

Further, in the purification step, according to the embodiment of the present invention in which a cat-derived erythropoetin is adsorbed by contacting with a carrier having a cation exchange group in a buffer solution having a buffering action in an acidic region, the cat-derived erythropoetin is further added. It can be purified efficiently.

In Experiment 2, the photograph after 1 hour has passed since the egg white solution was adjusted to pH and allowed to stand is shown. The correspondence between the sample number and the pH condition is as shown in Table 1. The elution chart of purification by cation exchange column chromatography under each pH condition in Experiment 4 is shown. The solid line shows the total protein amount, and the dotted line shows the fEPO recovery rate. Most of the total protein content is OTF (ovotransferrin). The horizontal axis indicates the amount of eluate in column volume (CV). In Experiment 4, when purification by cation exchange column chromatography was performed at pH 4.8, the molecular weight marker M, the pretreated egg white raw material P before purification, the non-adsorbed fraction F, the washed fraction W, and the eluted fraction. The results of SDS-PAGE of the fraction E and the total eluted fraction A are shown. The unit of the number of the molecular weight marker M is kilodalton (kDa).

<Cat-derived erythropoietin>
Hereinafter, erythropoietin may be referred to as "EPO". In addition, cat-derived erythropoietin may be described as "fEPO".

The DNA base sequence encoding fEPO is shown in SEQ ID NO: 1. The amino acid sequence encoded by the base sequence shown in SEQ ID NO: 1 is as shown in SEQ ID NO: 2, and since the amino acid residues 1 to 26 are signal sequences, they are also called fEPO preproteins. The amino acid sequence in which the signal sequence of the fEPO preprotein is removed and the 192nd amino acid group is deleted is the mature fEPO, and the amino acid sequence of the mature fEPO is as shown in SEQ ID NO: 3. The fEPO may also be a polypeptide partially containing the amino acid sequence of the fEPO preprotein of SEQ ID NO: 2 or a polypeptide partially containing the amino acid sequence of the mature fEPO of SEQ ID NO: 3.

The fEPO is not limited to the fEPO preprotein consisting of the amino acid sequence shown in SEQ ID NO: 2 and the mature fEPO consisting of the amino acid sequence shown in SEQ ID NO: 3, and active mutants thereof can also be used. As the active mutant, preferably, under the activity measurement conditions shown in the examples of the present specification, an fEPO preprotein consisting of the amino acid sequence shown in SEQ ID NO: 2 or a mature fEPO consisting of the amino acid sequence shown in SEQ ID NO: 3 was used. It is a polypeptide showing an activity of 10% or more, preferably 40% or more, more preferably 60% or more, still more preferably 80% or more of the activity. The above-mentioned active variant includes, for example, a polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 2 or 3, more preferably SEQ ID NO: 2. Or, in the amino acid sequence shown in 3, a polypeptide consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in total at the N-terminal and / or C-terminal thereof, or shown in SEQ ID NO: 2 or 3. A polypeptide consisting of an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more amino acid identity with respect to the amino acid sequence. Applicable. The active mutant of the fEPO preprotein consisting of the amino acid sequence shown in SEQ ID NO: 2 is more preferably selected from the N-terminal, C-terminal, portion of residues 1 to 26 of SEQ ID NO: 2 and residue 192. Includes mutations as listed above in at least one location. The mature fEPO consisting of the amino acid sequence shown in SEQ ID NO: 3 may have a methionine residue encoded by the start codon added to the N-terminal. In the present specification, the term "plurality" means, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more. Also, 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 29 or less, 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less. , 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less. .. "Amino acid identity" refers to SEQ ID NO: 2 or 3 when two amino acid sequences are aligned and a gap is introduced as needed to maximize the degree of amino acid matching between the two. The ratio (%) of the same amino acid residue to the total number of amino acid residues of the indicated protein. Amino acid identity can be calculated using a protein retrieval system by BLAST or FASTA (Karlin, S. et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 5873-5877; Altschul, SF et al., 1990, J. Mol. Biol., 215: 403-410; Pearson, WR et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448 ). Conservative amino acid substitution is desirable for amino acid substitution. “Conservative amino acid substitution” refers to a substitution between amino acids having similar properties such as charge, side chain, polarity, and aromaticity. Amino acids with similar properties include, for example, basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine), non-polar amino acids. It can be classified into sex amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched amino acids (leucine, valine, isoleucine), aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine), etc. can.

The fEPO is a transgenic cell in which a foreign gene encoding fEPO is introduced into a generally available animal cell (for example, CHO cell), a plant cell, a prokaryote, or yeast and genetically modified to express fEPO. Produced in, or in transgenic animals or plants genetically modified to express fEPO by introducing a foreign gene encoding fEPO, or produced using a cell-free protein synthesis system. Things can be used, but they are not limited to these. Examples of the transgenic animal include birds, and examples of the birds include poultry birds such as chickens, quail, turkeys, ducks, ducks, ostriches, geese, onagadori, pigeons, emu, geese, and holo holo butterflies, and chickens are particularly preferable. Transgenic birds are preferably those that accumulate fEPO in their eggs. A method for producing a transgenic bird expressing fEPO in an egg and obtaining fEPO from the egg is as described in Patent Document 1 and the like. The transgenic plant is not particularly limited, and a dicotyledonous plant may be used even if it is a monocotyledonous plant. Etc.), Gramineae (Asparagus, Yuri, Onion, Nira, Katakuri, etc.), Ginger (Ghost, Myoga, Ukon, etc.) Rapeseed, potash flower, broccoli, daikon, etc.), grass family (tomato, eggplant, potato, tobacco, etc.), legume family (soybean, pea, green beans, alfalfa, etc.), uri family (cucumber, melon, pumpkin, etc.), seri family (cucumber, melon, pumpkin, etc.) For carrots, celery, honeybees, etc.), grasses (lettuce, etc.), grasses (wata, okura, etc.), red-spotted plants (sugar beet, spinach, etc.), dicotyledons (eucalyptus, cloves, etc.), yanagiaceae (poplar, etc.) Examples include, but are not limited to, the plants to which they belong.

The fEPO polypeptide may be modified with one or more sugar chains or other groups. The type and number of sugar chains that modify fEPO are not particularly limited, and the type and number of sugar chains according to the expression system used (that is, transgenic cells, transgenic animals, transgenic plants, cell-free protein synthesis system, etc.) It is generally modified by. For example, when the expression system used is chicken, the sugar chain modification is mainly performed by a sugar chain in which 2 to 5 sugar chains containing β-N-acetylglucosamine residue are further bound to the trimannosyl core. be.

<Purified cat-derived erythropoietin>
In the present invention, "purified" feline-derived erythropoetin (fEPO) means fEPO that exists in a state where the fEPO concentration per protein amount is higher than that in the protein solution that is the raw material before purification, and is purified. It may be a crude product of any degree. For example, fEPO that has undergone a purification step using a cation exchanger is also included in the purified fEPO according to the present invention.

<Protein solution>
The protein solution used as a raw material in the method of the present invention is not particularly limited as long as it is a liquid substance containing fEPO, and may be in the form of fEPO dissolved in water. As such a protein solution, an fEPO-containing protein solution derived from an fEPO production system such as a transgenic cell expressing fEPO, a transgenic animal, a transgenic plant, or a cell-free protein synthesis system can be used. ..

The first preferred form of the protein solution used as a raw material is a protein solution prepared using transgenic avian eggs having an exogenous gene encoding fEPO. The egg can be in various forms such as a whole egg obtained by breaking an egg, an egg white separated from the whole egg, an egg yolk, and the like, and is preferably an egg white. The eggs may be frozen and then thawed. The protein solution prepared using the egg may be the egg itself or a solution obtained by diluting the egg with water or an aqueous solution. The protein solution prepared using the eggs is preferably sheared to be sufficiently homogenized. More preferably, the protein solution prepared using the egg is, for example, 3 parts by volume or less, preferably 2 parts by volume, based on the sheared egg itself or the egg and 1 part by volume of the egg. It is a mixed solution with water of 1 part or less, more preferably 1 part by volume or less.

A second preferred form of the protein solution used as a raw material is a protein solution prepared from a culture mixture formed by culturing transgenic cells (eg, CHO cells) having an exogenous gene encoding fEPO in a medium. be. As the protein solution according to the second preferred form, preferably, the culture mixture itself, the supernatant of the culture mixture, the crushed solution obtained by crushing the cells in the culture mixture, the supernatant of the crushed solution, and the like. Alternatively, a solution obtained by diluting one of these solutions with a concentrated solution, water or an aqueous solution can be exemplified.

<pH>
In the present invention, the pH value refers to a value measured using a pH meter with the liquid temperature at the time of measurement being 22.5 ± 0.5 ° C., unless otherwise specified. It is preferable to use the pH meter after performing two-point calibration at pH 4 and pH 7 using a commercially available standard reagent.

<pH adjustment process>
The pH adjusting step in the present invention is a step of adjusting the pH of the protein solution containing fEPO to a range of 4 or more and 7 or less.

By adjusting the pH of the protein solution containing fEPO to a range of 4 or more and 7 or less, surprisingly, some of the components contained in the protein solution other than fEPO are insolubilized and precipitated. Remarkably promoted. As a result, the proportion of fEPO in the dissolved components increases in the protein solution that has undergone the pH adjustment step. Therefore, fEPO can be efficiently purified by subjecting the protein solution that has undergone the pH adjustment step to a purification step described later. On the other hand, when the pH of the protein solution containing fEPO is set to a range of less than 4 or more than 7, precipitates due to components other than fEPO are difficult to form, and the effect of increasing the purification efficiency of fEPO cannot be obtained.

The range of pH in the pH adjusting step is more preferably 4.1 or more, 4.2 or more, 4.3 or more, 4.4 or more, 4.5 or more, and 4 from the viewpoint of promoting the formation of precipitates. 6.6 or more, 4.7 or more, 4.8 or more, 4.9 or more, 5.0 or more, and 6.5 or less, 6.4 or less, 6.3 or less, 6.2 or less, 6. 1 or less, 6.02 or less, 6.0 or less, 5.9 or less, 5.8 or less, 5.7 or less, 5.6 or less, 5.5 or less, 5.4 or less, 5.3 or less, 5. It is 2 or less and 5.1 or less. In particular, when the pH is 4.7 or higher, 4.8 or higher, 4.9 or higher or 5.0 or higher, and / or the pH is 6.1 or lower, 6.02 or lower, 6.0 or lower, 5. 9 or less, 5.8 or less, 5.7 or less, 5.6 or less, 5.5 or less, 5.4 or less, 5.3 or less, 5.2 or less, 5.1 or less, from the protein solution It is preferable because the formation of the precipitate of is particularly promoted.

Further, adjusting the pH of the protein solution to the above range in the pH adjusting step is a viewpoint that fEPO can be obtained in a high yield when purifying using a carrier containing a cation exchange group in the purification step described later. It is also preferable from. From this point of view, the above range of pH in the pH adjusting step is particularly 4.4 or more, 4.5 or more, 4.6 or more, 4.7 or more, 4.8 or more, 4.9 or more, 5.0 or more. It is preferable to do so. From the viewpoint of yield, the upper limit of pH in the pH adjusting step is not particularly limited, but preferably 6.5 or less, 6.4 or less, 6.3 or less, 6.2 or less, 6.1 or less, 6.0 or less, 5.9 or less, 5.8 or less, 5.7 or less, 5.6 or less, 5.5 or less, 5.4 or less, 5.3 or less, 5.2 or less.

Furthermore, adjusting the pH of the protein solution to the above range in the pH adjusting step is also preferable from the viewpoint of maintaining the activity of fEPO. From this point of view, the range of pH in the pH adjusting step is particularly 4.1 or more, 4.2 or more, 4.3 or more, 4.4 or more, 4.5 or more, 4.6 or more, 4.7 or more. It is preferably 4.8 or more, 4.9 or more, and 5.0 or more. From the viewpoint of activity, the upper limit of pH in the pH adjusting step is not particularly limited, but is preferably 6.5 or less, 6.4 or less, 6.3 or less, 6.2 or less, 6.1 or less, 6.0 or less. ..

As described above, in one embodiment of the present invention, the pH adjustment step promotes the formation of precipitates, improves the yield of purification of fEPO using a carrier containing a cation exchange group, and activates fEPO. It can have an advantageous effect on retention. That is, by performing the pH adjustment step and the subsequent purification step of fEPO under consistent pH conditions, the activity of fEPO can be maintained and the purification efficiency can be improved. Such an effect is an unexpected advantageous effect that is not suggested by the prior arts such as Patent Documents 4 and 6.

The above effect of the pH adjustment step is obtained when a protein solution prepared by using the above-mentioned first preferred form as a raw material protein solution, that is, a transgenic avian egg having an exogenous gene encoding fEPO is used. This is especially noticeable. It is known that the eggs of transgenic birds contain ovalbumin, ovotransferrin, ovomucoid, etc. as major proteins, and these major proteins have an isoelectric point in the acidic region (for example, the isoelectric point of ovoalbumin is In general, 4.6, the isoelectric point of ovotransferrin is 6.1, and the isoelectric point of ovomucoid is 3.9 to 4.5), whereas fEPO has the amino acid sequence shown in SEQ ID NO: 3. The isoelectric point is estimated to be 8.2. Therefore, by setting the pH of the protein solution prepared using the eggs of transgenic birds having an exogenous gene encoding fEPO to 4 or more and 7 or less, fEPO whose isoelectric point is in the alkaline region is dissolved. It is presumed that proteins in the acidic region of the isoelectric point are precipitated, but this estimation mechanism is not constrained.

The pH adjustment step is performed on the protein solution before the purification step described later. By performing the pH adjustment step first and then the purification step, the above-mentioned effect of increasing the purification efficiency of fEPO in the purification step can be obtained.

The pH can be adjusted by mixing a sufficient amount of the buffer solution and the protein solution, and if necessary, mixing an acid or a base to adjust the pH of the mixture to the target pH in the above range. .. More specifically, the pH of a mixture of a sufficient amount of buffer solution and protein solution is measured, and if the pH is outside the above range, an appropriate amount of acid or base is mixed to adjust the pH of the mixture. It can be adjusted to be in the range. The sufficient amount of the buffer solution is, for example, 1.5 parts by weight or more, preferably 2 parts by weight or more with respect to 1 part by volume of the protein solution, and the upper limit is not particularly limited, but typically 10 parts by weight. Hereinafter, more typically, it is 7 parts by weight or less. By using a sufficient amount of buffer solution, the viscosity of the protein solution can be sufficiently reduced, and it is preferable because it is easy to handle when it is used as it is for purification using a carrier containing a cation exchange group in the purification step. Further, also in the embodiment for removing the precipitates precipitated in the pH adjusting step, it is preferable because the precipitates can be easily removed by sufficiently reducing the viscosity of the protein solution. The viscosity of a mixture of a sufficient amount of buffer solution and protein solution in the pH adjustment step is 20 cP or less when measured with a B-type viscometer (for example, TVB-10 type viscometer manufactured by Toki Sangyo Co., Ltd.) at 15 ° C. , More preferably 10 cP or less, still more preferably 5 cP or less. In Patent Document 6, an acidic buffer solution of about 0.5% by weight to about 5% by weight is added to the egg white pool to form a mixed egg white having a pH of about 5 to 6.5. As described above, a method of mixing the egg white and the acidic buffer solution is disclosed, and it is described that the viscosity can be reduced by using the acidic buffer solution in this pH range. However, as in Patent Document 6, the viscosity of egg white to which a small amount of acidic buffer is added is very high, and it is not easy to handle when removing precipitates or performing column chromatography.

The buffer solution is not particularly limited, and for example, one containing at least one selected from glycine, phthalic acid, citric acid, succinic acid, acetic acid and phosphoric acid as a buffer component can be used. Further, those having low biotoxicity suitable for the production of pharmaceuticals are preferable, and for example, an acetate buffer solution is preferable because it is easy to use. Further, in view of the isoelectric point of fEPO (fEPO consisting of the amino acid sequence of SEQ ID NO: 3 is estimated to have an isoelectric point of 8.2 from the amino acid sequence), a buffer solution having a buffering action in an acidic region is preferably used. Can be used. The buffering action is an action in which the pH hardly changes even if an acid or a base is added or the concentration is changed due to evaporation or dilution. The acidic region preferably includes the above pH range. The concentration of the buffer component of the buffer solution is preferably 1 M or less, more preferably 500 mM or less, more preferably 400 mM or less, more preferably 300 mM or less, more preferably 200 mM or less, more preferably 100 mM or less, and even more preferably 90 mM or less. , 80 mM or less is more preferable. The protein may be denatured when a high concentration of buffer component comes into contact with the protein. A buffer solution having a relatively low concentration of the buffer component as described above is preferable because it causes less damage to the protein containing fEPO when mixed with the protein solution. In Patent Document 6, it is described that an acidic buffer solution containing about 5 M to about 6 M sodium acetate is mixed with egg white, but such a buffer solution containing high concentration of acidic sodium denatures egg white. It is highly probable. In the present embodiment, the upper limit of the concentration of the buffer component of the buffer solution is not particularly limited, but is typically 1 mM or more, more preferably 10 mM or more, still more preferably 20 mM or more. The concentration of the buffer component is a concentration converted as being present in the form of a free form, regardless of whether the buffer component is contained in the form of a salt or a form of a free form.

The pH-adjusted protein solution preferably contains the protein at a concentration of preferably 1 mg / mL or more, preferably 100 mg / mL or less, more preferably 5 mg / mL or more, and more preferably 15 mg / mL or less. The pH-adjusted protein solution has a fEPO of preferably 12.3 μg / mL or more, more preferably 16.6 μg / mL or more, preferably 60.0 μg / mL or less, and more preferably 41.7 μg / mL or less. Contains in concentration. The protein concentration or fEPO concentration is particularly high when a protein solution prepared using the above-mentioned first preferred form as a raw material protein solution, that is, a transgenic avian egg having an exogenous gene encoding fEPO is used. Suitable. Here, the fEPO concentration refers to a concentration based on the weight of fEPO, which is composed of the amino acid sequence shown in SEQ ID NO: 3, is not chemically modified by a water-soluble long-chain molecule or the like, and is converted as a polypeptide of mature fEPO. ..

The protein concentration is measured by the absorbance at 280 nm using a quartz cell having an optical path length of 1 cm using a spectrophotometer using water as a blank. At this time, the analytical sample is diluted with water and measured so that the absorbance does not exceed the upper limit of the analytical sensitivity of the spectrophotometer, and the background correction is performed using the value of the absorbance at 320 nm. Specifically, the protein concentration (mg / mL) is calculated as (absorbance at 280 nm-absorbance at 320 nm) × dilution ratio × 0.959.

As a method for measuring the fEPO concentration (ng / mL), an EPO-ELISA kit (manufactured by Roche) is used to measure the pretreated egg white raw material and the fEPO standard product (manufactured by the company). A calibration curve of 4.0 to 1.1 ng / mL is prepared from the standard product, and the erythropoietin concentration in the protein solution is calculated using the following formula. Formula: Calculate as concentration (ng / mL) = (average absorbance-intercept) / slope x dilution rate.

<Precipitate removal process>
The protein solution whose pH has been adjusted by the pH adjustment step may be used as it is for the treatment in the purification step, or the precipitate removal which removes the precipitate from the pH-adjusted protein solution in the pH adjustment step before the purification step. The process may be performed. The precipitate in the pH adjustment step is considered to be a protein component as described above.

The precipitate removal step is a step of removing the precipitate from the pH-adjusted protein solution by any means such as filtration and centrifugation, and recovering the protein solution having a reduced amount of precipitate. By performing the precipitate removing step, the amount of unnecessary protein can be reduced and the purification efficiency can be improved, and the handling of the protein solution in the purification step becomes easy.

Examples of the method for separating the precipitate by filtration include filtration using a filter having an average pore size of 0.5 μm or more and 25 μm or less, and more preferably an average pore size of 0.2 μm or more and 5 μm or less. Examples of the method for separating the precipitate by centrifugation include a method for centrifuging from 5,000 × g to 10,000 × g.

<Refining process>
In the purification step, the pH-adjusted protein solution in the pH-adjusting step was subjected to a precipitate removal step as necessary, and then brought into contact with a carrier equipped with a cation exchange group to adsorb fEPO, and then adsorbed. It involves eluting fEPO from the carrier.

Strong cation exchange group including a carboxyl group _- as the cation exchange group a sulfonic acid group (-SO ³⁾ (-COO ^-), etc. Weak cation exchange groups comprising the like. Specific examples of the strong cation exchange group containing a sulfonic acid group include sulfopropyl (SP) and the like. Specific examples of the weak cation exchange group containing a carboxyl group include carboxymethyl (CM).

The carrier to which the cation exchange group is linked is not particularly limited as long as it is a carrier generally used in the field of ion exchange chromatography, and for example, a styrene-based carrier, an agarose-based carrier, and a cellulose-based carrier can be used. Examples of the styrene-based carrier include a carrier composed of a crosslinked polymer obtained by copolymerizing styrene and divinylbenzene, and examples of commercially available products include trade names MiniBeads, MonoBeads, and SOURCE15 commercially available from GE Healthcare Japan Co., Ltd. , SOURCE30 can be exemplified. Examples of commercially available agarose-based carriers include the trade names Sepharose High Performance, Sepharose Fast Flow, Sepharose 4 Fast Flow, Sepharose XL, and Sepharose Big Base, which are commercially available from GE Healthcare Japan Co., Ltd. .. Examples of the cellulosic carrier include carriers of the trade name Cellfine MAX series commercially available from JNC Corporation.

By using a protein solution whose pH has been adjusted to 4 or more and 7 or less by the above pH adjustment step as the protein solution to be brought into contact with a carrier having a cation exchange group (hereinafter referred to as "cation exchanger"), It is preferable because the yield of fEPO in purification with a cation exchanger can be increased. From this point of view, the pH of the protein solution to be brought into contact with the cation exchanger is particularly 4.1 or higher, 4.2 or higher, 4.3 or higher, 4.4 or higher, 4.5 or higher, 4.6 or higher, 4. It is preferably 7 or more, 4.8 or more, 4.9 or more, and 5.0 or more, and among these ranges, it is particularly preferable that the pH value is 4.4 or more or higher. From the viewpoint of this yield, the upper limit of the pH of the protein solution to be brought into contact with the cation exchanger is not particularly limited, but is preferably 6.5 or less, 6.4 or less, 6.3 or less, 6.2 or less, 6.1. Below, 6.0 or less, 5.9 or less, 5.8 or less, 5.7 or less, 5.6 or less, 5.5 or less, 5.4 or less, 5.3 or less, 5.2 or less.

Further, it is preferable to use a protein solution whose pH is adjusted to 4 or more and 7 or less by the above pH adjusting step as the protein solution to be brought into contact with the cation exchanger from the viewpoint of maintaining the activity of fEPO. From this point of view, the pH of the protein solution to be brought into contact with the cation exchanger is particularly 4.1 or higher, 4.2 or higher, 4.3 or higher, 4.4 or higher, 4.5 or higher, 4.6 or higher, 4. It is preferably 7 or more, 4.8 or more, 4.9 or more, and 5.0 or more, and among these ranges, it is particularly preferable that the pH value is 4.8 or more or higher. From the viewpoint of this activity, the upper limit of the pH of the protein solution to be brought into contact with the cation exchanger is not particularly limited, but is preferably 6.5 or less, 6.4 or less, 6.3 or less, 6.2 or less, 6.1 or less. , 6.0 or less.

In this way, by contacting the protein solution adjusted to a predetermined pH range in the pH adjusting step with the cation exchanger, the yield of purification of fEPO using a carrier containing a cation exchange group can be improved, and the yield of fEPO can be improved. It can exert an advantageous effect in maintaining activity. As described above, the pH adjustment step can promote the formation of precipitates. As described above, by performing the pH adjustment step and the subsequent purification step of fEPO under consistent pH conditions, the activity of fEPO can be maintained and the purification efficiency can be improved.

When the pH-adjusted protein solution is brought into contact with the cation exchanger, first, a bed formed by filling a column or the like with the cation exchanger is adsorbed so as to have a pH adjusted to be lower than the isoelectric point of fEPO. It is preferably filled with a buffer solution for equilibration. The adsorption buffer is substantially the same as the buffer used in the pH adjustment step (preferably, the concentration of the buffer component of the buffer is also substantially the same), for example, acetate buffer, phosphate buffer. , Citrate buffer, etc. However, in view of the isoelectric point of fEPO, an adsorption buffer having a buffering action in an acidic region can be preferably used. The pH value of the acidic region can be selected from the same range as the above pH value of the protein solution to be contacted with the cation exchanger, and is preferably substantially the same as the protein solution to be contacted with the cation exchanger. The pH value can be the same. The buffering action is an action in which the pH hardly changes even if an acid or a base is added or the concentration is changed due to evaporation or dilution. The isoelectric point of fEPO is estimated to be 8.2 from the amino acid sequence shown in SEQ ID NO: 3, and it is considered that fEPO is positively charged under acidic pH conditions. Therefore, when the protein solution is brought into contact with the cation exchanger in the adsorption buffer having a buffering action in the acidic region, fEPO can be adsorbed to the cation exchanger with high probability, and fEPO is purified. can do. This effect is particularly remarkable when a protein solution prepared by using the above-mentioned first preferred form as a raw material protein solution, that is, a transgenic avian egg having an exogenous gene encoding fEPO is used. As described above, most of the major proteins contained in the eggs of transgenic birds have an isoelectric point in the acidic region and are difficult to be adsorbed on the cation exchanger under acidic conditions, so that they have a buffering effect in the acidic region. It is considered that fEPO mixed with the main protein can be selectively adsorbed on the cation exchanger in the adsorbing buffer solution.

The pH-adjusted protein solution is loaded onto the bed of the equilibrated cation exchanger to adsorb fEPO to the cation exchanger and wash the bed if necessary. Then, the elution buffer solution in which salts are added to the adsorption buffer solution to increase the ionic strength stepwise or continuously is passed through the bed of the cation exchanger on which the fEPO is adsorbed to elute the fEPO. , FEPO-containing liquid. The ionic strength of the elution buffer is preferably 100 mM or more and 300 mM or less. Purification using a cation exchanger is preferably carried out within a temperature range of 20 ° C. or higher and 25 ° C. or lower. The pH of the cleaning buffer solution used for cleaning and the adsorption buffer solution can be substantially the same as the pH value of the protein solution in contact with the cation exchanger.

Purification using the cation exchanger is preferably performed first as the fEPO purification step other than the precipitate removal step. The reason is that when the protein solution used as a raw material contains a large amount of albumin, the purification ratio can be increased by removing it first.

In the purification step of the present invention, in addition to the purification using the cation exchanger, a step of purifying fEPO by further protein purification treatment may be combined. As other purification treatments, salting out, adsorption chromatography, ion exchange chromatography, size exclusion chromatography, antibody column method and the like can be used alone or in combination. In addition, other protein purification treatments not exemplified may be performed. Examples of the adsorption chromatography include dye affinity chromatography, heparin affinity chromatography, metal ion affinity chromatography and the like. Examples of the dye affinity chromatography include those using a solid phase carrier to which a triazine dye such as Cibacron Blue 3G is linked. However, the method of the present invention more preferably does not include the step of purifying by dye affinity chromatography. This is because it is possible to prevent the dye from being mixed with the target purified fEPO by not including the step of purifying by dye affinity chromatography. As the ion exchange chromatography, in addition to the above-mentioned cation exchange chromatography, anion exchange chromatography can also be used.

When performing a plurality of purification treatments, the buffer solution for dissolving the protein containing fEPO can be exchanged between the treatments, if necessary. The buffer solution can be replaced by the usual method using ultrafiltration or dialysis.

<Water-soluble long-chain molecule addition step>
More preferably, the present invention further includes a water-soluble long-chain molecule addition step of chemically modifying fEPO purified in the purification step with a water-soluble long-chain molecule to obtain a water-soluble long-chain molecule-added fEPO.

The water-soluble long-chain molecule is not particularly limited, and for example, PEG (polyethylene glycol), polyamino acid, polypropylene glycol and the like can be used. These can produce reaction precursors and can be added to proteins by a synthetic reaction. Of these, PEG is non-antigenic and non-toxic, so it is also effective in reducing the antigenicity of the modified protein and suppressing the expression of anti-protein antibody as a side effect.

It has been reported that when fEPO is administered to rodents such as mice and rats and the hematopoietic effect is confirmed, the increase in reticulocytes is maximized on the 4th day after a single administration in blood, and the effect disappears on the 7th day. However, it is known that the addition of PEG inhibits the metabolism in the liver, prolongs the life span in the blood, prolongs the duration of the drug effect, and doubles the effect for 14 days in rat hematopoiesis experiments.

As the molecular weight of the added water-soluble long-chain molecule such as PEG increases, the blood life of the water-soluble long-chain molecule-fEPO complex increases. However, since the addition of an extremely high molecular weight water-soluble long-chain molecule inhibits the hematopoietic activity of fEPO (WO02 / 032957), the weight average molecular weight of the water-soluble long-chain molecule is 5 kDa or more and 40 kDa or less is an in vivo hematopoietic effect. Is preferable, more preferably 10 kDa or more, and more preferably 30 kDa or less. More preferably, it is 20 kDa.

The fEPO has at least three binding sites for water-soluble long-chain molecules, and the water-soluble long-chain molecule that binds to one fEPO polypeptide can be 1 (mono), 2 (di), or 3 (tri) molecules. However, since PEG binds to a plurality of sites, the receptor binding ability of EPO is inhibited and the in vivo activity is lowered, a mono- or di-form is preferable, and a di-form is particularly preferable.

In the present invention, "chemical modification" refers to chemically changing a specific functional group to change functions such as activity and reactivity, and when fEPO is chemically modified with a water-soluble long-chain molecule such as PEG. Reacts a functional group (for example, a primary amino group) of a polypeptide constituting fEPO with a functional group of a water-soluble long-chain molecule to form a covalent bond, and is water-soluble in the polypeptide constituting fEPO. Refers to the addition of long-chain molecules.

As a method of chemically modifying fEPO with water-soluble long-chain molecules, fEPO and water-soluble long-chain molecules are mixed at a molar ratio of about 1: 1 to 10, and then mixed at 4 to 37 ° C. for 30 to 180. Examples thereof include a method of reacting for 1 minute, adding about 1/10 of a 100 mM glycine solution as a reaction terminator, and stopping the reaction while mixing at 4 to 37 ° C. for 1 hour.

The water-soluble long-chain molecule-modified fEPO has an additional number of water-soluble long-chain molecules of 1 or 2 or more, and is apparent in an aqueous solvent measured by gel filtration column chromatography of one water-soluble long-chain molecule-modified molecule. The molecular weight of is preferably 100 kDa to 900 kDa, the number of water-soluble long-chain molecules added is 1, and the apparent molecular weight is 100 kDa to 500 kDa, or the number of water-soluble long-chain molecules added is 2. It is more preferable that the apparent molecular weight is 100 kDa to 500 kDa. The apparent molecular weight is measured by the gel filtration column chromatography using a low-pressure chromatographer AKTA explorer 100 (manufactured by Amersham) and a gel filtration column Superdex 200 10/300 (manufactured by Amersham).

Generally, a method for covalently attaching a water-soluble long-chain molecule to a protein includes a chemical reaction with a polyol, lactol, amine, carboxylic acid or carboxylic acid derivative which is an oxidatively active functional group of the protein or sugar chain. There is also a method using a sulfonate ester activated polymer, for example, a sulfonate ester activated PEG. When it is added to EPO, it can be added by these methods.

Hereinafter, a preferable form when the water-soluble long-chain polymer is PEG will be described.

As the PEGylation reaction precursor used for covalently bonding PEG to a protein, one in which one end of a long-chain molecule is methoxylated can be used. Further, a PEG in which the non-methoxyinated terminal is modified into a group capable of forming a covalent bond by undergoing a nucleophilic reaction by a nucleophilic group such as an amino group possessed by fEPO (for example, succinimidyl fatty acid esterified) has been developed. Among them, the following formula:
CH _3- O- (CH ₂ CH ₂ O) _n -XY
(During the ceremony,
Y represents a leaving group such as a succinimidyloxy group.
X is-(CH ₂ ) _m- C (= O)-
It is a group represented by
m is an integer of 1 or more, 8 or less, more preferably 4 or more, more preferably 6 or less, and most preferably 5.
n is an integer indicating the degree of polymerization)
The one represented by is preferable from the viewpoint of reactivity. It is known that when the PEGylation reaction precursor represented by the above formula is reacted with human EPO, it is selectively added to the amino group of the N-terminal or lysine residue. Since there are multiple lysine residues in EPO, the number of PEGs added increases as the reaction progresses, resulting in a mixture of isomers with different numbers of additions. It is preferred that alanine 27, lysine 71 and / or lysine 78 are PEGylated in the fEPO consisting of SEQ ID NO: 2, and alanine 1, lysine 45 and / or lysine 52 are PEGylated in the mature fEPO consisting of SEQ ID NO: 3, respectively.

<Virus removal process>
In the method of the present invention, when the above-mentioned water-soluble long-chain molecule addition step is performed, more preferably, the water-soluble long-chain molecule addition fEPO obtained in the water-soluble long-chain molecule addition step is passed through a filter to remove the virus. It further includes a virus removal step. In the water-soluble long-chain molecule addition step, a liquid composition in which the water-soluble long-chain molecule-added fEPO is dissolved is obtained, and the virus removal step can be performed by passing the liquid composition through the filter.

Here, as the filter, a filter composed of a non-woven fabric, a hollow fiber membrane, a porous film, or the like can be used. The filter preferably has pores with a width in the range of 1 to 1,000 nm.

Examples of the virus removed by the virus removal step include human parvovirus, mouse microvirus, porcine parvovirus, encephalomyelitis virus, hepatitis virus, human immunodeficiency virus and the like.

<Formulation process>
When the above-mentioned water-soluble long-chain molecule addition step is performed, the method of the present invention more preferably contains the water-soluble long-chain molecule-added fEPO obtained in the water-soluble long-chain molecule addition step, and contains the osmotic pressure of the body fluid of a mammal. And further include a formulation step of preparing a liquid composition having the same osmotic pressure and pH as pH. Here, "same" includes "substantially the same".

Mammals refer to mammals to which fEPO is administered, such as cats and dogs. An fEPO-containing liquid composition having the same osmotic pressure and pH as the osmotic pressure and pH of the body fluid of the target mammal is preferable because it does not easily cause pain when subcutaneously administered to the target mammal. For example, when producing a liquid composition suitable for administration to a mammal having a body fluid having an osmotic pressure of about 280 mOsm / KgH _{2 O and a pH of about 7.4, it is prepared in the formulation step.} the liquid composition, the osmotic pressure of preferably 200 mOsm / KGH ₂ O or more, 400 mOsm / KGH ₂ O or less, more preferably 250 mOsm / KGH ₂ O or more, and more preferably not more than 300mOsm / KgH ₂ O, pH is preferably Is 7.0 or more, 8.0 or less, more preferably 7.3 or more, and more preferably 7.7 or less. When the osmotic pressure of the body fluid of a mammal is XmOsm / KgH ₂ O, the osmotic pressure of the liquid composition is preferably (X ± 100) mOsm / KgH ₂ O, more preferably (X ± 50) mOsm /. It can also be KgH _{2 O.} When the pH of the body fluid of the mammal is Y, the pH of the liquid composition can be preferably (Y ± 0.5), more preferably (Y ± 0.1). Here, the osmotic pressure and pH refer to the values measured at 20 to 25 ° C.

<Form of erythropoietin derived from cats>
The purified fEPO, the water-soluble long-chain molecule-added fEPO and the liquid composition produced by the method of the present invention may be provided in any form, for example, a drug composition for non-human animals. It may be in the form of an intermediate product for the production of an fEPO-containing drug composition, for example, an fEPO solution containing fEPO in water.

The purified fEPO, the water-soluble long-chain molecule-added fEPO and the liquid composition produced by the method of the present invention include a solvent such as water, an excipient, in addition to the purified fEPO or the water-soluble long-chain molecule-added fEPO. It may be a combination of at least one component selected from the group consisting of a disintegrant, a binder, a stabilizer, a pH adjuster, an osmotic pressure adjuster and a surfactant, and the component is preferably a combination. It is a pharmacologically acceptable component for non-human animals to be administered. The purified fEPO, the water-soluble long-chain molecule-added fEPO and the liquid composition produced by the method of the present invention are the purified fEPO or the water-soluble long-chain molecule-added fEPO combined with other additives. The additives may include, for example, lubricants, coating agents, colorants, anti-aggregation agents, absorption promoters, solubilizers, health food materials, dietary supplement materials, vitamins, flavors, sweeteners, preservatives. At least one additive selected from the group consisting of agents, preservatives and antioxidants can be used. The additive is preferably a pharmacologically acceptable additive for the non-human animal to be administered.

The method of the present invention can further include the step of preparing a solution, gel or powder containing purified fEPO or water-soluble long-chain molecule-added fEPO and then forming it into various pharmaceutical forms. Here, as the form of the pharmaceutical product, for example, an injection, a drip, an injection, an external liquid, a patch, a coating agent (cream, ointment, etc.), an inhalant, a spray, a suppository, a rectal capsule. Examples thereof include a subcutaneously implantable sustained-release preparation, a micelle preparation, a gelled preparation, a liposome preparation, and a form of a preparation for parenteral administration such as a pessary for intravaginal administration, and a form of a preparation for oral administration. Applications of injections and infusions include intravenous, subcutaneous, intradermal, intramuscular, intramuscular, intranasal, eye drops, intracerebral, intraperitoneal, lesions and the like. The injection may be in the form of a prefilled syringe formulation. Applications of the injectable agent include in the rectum and vagina. Examples of various preparations for oral administration include solid preparations such as tablets, pills, capsules, powders, fine granules and granules, and liquid preparations such as extracts, elixirs, syrups, tinctures and limonades. Be done. Among these forms of drugs, a preparation in which fEPO is dissolved in a liquid is included in the above-mentioned "fEPO-containing liquid composition".

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples. If the product name is stated, follow the instructions in the attached manual unless otherwise specified.

<Experiment 1>
The fEPO-containing egg white was prepared by a transgenic chicken by a modified method based on the method described in JP-A-2007-89578, and the main points of the process are described below.

In the following tests, the amount of fEPO is an amount based on the weight converted as a polypeptide (mature fEPO) consisting of the amino acid arrangement shown in SEQ ID NO: 3. The fEPO produced in egg white is a polypeptide (mature fEPO) consisting of the amino acid sequence shown in SEQ ID NO: 3.

1. 1. Preparation of egg white containing EPO derived from cats using transgenic chickens 1.1. Microinjection of retroviral vector into chicken embryos and artificial hatching The retroviral vector for fEPO expression was microinjected into chicken embryos to prepare transgenic chickens expressing fEPO. Microinjection and artificial hatching were performed under sterile conditions.

The DNA base sequence encoding fEPO (total length of preprotein) is shown in SEQ ID NO: 1.

A solution containing a retroviral vector for fEPO expression prepared by the method of Example 5 of JP-A-2007-89578 was used. Virus titers of the solution was 1.6 × ¹⁰ 9 cfu / ml. The method for measuring the virus titer is as described in Example 3 of JP-A-2007-89578.

The outside of fertilized chicken eggs (manufactured by Shiroyama Chicken Farm) was sterilized with a disinfectant solution (manufactured by Showa Frankie Co., Ltd.) and ethanol. Incubator P-008 (B) type (manufactured by Showa Frankie Co., Ltd.) was set to an environment of 38 ° C. and humidity of 50 to 60%, and the time when the power was turned on was set as the incubation start time (0 hours), and thereafter 15 Incubation was carried out while turning 90 ° every minute.

About 55 hours after the start of incubation, the eggs were taken out from the incubator and a hole of about 1 mm was made in the blunt end. Subsequently, a hole having a diameter of about 7 to 10 mm was made slightly above the center of the side surface of the egg. Under the stereomicroscope system SZX12 (manufactured by Olympus), about 2 μl of the virus solution prepared by the procedure described in Example 5 of Japanese Patent Application Laid-Open No. 2007-89578 is injected into the femtochip II (manufactured by Eppendorf), and the femtojet (manufactured by Eppendorf) is injected. Was injected into the heart of a chicken embryo through the hole.

After injecting the virus solution, the hole was closed with Scotch Tape BK-15 (manufactured by Sumitomo 3M Ltd.) and returned to the incubator to continue incubation. The incubator turnover was changed to 30 ° turnover every 30 minutes. One week after the start of incubation, the incubator was supplied with oxygen at 60 cc / min to incubate. On the 19th day from the start of incubation, the eggs were stopped and waited for them to hatch naturally. The naturally hatched and born chicks were bred and grown to obtain female adults of transgenic chickens. As feeds, young chicks SX Safety and Neo Safety 17 (manufactured by Toyohashi Feed Co., Ltd.) were used. The expression of fEPO in the egg white spawned by transgenic chickens was confirmed by the cell proliferation assay by BaF / EPOR described later. It was confirmed that the fEPO activity in egg white was 1.4 × 10 ^{4 U / mL.}

Eggs of chickens whose fEPO activity was confirmed in the egg white were collected, split using a diamond cutter type egg breaking machine (manufactured by Mitaka Electric Co., Ltd.), and egg white and egg white using an egg white separation slit (manufactured by Mitaka Electric Co., Ltd.). Egg yolk was separated and only egg white was collected. The collected egg white was sheared by passing an egg white strainer (manufactured by Sankyo Giken Co., Ltd.) having a diameter of 1 mm. 10 to 20 L of sheared egg white was collected in a tank having a capacity of 20 L, and then stirred at 750 rpm for 5 minutes using a stirrer to mix. The egg whites after stirring were subdivided into containers and then cryopreserved using a freezer at −80 ° C. (CLN-50CD1 manufactured by Nippon Freezer Co., Ltd.).

The above operation was performed a plurality of times to prepare an amount of fEPO-containing egg white required for the following purification treatment and freeze it.

1.2. Measurement of Cat-Derived Erythropoietin Activity The activity of fEPO in egg white was carried out by a cell proliferation assay using BaF / EPOR, which is an EPO-dependent cell line (Japanese Patent Laid-Open No. 10-94393). In the cell proliferation assay, epodin (manufactured by Chugai Pharmaceutical Co., Ltd.) was used as the standard erythropoietin, and a calibration curve for proliferation was drawn, and the erythropoietin activity of an unknown sample was measured based on the calibration curve. As a medium for BaF / EPOR cells, RPMI1640 liquid medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 5% fetal bovine serum (Fetal Bovine Serum, FBS) and 50 units / ml penicillin and streptomycin was used. Epodin was added to a final concentration of 1 IU / ml during normal BaF / EPOR cell culture. Cells in the logarithmic proliferation phase were used in the cell proliferation assay.

Epodin in the medium was first removed for cell proliferation assay on BaF / EPOR cells. The cultured BaF / EPOR cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was removed, and 10 ml of a medium containing no epodin was added to the precipitate and suspended. The same operation was performed three times to remove the epodin in the medium. Cells were counted and diluted in Epodin-free medium to a concentration of 55555 Cells / ml. 90 μl was seeded in each well of the 96-well microtiter plate. To this, add 10 μl of Epodin diluted in the medium to 25, 16, 10, 6.4, 4.0, 2.5, 1.6, 1.0 IU / ml, and hang it uniformly. It became turbid (final concentrations of erythropoietin were 2.5, 1.6, 1.0, 0.64, 0.4, 0.25, 0.16 and 0.1 IU / ml, respectively).

The sample used for the assay was serially diluted 2 to 4 times with a medium so that it was within the measurement range of the calibration curve, and 10 μl was added to the seeded cells and suspended uniformly. Three points of the same standard sample and unknown sample were measured. After culturing for 2 days, 10 μl of Cell Counting Kit-8 (manufactured by Doujin Kagaku Kenkyusho) solution was added to each well. After the color reaction was carried out for 1 to 4 hours, 10 μl of 0.1 mol / l hydrochloric acid was added to stop the reaction, and the absorbance at 450 nm was measured using a microplate reader. The measurement results of the standard sample were logarithmically approximated to obtain an approximate expression. The activity of the unknown sample was converted from the obtained approximate formula. The activity of fEPO corresponding to 1 IU of epodin is defined as 1 U.

The egg white used for the activity measurement was prepared so as to be uniform as a whole by ultrasonic waves or a physical method. The prepared sample was cryopreserved at −80 ° C. until the activity was measured.

2. Pretreatment of egg white As a pretreatment for purifying fEPO from egg white containing fEPO obtained in the above procedure 1, the following treatment was performed on the egg white.

2.1. Thawing The frozen egg whites obtained in 1 above and stored in a freezer at -80 ° C were taken out together with the container, thawed at a temperature of 15 ° C for 14 hours using a thawing machine, and used for the subsequent treatment.

2.2. Shearing The egg white after thawing was sheared according to the following procedure.

The collected egg white was weighed in a tank having a capacity of 100 L in an amount of 40.8 kg, and then sheared by passing the liquid through an egg white strainer (manufactured by Sankyo Giken Co., Ltd.) having a diameter of 1 mm, and collected in a tank having a capacity of 100 L. Further, purified water was passed through the strainer for washing, and a total of about 72 L (about 72 kg) of egg white liquid including the washing liquid was collected.

2.3. pH adjustment As a pH adjustment buffer solution, a 50 mM acetic acid-sodium acetate buffer solution was prepared by the following procedure.

Approximately 2 L of purified water is added in a 5 L volume of jug, and 1149.6 g of sodium acetate trihydrate (Merck Co., Ltd.) and 187.2 g of glacial acetic acid (Merck Co., Ltd.) are added to the water. The mixture was sufficiently stirred and adjusted to 4 L to prepare a concentrated buffer solution.

136 L of purified water was added to a tank having a capacity of 500 L, 4 L of the concentrated buffer solution was added to the water, and the volume was adjusted to 158 kg using purified water and stirred sufficiently.

2.2. Of the above Experiment 1 while adding 158 kg of the buffer solution to a tank having a capacity of 500 L and stirring at 190 rpm. About 72 kg of egg white liquor obtained by the shearing treatment of No. 1 was added, and then purified water was added to adjust the total amount to 240.0 kg. Then, glacial acetic acid was added to adjust the pH to 5.1 to obtain a mixed solution (hereinafter referred to as pH-adjusted egg white solution). The pH-adjusted egg white liquor became cloudy immediately after the acetic acid buffer and the egg white were mixed.

The pH measurement was carried out using a pH meter (HORIBA, Ltd.) that was calibrated at two points at pH 4 and pH 7 using a calibration solution manufactured by Nacalai Tesque. The solution temperature at the time of measurement was 22.5 ± 0.5 ° C. Unless otherwise specified, all pH values herein are values measured under these conditions.

2.4. Membrane filtration Membrane filtration of pH-adjusted egg white liquor that caused cloudiness was performed according to the following procedure.

2.3. The pH-adjusted egg white liquor prepared in the above was pumped and passed through a stainless steel wire mesh having a mesh diameter of 100 to 300 μm to remove large agglomerates. Then, the filtered liquid was filtered using a microfiltration membrane (depth filter having a pore size of 0.2 to 5 μm, manufactured by Merck Millipore) at a flow rate of about 20 to 30 LMH, and the filtered liquid was collected in a tank. The liquid remaining on the microfiltration membrane was recovered by flowing 160 kg of an extrusion buffer solution.

The filtrate collected in the tank was stirred and homogenized, and then aseptically filled in a bag through a 0.2 μm (Merck Millipore) filter.

As the buffer solution for extrusion used above, an acetic acid-sodium acetate buffer solution (pH 5.1) was prepared by the following procedure. 1000 g of purified water is added to a 1 L volume beaker, and 383.2 g of sodium acetate trihydrate (Merck Co., Ltd.) and 63.68 g of glacial acetic acid (Merck Co., Ltd.) are sufficiently added to the water. Stirred. After visually confirming that the powder was completely dissolved, stirring was stopped, mixed with 79.0 kg of purified water, and the pH was measured to confirm that the pH was 5.10 ± 0.05. This operation was repeated to prepare a total of 160 kg of extrusion buffer solution.

The protein concentration of the egg white treatment solution after membrane filtration (hereinafter referred to as “pre-treatment egg white raw material”) was 7.4 mg / mL, the fEPO concentration was 15.9 μg / mL or more, and the pH value was 5.12.

Method for measuring protein concentration: After pretreatment, the egg white raw material is diluted with purified water and the precipitate is removed by centrifugation or filter filtration. Using a spectrophotometer and a quartz cell with an optical path length of 1 cm, the absorbance at wavelengths of 280 nm and 320 nm is measured, and the amount of protein is calculated from the following formula. Formula: Protein amount (mg / mL) = (Abs280-Abs320) x 0.959 x dilution factor Measurement method of fEPO concentration: Egg white raw material after pretreatment and PEGylation using EPO-ELISA kit (manufactured by Roche) Measure mature fEPO standard products (managed by the company) that have not been used. A calibration curve of 4.0 to 1.1 ng / mL is prepared from the standard product, and the concentration of erythropoietin in the pretreated egg white raw material is calculated using the following formula. Formula: Concentration (ng / mL) = (average absorbance-intercept) / slope x dilution rate

<Experiment 2>
The relationship between pH and the amount of precipitate in the chicken egg white solution was investigated.
1. 1. Method The fEPO-containing chicken egg white prepared by the procedure described in "1.1. Preparation of egg white containing cat-derived EPO using transgenic chicken" in Experiment 1 was cryopreserved at -20 ° C and then incubated at 15 ° C. Incubated overnight and thawed.

The thawed egg white was sheared with a strainer in the same manner as in "2.2. Shearing" of Experiment 1.

1500 ml of a 53 mM sodium acetate aqueous solution was prepared. To 306 g of sheared egg white (egg white specific density 1.02), 1500 ml of the 53 mM sodium acetate solution was added (final concentration 44 mM), and the mixture was stirred with a stirrer to prepare an egg white solution.

100 ml of the egg white solution was dispensed, and glacial acetic acid was added as needed to adjust the pH to each value shown in Table 1 or Table 2 below.

After adjusting the pH, suspend again and O. D600 was measured.

O. After measuring D600, it was transferred to another glass container.

All samples were individually transferred to a glass container and then homogenized by inversion mixing at the same time. After homogenization, the mixture was allowed to stand for 1 hour, and then a photograph was taken.

2. Results The measurement results of the turbidity (OD600) of the egg white solution in each sample are shown in Table 1 below.

In addition, a photograph of each sample (see Table 1 for the sample number) after homogenization and standing for 1 hour is shown in FIG.

From the above results, it was estimated that the amount of precipitate formed changed between pH 4.00 and pH 4.91. Therefore, using another lot of egg white, the turbidity (OD600) of the egg white solution was measured by the same procedure as in Samples 1 to 8 except that the pH of the egg white solution was set to 4.00, 4.40, 4.79. bottom. The results are shown in Table 2.

From the above results, it was clarified that a precipitate was efficiently formed from egg white at pH 4 to 7. O. Since the value of D600 is maximized, it can be said that pH around 5.1 is optimal for removing the precipitate. Further, it was clarified that the precipitate was efficiently formed in the range of pH 4.4 to 6.1, particularly in the range of pH 4.7 to 6.02.

<Experiment 3>
Ovalbumin, ovomucoid and the like are contained as major proteins in chicken egg white. It is known that the isoelectric point (pI) of ovalbumin is 4.6 and the pI of ovomucoid is 3.9 to 4.5 (Biochemical Dictionary 4th Edition, Tokyo Kagaku Dojin). On the other hand, fEPO consisting of the amino acid sequence of SEQ ID NO: 3 is estimated to have a pI of 8.2 from the amino acid sequence. Each protein has a different charge state depending on the pH of the solution, and is negatively charged under pH conditions higher than pI and positively charged under pH conditions lower than pI. As described above, since the pI differs between the main protein contained in chicken egg white and fEPO, it is considered that the main protein in egg white such as ovalbumin and ovomucoid can be separated from fEPO by ion exchange chromatography.

Therefore, the conditions under which fEPO binds to the cation exchanger and the conditions under which ovalbumin can be removed were examined by the batch method.

1. 1. Method 1.1. PH adjustment of egg white solution A 50 mM sodium acetate solution of pH 3.5, 4.4, 5.0, 5.6 was prepared and mixed with thawed and sheared egg white in the same manner as in Experiment 2. , Each pH-adjusted egg white liquor having a pH of 3.5, 4.4, 5.0, and 5.6 was prepared. The pH-adjusted egg white liquor was centrifuged to remove the precipitate before being used in the experiment.

1.2. Adsorption and recovery on cation exchange column carrier The 50 mM acetate buffer described in this column was used at the same pH as the pH-adjusted egg white solution. 500 μl of a cation exchange column carrier (SP sepharose FastFlow, GE Healthcare) suspended in ethanol was centrifuged (1000 g, 3 minutes), and the supernatant was discarded. 1 ml of 1 M NaCl was added, the suspension was performed, and the mixture was centrifuged (1000 g, 3 minutes), and the supernatant was discarded. 1 ml of 50 mM acetate buffer was added, and the suspension was centrifuged (1000 g, 3 minutes), and the supernatant was discarded. Again, 1 ml of 50 mM acetate buffer was added, suspended and centrifuged (1000 g, 3 minutes), and the supernatant was discarded. For precipitation, 500 μl of 50 mM acetate buffer and 1.1. 250 μl of the pH-adjusted egg white solution prepared in the column was added, and the mixture was suspended and shaken at room temperature for 1 hour. After shaking, centrifugation (1000 g, 3 minutes) was performed. 1 ml of 50 mM acetate buffer was added to the precipitate, and the mixture was shaken at room temperature for 1 hour. 1 ml of 1M NaCl was added and shaken at room temperature for 1 hour. After shaking, centrifugation (1000 g, 3 minutes) was performed, and the supernatant was collected as an "adsorption fraction". The fEPO measurement of each collected fraction was performed by a cell proliferation assay by BaF / EPOR (see the column of "1.2. Measurement of activity of erythropoietin derived from cat" in Experiment 1).

Whether or not ovalbumin was removed was evaluated by the following procedure: Equal amounts of each sampling solution and 10 mM DTT-containing sample loading buffer were mixed and heat-treated at about 98 ° C. for 5 minutes. This was electrophoresed on a 12.5% concentration polyacrylamide gel and then silver stained using a silver staining kit (manufactured by Invitrogen) to determine the presence or absence of an ovalbumin band.

2. Results The results are shown in Table 3.

◯ for the adsorption fraction indicates that fEPO was detected and that the amount of fEPO in the pretreated egg white raw material was 50% or more when the amount of fEPO was 100%.

Regarding the removal of ovalbumin, x means that ovalbumin has not been removed at all in the adsorption fraction, Δ means that it has been removed to some extent, and ○ means that it has been largely removed.

It was clarified that the cation exchange column carrier adsorbs not only fEPO but also ovalbumin at pH 3.5, whereas it can adsorb fEPO and remove ovalbumin at higher pH conditions. Although there was no difference in the amount of fEPO adsorbed, pH 4.4 or higher is preferable in order to achieve the purpose of removing ovalbumin.

<Experiment 4>
Using the AKTA chromatography system, the effect of pH conditions on the recovery rate of fEPO in cation exchange column chromatography was confirmed in a narrower range than in Experiment 3.

As the 50 mM acetate buffer described in the "2.3 pH adjustment" column of Experiment 1, a pH of 4.8, 4.9, 5.0, 5.2 was used instead of 5.1. After pretreatment, an egg white raw material was prepared. Therefore, the pH of the obtained pretreated egg white raw material is adjusted to 4.8, 4.9, 5.0, and 5.2, respectively. All purification by cation exchange column chromatography was performed under a temperature condition of 20 ° C.

The XK26 column (manufactured by GE Healthcare) was filled with SP sepharose FastFlow (SPFF: GE Healthcare) at a bed height of 15 cm and a compression rate of 113%.

The following buffer solutions were prepared at pH 4.8, 4.9, 5.0, 5.2, and experiments were conducted using a buffer solution having the same pH as the pretreated egg white raw material.
Solution A: 50 mM acetic acid-sodium acetate buffer B solution: 50 mM acetic acid-sodium acetate buffer / 70 mM sodium chloride Solution C: 50 mM acetic acid-sodium acetate buffer / 150 mM sodium chloride D solution: 50 mM acetic acid-sodium acetate buffer / 1M Sodium acetate

After equilibration by passing the solution A through the solution, the pretreated egg white raw material having a total protein amount of 88 mg was loaded on 1 ml of the carrier volume. The linear flow velocity under load was 30 cm / h. Solution B was passed through 3 CV to wash away proteins that were not adsorbed and weakly bound to the carrier. The main fraction was eluted with 5 CV of C solution, and 1 CV was collected at a time. After recovery, they were mixed so as not to foam and sampled. The residual protein component remaining on the column was eluted with 3 CV solution D.

The following analysis was performed on the non-adsorbed fraction (flow-through), the washed fraction, the eluted fraction, and the total eluted fraction when egg white was added.

<Analysis>
Protein amount: The protein concentration was measured by the same procedure as described in "2.4. Membrane filtration" of Experiment 1 above. The total protein amount was calculated by multiplying the protein concentration by the amount of the recovered liquid.

SDS-PAGE: Equal amounts of each sampling solution and 10 mM DTT-containing sample loading buffer are mixed and heat-treated at about 98 ° C. for 5 minutes. After electrophoresis of this on a 12.5% concentration polyacrylamide gel, silver staining was performed using a silver staining kit (manufactured by Invitrogen) to confirm the presence or absence of OVA.

ELISA: The fEPO concentration of the loading solution and the eluted fraction was measured by the same procedure as described in "2.4. Membrane filtration" of Experiment 1 above. The fEPO recovery rate was calculated by dividing the fEPO concentration contained in the elution fraction by the amount of fEPO contained in the egg white raw material loaded with the amount of recovered liquid.

FIG. 2 shows an dissolution chart. The solid line shows the total protein amount, and the dotted line shows the fEPO recovery rate. Most of the total protein amount is impurity protein such as OTF (ovotransferrin), and the relative amount of fEPO is about 3%.

FIG. 3 shows a molecular weight marker M, a pretreated egg white raw material P before purification, a non-adsorbed fraction F, a washed fraction W, and an eluted fraction when purification by cation exchange column chromatography was performed at pH 4.8. The results of SDS-PAGE of the fraction E and the total eluted fraction A are shown.

The following table shows the fEPO yield for each pH condition at the time of adsorption.

As shown in FIG. 3, even under the condition of pH 4.8, ovalbumin (OVA), which is a major impurity, is contained in the non-adsorbed fraction F and the washed fraction W, and is not mixed in the eluted fraction E. Was confirmed. The recovery rate of fEPO was as high as 74% or more at pH 4.8 to 5.2, and 78% or more at pH 4.9 to 5.2, resulting in a higher recovery rate of fEPO.

<Experiment 5>
2.1. Of Experiment 1 above. The egg white liquor obtained through the thawing treatment of the above experiment 1 and 2.4. The viscosity of 2 lots of the pretreated egg white raw material obtained through the membrane filtration treatment was measured at 15 ° C. using a TVB-10 type viscometer manufactured by Toki Sangyo Co., Ltd.
Egg white liquor viscosity: 49.2 cP (15 ° C)
Viscosity of egg white raw material after pretreatment: 3.1 cP, 2.9 cP (15 ° C)

This result indicates that the egg white liquor that has not undergone the steps of pH adjustment and membrane filtration has a very high viscosity, and the viscosity is reduced by the steps of pH adjustment and membrane filtration.

<Experiment 6>
The effect of pH on fEPO activity was investigated by the following procedure.

As the purified fEPO solution, a solution containing 61.5 μg / mL of fEPO in terms of mature fEPO that was not PEGylated was used. This purified fEPO solution is a non-PEGylated purified fEPO solution obtained in the step of “3. Further purification” of Experiment 7 below.

The following buffer solutions were prepared as buffer solutions for pH control.

Buffer solution for pH 4.0: Acetic acid was added to a 50 mM sodium acetate aqueous solution to adjust the pH to 4.0.

Buffer solution for pH 5.0: Acetic acid was added to a 50 mM sodium acetate aqueous solution to adjust the pH to 4.0.

Buffer solution for pH 6.0: 1N hydrochloric acid was added to phosphate buffered saline (PBS) to adjust the pH to 6.0.

The purified fEPO solution was diluted 200-fold with a buffer solution for pH 4.0, and a diluted solution diluted 400-fold with a buffer solution for pH 5.0 and a buffer solution for pH 6.0 was prepared.

The diluted solution was allowed to stand at room temperature for 3 days, and after 3 days, 1.2. The erythropoietin activity of the diluent was measured by the cell proliferation assay by BaF / EPOR described in the column. The activity was expressed as erythropoietin activity (U) as epodin per 1 mL of purified fEPO solution before dilution. Three measurements were taken under each pH condition, and the average value is shown in the following table.

This result is because the activity of fEPO is easily maintained when the pH is 5 or more, and fEPO is unlikely to be deactivated when the pH is around 5.0.

Then, from Tables 3 to 5 above, if the pH of the protein solution when loading the chromatography in the purification step is 4.4 to 6.0, ovalbumin may be removed and the recovery rate of fEPO may be increased. It became clear. It was also found that when the pH is 4.8 to 5.2, the recovery rate is further improved while maintaining the activity.

<Experiment 7>
From the results of the above experiments, it is preferable to adjust the pH of egg white to pH 5.1, perform membrane filtration, and then perform cation exchange chromatography at pH 5.1 as a condition for purifying fEPO from egg white. They estimated. Therefore, in Experiment 7 below, 2.4 of Experiment 1 The pretreated egg white raw material (the egg white raw material obtained by membrane filtration after adjusting the egg white to pH 5.1) prepared in the column is purified by cation exchange chromatography at pH 5.1 and further PEGylated to prepare a PEGylated fEPO preparation. bottom.

1. 1. Purification by cation exchange chromatography <Process outline>
In this step, 2.4 of Experiment 1. The pretreated egg white raw material prepared in the column was loaded on a cation exchange chromatography carrier to adsorb fEPO. After washing the impurities, fEPO was crudely purified by eluting fEPO. The goal of this step is to remove the main impurity, ovalbumin (hereinafter referred to as OVA).

<Main equipment>
-Chromatography system: AKTA chromatography system (manufactured by GE Healthcare)
Ancillary equipment: A, B pump, sample pump, electrical conductivity meter, absorbance monitor (280 nm and 215 nm), thermometer, pressure gauge / purification column: BPG300 column (manufactured by GE Healthcare)

<Purification carrier>
・ SP sepharose FastFlow (SPFF: GE Healthcare)
Bed height 15 cm, compression rate 113%

<Packing method for carrier to column>
The liquid was passed through the column during packing in the direction from the top to the bottom of the column (hereinafter referred to as Down Flow). The packing work was carried out at room temperature (hereinafter, the range of room temperature is 20.0 to 25.0 ° C.), and the column evaluation and column storage conditions were also set to 20 to 25 ° C.
Packing liquid: Water Carrier compressibility (natural sedimentation volume / filling volume): 1.13
Final bed height: 15 cm

<Buffer solution>
The following buffer was used.
Solution A: 50 mM acetic acid-sodium acetate buffer (pH = 5.10)
Solution B: 50 mM acetate-sodium acetate buffer (pH = 5.10) / 70 mM sodium chloride Solution C: 50 mM acetate-sodium acetate buffer (pH = 5.10) / 150 mM sodium chloride Solution D: 50 mM acetate-sodium acetate Buffer solution (pH = 5.10) / 1M sodium chloride

Each of the prepared buffers was used within 72 hours after performing 0.2 μm filter filtration.

For pH measurement, a calibration solution manufactured by Nakarai Co., Ltd. was used, and two-point calibration of pH = 4 and pH = 7 was carried out. The solution temperature at the time of pH measurement was 22.5 ± 0.5 ° C.

<Reagent>
The following reagents were used.
Sodium acetate trihydrate (Merck Co., Ltd.)
Glacial acetic acid (Merck Co., Ltd.)
Sodium chloride (Merck Co., Ltd.)

<Preparation method>
Solution A: 479.00 g of sodium acetate and 79.600 g of acetic acid were added, dissolved and mixed with respect to 100.00 kg of water, and the pH and electrical conductivity were confirmed.
Solution B: 479.00 g of sodium acetate and 409.20 g of sodium chloride were dissolved in 100.00 kg of water, 69.970 g of acetic acid was added, and after mixing, the pH and electrical conductivity were confirmed.
Solution C: Dissolve 718.50 g of sodium acetate in 150.00 kg of water, dissolve 1315.50 g of sodium chloride, add 97.650 g of acetic acid, mix, and check the pH and electrical conduction. rice field.
Solution D: Dissolve 958.00 g of sodium acetate in 200.00 kg of water, dissolve 11688.0 g of sodium chloride, add 88.480 g of acetic acid, mix, and check the pH and electrical conductivity. rice field.

<Process monitoring>
The flow velocity, liquid volume, electrical conductivity, absorbance (280 nm), pressure (less than or equal to the filling pressure), and temperature contained in the chromatosystem were monitored.

The flow velocity was confirmed by actually measuring the outlet flow rate for both the sample pump and the system pump. Regarding the temperature, it was confirmed that the buffer solution and the sample used were 20-25 ° C. before the start of chromatography.

<Operation procedure>
The temperature at the time of purification was 20-25 ° C. The linear flow velocity was 30 cm / h when the pretreated egg white raw material was loaded and 60 cm / h when fractionated. For the purpose of preventing column clogging due to the generation of agglomerates, the egg white raw material after the pretreatment was filtered by 0.2 μm, and a 0.2 μm filter was installed immediately before the column.

1) Equilibration In order to replace the preservative solution in the column, the following operation was performed after passing 1 column volume (hereinafter referred to as CV) of water.

After passing the liquid D (3 CV or more), the liquid A was passed (3 CV or more), and it was confirmed that the electric conductivity was stable.

2) Load of post-treatment egg white raw material A pre-treatment post-treatment egg white raw material of 88 mg as a total protein amount was loaded on 1 ml of the carrier volume (*). To prevent column clogging, the pretreated egg white raw material was filtered by 0.2 μm. After the pretreatment, the egg white raw material was stored at 4 ° C., but was returned to 20-25 ° C. before being added to the column. The linear flow velocity was set to 30 cm / h, and the egg white raw material was loaded after the pretreatment. The linear flow velocity during fractionation (washing / elution / total elution) was 60 cm / h.
* The carrier volume is the carrier volume after compression and filling, and is the column cross-sectional area x bed height.

3) Washing After pretreatment After loading the egg white raw material, solution B was passed through 3 CV in order to wash away proteins that were not adsorbed and weakly bound to the carrier.

4) Elution The main fraction was eluted with 5 CV C solution and collected together. After recovery, they were mixed so as not to foam, sampled, and the following process control test was carried out. The rest was stored at 4 ° C until use.

5) Total elution The residual protein component remaining on the column was eluted with 3 CV solution D.

<Sampling>
When the egg white was added, the non-adsorbed fraction (flow-through), the washed fraction, the eluted fraction, and the total eluted fraction were sampled after stirring the liquid well, and the following process control test was carried out.

<Process control test>
A process control test was conducted for the following items. The process control test was carried out after the chromatography was completed and before the next step, extrafiltration.

The eluted fraction obtained in this step was designated as "SPFF eluted fraction".

Protein amount: The protein concentration is measured by the same procedure as described in "2.4. Membrane filtration" of Experiment 1 above. The amount of protein in the eluted fraction was 2.4 mg / mL.

SDS-PAGE: Equal amounts of each sampling solution and 10 mM DTT-containing sample loading buffer are mixed and heat-treated at about 98 ° C. for 5 minutes. This is electrophoresed on a 12.5% concentration polyacrylamide gel and then silver stained using a silver staining kit (manufactured by Invitrogen). As a result, the OVA in the loaded sample was not in the eluted fraction.

ELISA: The fEPO concentration of the load solution and the eluted fraction is measured by the same procedure as described in "2.4. Membrane filtration" of Experiment 1 above. These concentrations are integrated with the load weight or the elution fraction weight to obtain the relative value of the fEPO amount of the elution fraction with respect to the load amount. As a result, the amount of fEPO in the eluted fraction was 91% or more of the amount of fEPO loaded on the cation exchange chromatography column.

2. Concentration / salt exchange process by ultrafiltration 1. The cation exchange chromatography eluate (SPFF elution fraction) obtained in the above was concentrated and salt-exchanged with an ultrafiltration membrane.

As the ultrafiltration membrane, an ultrafiltration membrane (regenerated cellulose fraction molecular weight 5k), a pericon ultrafiltration membrane manufactured by Merck Co., Ltd., and a V-screen membrane were used. An ultrafiltration membrane of ^{0.4 m 2} or more was used per 11.6 L of the treatment liquid.

As the buffer for ultrafiltration, 4.95 mM phosphate buffer (pH = 6.75) and 20 ppm CaCl ₂ were used for salt exchange.

The protein concentrations of the recovered solution after concentration and salt exchange (recovered solution after ultrafiltration) were 9.74 mg / mL and 9.07 mg / mL, respectively, in the two tests. The total amount of protein was measured by the same procedure as described in "2.4. Membrane filtration" of Experiment 1 above.

3. 3. Further purification 2. A purified fEPO fraction (hereinafter referred to as a purified EPO fraction) was obtained from the recovered liquid obtained in 1) through a plurality of known purification steps.

The purified EPO fraction contains 50 mM phosphate buffer (pH 8.35) as buffer.

The purified EPO fraction had a pH of 8.35, was colorless and transparent, and contained no insoluble substances.

fEPO concentration: The purified EPO fraction is diluted with a phosphate buffer solution, the absorbance at wavelengths of 280 nm and 320 nm is measured using a spectrophotometer and a quartz cell with an optical path length of 1 cm, and the fEPO concentration is calculated from the following equation. Formula: fEPO concentration (mg / mL) = (Abs280-Abs320) x 0.959 x dilution ratio

As a result, the fEPO concentration was 2.3 mg / mL. The fEPO concentration obtained by the above formula is a concentration based on the weight converted as a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 and not chemically modified by a water-soluble long-chain molecule or the like.

SDS-PAGE / WB: Purified EPO fraction is mixed with 10 mM DTT-containing sample loading buffer and heat-treated at about 98 ° C. for 5 minutes. This is electrophoresed on a 12.5% concentration polyacrylamide gel and transferred to a PVDF membrane using a transfer electrophoresis tank. As a result of detecting the protein using the anti-fEPO monoclonal antibody, each test solution (manufactured by TOYOBO) and the detection reagent (manufactured by GE), a band was confirmed at a position of about 30 kDa.

SDS-PAGE (CBB staining): The purified EPO fraction is mixed with 10 mM DTT-containing sample loading buffer and heat-treated at about 98 ° C. for 5 minutes. This is electrophoresed on a 15% concentration polyacrylamide gel and then CBB stained. As a result, a single band was confirmed.

HPLC-RP: Using a column for HPLC and reverse phase (manufactured by GRACE), mobile phase A (ultrapure water / trifluoroacetic acid = 1000/1) and mobile phase B (ultrapure water / acetonitrile / trifluoroacetic acid =) Analyze with a gradient of 100/900/1) at 0.75 mL / min for 70 minutes. The chart of the purified EPO fraction is compared with that of the control (phosphate buffer), the peak is visually detected, and the peak calculation is performed manually. As a result of determining the other peak areas with respect to the peak area of fEPO, no peak of 0.5% or more was observed.

HPLC-SEC: Analysis is performed for 70 minutes at 0.5 mL / min of mobile phase (0.05% polysorbate / phosphate buffer) using HPLC and a size exclusion column (manufactured by Tosoh Corporation). The chart of the purified EPO fraction is compared with that of the control (phosphate buffer), the peak is visually detected, and the peak calculation is performed manually. As a result of determining the other peak areas with respect to the peak area of fEPO, no peak of 2.0% or more was observed.

Biological activity measurement: 1.2. Of Experiment 1 above. The biological activity was determined according to the method described in the activity measurement of cat-derived erythropoietin described in the column. The purified EPO fraction was diluted (1,000,000 to 3375,000 times) and added to the culture broth. After culturing for 2 days 37 ° C. 5% CO ₂ atmosphere, Cell Counting Kit 8 by adding (Dojin Chemical) and cell numbers were determined. Creating a standard curve, read the activity, the results of calculating the specific activity of the preparation was 2.8 × ¹⁰ 5 U / mg.

ELISA: OVA measurement kit (manufactured by Morinaga Seigaku Co., Ltd.), ovotransferrin measurement kit (manufactured by ICL), and lysozyme measurement kit (manufactured by CUSABIO), except that OVA (manufactured by SIGMA) is used as a standard product. The amount of each substance in the purified EPO fraction is quantified according to the procedure of. As a result, OVA was 369 ppm, ovotransferrin was 1 ppm, and lysozyme was 62 ppm with respect to the amount of fEPO.

4. PEGylation of fEPO 3. The following PEGylating agent was added to the purified EPO fraction obtained in.
SUNBRIGHT ME-200HS (PEGylating agent for linear PEG with an average molecular weight of 20K, manufactured by NOF CORPORATION)

The purified EPO fraction and the PEGylating agent were mixed at a ratio of fEPO and the PEGylating agent in a molar ratio of 1: 5, and then reacted at 4 ° C. for 2 hours while mixing. After the reaction for 2 hours, 1/10 amount of 100 mM glycine solution was added as a reaction terminator, and the reaction was stopped while mixing at 4 ° C. for 1 hour.

5. Separation and purification of PEGylated fEPO 4. The PEGylation reaction solution obtained in 1) was diluted with 10 times the amount of 50 mM acetate-sodium acetate buffer (pH 4.5), filtered through a 0.2 μm filter, and then mono-PEGyl, diPEGyl, oligoPEGyl and unreacted. In order to separate and recover PEG and unreacted EPO, separation and purification were performed using a cation exchange column.

The following operation was performed at 20-25 ° C.

The diluted solution was loaded on a cation exchange column (MacroCapSP: manufactured by GE Healthcare Japan Co., Ltd.). The loaded column was washed with 50 mM acetate-sodium acetate buffer (pH 4.5), followed by step elution with 50 mM acetate-sodium acetate buffer, 0.15 M NaCl (pH 4.5) to give the elution fraction. Recovered. The recovered elution fraction was uniformly stirred to obtain "MCSP elution fraction". Then, the residual protein was eluted with 50 mM acetate-sodium acetate buffer, 1 M NaCl (pH 4.5).

The "MCSP-eluted fraction" corresponds to the fraction in which the peaks of the mono-PEG form and the diPEG-form are recovered.

<Sampling>
The PEGylation reaction solution and the elution fraction were sampled after stirring the solution well, and a process control test was carried out.

SDS-PAGE: Equal amounts of each sampling solution and 10 mM DTT-containing sample loading buffer are mixed and heat-treated at about 98 ° C. for 5 minutes. This is electrophoresed on a polyacrylamide gradient gel having a concentration of 4 to 20%, and then silver-stained using a silver staining kit (manufactured by Invitrogen). As a result, bands were recognized at 60 kDa and 120 kDa.

6. Drug substance preparation A buffer solution containing the same components in water and pH-adjusted except that PEGylated fEPO and Tween80 were not contained among the components shown in Table 6 was used as the drug substance buffer solution. 5. Above. The MCSP-eluted fraction obtained in (1) was salt-exchanged with the drug substance buffer by ultrafiltration, and the protein concentration was adjusted to 0.27 mg / mL.

The virus was removed by passing the solution after the above ultrafiltration through a hollow fiber membrane having an average pore size of 20 nm.

The solution after removing the virus was filtered by a 0.2 μm filter and aseptically filled in a bag as the drug substance.

7. Formulation The above 6. A liquid composition having the following composition was produced using the drug substance prepared in 1.

After adding 0.5 g of Tween 80 to the volumetric flask, a drug substance buffer solution was added so as to make a total of 100 mL, and the mixture was well stirred to prepare Tween 80 solution. Accurately weighed 468.9 mL of the drug substance and charged it into a 2 L graduated cylinder. 13 mL of Tween 80 solution was accurately weighed and placed in the same 2 L volumetric cylinder, and then the drug substance buffer solution was added to increase the volume to 1300 mL. The composition of the PEGylated fEPO solution preparation thus obtained is shown in Table 6. Filtration was performed using Millipack 20 (manufactured by Merck & Co., Inc.), and glass vials were filled with 1.1 mL each. It was sealed with a rubber stopper and an aluminum cap.

fEPO concentration: 3. above. The fEPO concentration measured by the same procedure as described in the above was 0.11 mg / mL.

SDS-PAGE (CBB staining): 3. above. SDS-PAGE (CBB staining) was performed in the same procedure as described in the above section. As a result, bands were confirmed at 60 kDa and 120 kDa.

HPLC-RP: 3. above. HPLC-RP was performed in the same procedure as described in. As a result of determining the other peak areas with respect to the peak area of the PEG compound, no peak of 0.5% or more was observed.

HPLC-SEC: 3. above. HPLC-SEC was performed in the same procedure as described in. As a result of obtaining relative values from the peak areas of aggregates, tri-PEGs, mono-PEGs and diPEGs, the proportion of aggregates was less than 2.0%.

Biological activity measurement: 1.2. Of Experiment 1 above. The biological activity was determined according to the method described in the activity measurement of cat-derived erythropoietin described in the column. As a result of calculating the specific activity of the pharmaceutical product, it was 1.8 × 10 ⁴ U / mg.

All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (4)

A method for producing purified cat-derived erythropoietin.
A protein solution containing cat-derived erythropoetin prepared using the egg white of a transgenic bird having an exogenous gene encoding cat-derived erythropoetin, and an acidic region of 1.5 parts by volume or more with respect to 1 part by volume of the protein solution. The pH of the protein solution is adjusted to a range of 4.7 or more and 6.0 or less by mixing with a buffer solution having a buffering action in an acidic region, which contains a component having a buffering action at a concentration of 1 M or less. Adjustment process and
In the pH adjusting step, the pH-adjusted protein solution is brought into contact with a carrier equipped with a cation exchange group to adsorb cat-derived erythropoetin, and then the adsorbed cat-derived erythropoetin is eluted from the carrier. only including,
Prior to the purification step, a precipitate removing step of removing the precipitate from the pH adjusted protein solution in the pH adjusting step is further included.
Does not include the step of purification by dye affinity chromatography,
Method. A step of producing purified cat-derived erythropoietin by the method according to claim 1.
Water-soluble long-chain molecule-added cat-derived erythropoetin, which comprises a step of chemically modifying the purified cat-derived erythropoetin with a water-soluble long-chain molecule to obtain water-soluble long-chain molecule-added cat-derived erythropoetin. Production method. The method according to claim 2 , further comprising a virus removal step of removing a virus by passing a water-soluble long-chain molecule-added cat-derived erythropoietin obtained in the water-soluble long-chain molecule addition step through a filter. A formulation step of preparing a liquid composition containing erythropoetin derived from a water-soluble long-chain molecule-added cat obtained in the water-soluble long-chain molecule addition step and having the same osmotic pressure and pH as the osmotic pressure and pH of a mammalian body fluid. The method according to claim 2 or 3 , further comprising.

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