WO2017169978A1 - Method for producing purified feline erythropoietin - Google Patents

Abstract

The purpose of the present invention is to provide an improved means for producing purified feline erythropoietin (fEPO). This method for producing purified fEPO includes: a pH adjustment step for adjusting the pH of a protein solution including fEPO to within a range of 4-7; and a purification step for bringing the protein solution, the pH of which was adjusted in the pH adjustment step, into contact with a carrier provided with cation exchange groups, adsorbing the fEPO, and then eluting the fEPO from the carrier.

Description

Method for producing purified cat-derived erythropoietin

The present invention relates to a method for producing a purified feline-derived erythropoietin that can be used as an active ingredient in veterinary medicine.

Cats have long been an animal that has been attached to humans as pets, but recently, the status of being a member of human society has been established as an “animal as a companion, companion, partner”. On the other hand, it has been used as a laboratory animal in medicine, pharmacy, veterinary medicine, psychology, etc., and recently it has also been used for safety testing and efficacy testing of pharmaceuticals. Thus, in the situation where the social importance of cats is increasing, there is a high interest in cat diseases and infectious diseases, and effective treatment methods are desired. In recent years, pharmaceutical proteins have attracted attention in the treatment of cat diseases, and mainly human pharmaceutical proteins have been diverted. However, since the protein for human use has a different amino acid sequence from the in vivo protein inherent in cats, it may have different effects in vivo. In addition, differences in amino acid sequence can cause allergies, and worse, anaphylactoid symptoms. Therefore, since it cannot withstand high-frequency administration, development of a protein for pharmaceutical use inherent in cats is required.

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

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

In Example 9 of Patent Document 1, the egg white is diluted with water, adjusted to pH 5.0, stirred for 15 minutes or more, centrifuged, and then subjected to pretreatment to adjust the supernatant to pH 7.0. In addition, it is possible to purify cat-derived erythropoietin by performing blue sepharose chromatography, heparin chromatography, desalting column, anion exchange column chromatography and gel filtration chromatography in this order on the pretreated egg white liquid. It is disclosed.

In Paragraph 0026 of Patent Document 2, egg white is diluted with a buffer as a pretreatment, adjusted to pH, then applied to a blue sepharose column, dialyzed, and fractionated with a cation exchange column (SP column). It is disclosed that after concentration, after treatment with a desalting column, feline erythropoietin having a purity of 99% or more is obtained by purification of an anion exchange column (DEAE Sepharose column).

Patent Document 3 discloses a method in which a variant of a cat-derived erythropoietin with a modified amino acid sequence is expressed using cells such as CHO (Chinese hamster ovary) cells and purified from the cell culture medium or cell lysate. ing. Examples of methods for purification include affinity chromatography, anion exchange chromatography, cation exchange chromatography and the like. In Example 8 of Patent Document 3, the culture supernatant of a cell expressing a variant of cat-derived erythropoietin was adjusted to pH 8.5, and phenylboronate chromatography, anion exchange chromatography and hydrophobic interaction chromatography were performed in this order. Performing and purifying a variant of cat-derived erythropoietin.

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

In Patent Document 6, as a method for pretreating egg white for chromatographic treatment, an acid buffer of about 0.5 wt% to about 5 wt% with respect to the egg white is added to the egg white pool, and the pH is about 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 containing about 5M to about 6M sodium acetate as the acidic buffer.

JP 2007-89578 A JP 2010-1111595 A Special table 2012-504136 gazette Special table 2010-509327 JP 2010-511378 A International Publication WO2015 / 164320

However, the purification methods specifically disclosed in Patent Documents 1 and 2 use blue sepharose chromatography. The column used in blue sepharose chromatography is one in which dye molecules are immobilized on a carrier, but there is no risk that these molecules will be mixed into the purified feline erythropoietin product.

Further, Patent Documents 4 and 5 do not describe any production and purification of cat-derived erythropoietin. Further, Patent Document 4 discloses that the pH of a protein solution is adjusted in the acidic region before the cation exchange chromatography treatment and that a buffer having a buffering action in the acidic region is used when loading the column. Not. In the method of Patent Document 5, since purification is performed using dye affinity chromatography, there is no risk that the dye molecules are mixed into the purified product of erythropoietin, as in Patent Documents 1 and 2.

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

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. In addition, Patent Document 3 describes not only a protein purification method using affinity chromatography but also a protein purification method that does not use the protein purification method. However, when affinity chromatography is not used, it is generally necessary to increase the purification ratio. There is a problem of requiring stage chromatography, and there is room for improvement.

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

As means for solving the above problems, the present invention discloses the following invention.
(1) A method for producing a purified cat-derived erythropoietin,
A pH adjusting step for adjusting the pH of the protein solution containing cat-derived erythropoietin to a range of 4 or more and 7 or less;
a purification step comprising contacting the protein solution adjusted in the pH adjustment step with a carrier having a cation exchange group to adsorb cat-derived erythropoietin, and then eluting the adsorbed cat-derived erythropoietin from the carrier; Including methods.
(2) The method according to (1), further comprising a precipitate removing step of removing the precipitate from the protein solution whose pH has been adjusted 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 adjustment step.
(4) In the pH adjustment step, the protein solution is mixed with a buffer solution having a buffering action in the acidic region containing a component having a buffering effect in the acidic region at a concentration of 1 M or less. (1) to (3) The method in any one of.
(5) In the pH adjustment step, the protein solution is mixed with 1.5 parts by volume or more of a buffer solution having a buffering action in an acidic region with respect to 1 part by volume of the protein solution. ) Any one of the methods.
(6) The protein solution used in the pH adjustment step is a protein solution prepared using an egg of a transgenic bird having an exogenous gene encoding a cat-derived erythropoietin (1) to (5) The method described.
(7) producing a purified cat-derived erythropoietin by the method according to any one of (1) to (6);
Water-soluble long-chain molecule-added cat-derived erythropoietin comprising a water-soluble long-chain molecule-added step of chemically modifying the purified cat-derived erythropoietin with a water-soluble long-chain molecule to obtain a water-soluble long-chain molecule-added cat-derived erythropoietin Production method.
(8) The method according to (7), further comprising a virus removal step of removing the virus by passing the water-soluble long-chain molecule-added cat-derived erythropoietin obtained in the water-soluble long-chain molecule addition step through a filter.
(9) A preparation comprising a water-soluble long-chain molecule-added cat-derived erythropoietin obtained in the water-soluble long-chain molecule addition step, and preparing a liquid composition having the same osmotic pressure and pH as the body fluid of a mammal The method according to (7) or (8), further comprising a conversion step.

This specification includes the disclosure 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, feline-derived erythropoietin can be efficiently purified by using a pH adjustment step and a purification step using a carrier having a cation exchange group in combination.

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

Furthermore, in the purification step, the cat-derived erythropoietin is further adsorbed on the cat-derived erythropoietin by contacting with a carrier having a cation exchange group in a buffer solution having a buffering action in the acidic region. It can be purified efficiently.

In Experiment 2, the pH of the egg white solution is adjusted and allowed to stand, and a photograph after 1 hour is shown. Table 1 shows the correspondence between sample numbers and pH conditions. The elution chart of the refinement | purification by the cation exchange column chromatography in each pH conditions in Experiment 4 is shown. The solid line indicates the total protein amount, and the dotted line indicates the fEPO recovery rate. The majority of the total protein is OTF (ovotransferrin). The horizontal axis shows the amount of eluate in column volume (CV). In Experiment 4, when the 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 fraction E and total elution 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”. Cat-derived erythropoietin is sometimes referred to 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 among these, the first to 26th amino acid residues are signal sequences, so they are also referred to as fEPO preproteins. The signal sequence of the fEPO preprotein is removed and the amino acid sequence from which the 192nd amino acid group is deleted is mature fEPO. The amino acid sequence of mature fEPO is as shown in SEQ ID NO: 3. The fEPO may also be a polypeptide that partially includes the amino acid sequence of the fEPO preprotein of SEQ ID NO: 2 or a polypeptide that partially includes the amino acid sequence of the mature fEPO of SEQ ID NO: 3.

As fEPO, not only 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, but also active mutants thereof can be used. Preferably, the activity mutant used fEPO preprotein consisting of the amino acid sequence shown in SEQ ID NO: 2 or mature fEPO consisting of the amino acid sequence shown in SEQ ID NO: 3 under the activity measurement conditions shown in the examples of the present specification. The polypeptide exhibits an activity of 10% or more, preferably 40% or more, more preferably 60% or more, and still more preferably 80% or more. Examples of the active mutant include a polypeptide comprising 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 a polypeptide comprising an amino acid sequence in which 1 to a plurality of amino acids are added, deleted or substituted in total at the N-terminal and / or C-terminal in the amino acid sequence shown in 3 or SEQ ID NO: 2 or 3 A polypeptide comprising 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 to the amino acid sequence. Applicable. More preferably, the active variant of the fEPO preprotein consisting of the amino acid sequence shown in SEQ ID NO: 2 is selected from the N-terminus, C-terminus, the portion of residues 1 to 26 and 192 residues of SEQ ID NO: 2. It contains mutations as listed above in at least one place. 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 at the N-terminus. In this specification, “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 (aligned), and gaps are introduced as necessary to maximize the degree of amino acid identity between the two. The ratio (%) of the same amino acid residue to the total number of amino acid residues of the protein shown. Amino acid identity can be calculated using a BLAST or FASTA protein search system (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. ). The amino acid substitution is preferably conservative amino acid substitution. “Conservative amino acid substitution” refers to substitution between amino acids having similar properties such as charge, side chain, polarity, aromaticity and the like. 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), nonpolar Can be classified into functional amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched chain amino acids (leucine, valine, isoleucine), aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine), etc. it can.

As fEPO, transgenic cells that have been genetically modified to express fEPO by introducing a foreign gene encoding fEPO into a host of generally available animal cells (for example, CHO cells), plant cells, prokaryotes, and yeast. Manufactured using a cell-free protein synthesis system, a transgenic animal or transgenic plant that has been genetically modified to express fEPO by introducing a foreign gene encoding fEPO Although a thing etc. can be utilized, it is not limited to these. Examples of the transgenic animal include birds, and birds are preferably poultry birds such as chickens, quails, turkeys, ducks, ducks, ostriches, geese, long-tailed birds, pigeons, emu, pheasants and guinea fowls, with chickens being particularly preferred. Transgenic birds are preferably those that accumulate fEPO in the egg. 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 even monocotyledonous plants may be dicotyledonous plants. Examples of monocotyledonous plants include grass family (rice, barley, wheat, corn, sugarcane, buckwheat, sorghum, millet, millet. Etc.), liliaceae (asparagus, lily, onion, leek, katsuri etc.), plants belonging to the ginger family (ginger, myoga, turmeric, etc.), and dicotyledonous plants include, for example, cruciferous (Arabidopsis, cabbage, Rapeseed, cauliflower, broccoli, radish, etc.) Eggplant (tomato, eggplant, potato, tobacco, etc.), legume (soybean, pea, green beans, alfalfa, etc.), cucurbitaceae (cucumber, melon, pumpkin, etc.), celery family ( Carrots, celery, bees, etc.), asteraceae (lettuce, etc.), mallow (cotton, okra, etc.) Chenopodiaceae (sugar beet, spinach, etc.), Myrtaceae (Eucalyptus, clove, etc.), including but plants belonging to the Salicaceae (Populus, etc.), but are not limited to.

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 number and type of sugar chains according to the expression system used (ie, transgenic cells, transgenic animals, transgenic plants, cell-free protein synthesis systems, etc.). Is generally modified by For example, when the expression system used is a chicken, the sugar chain modification is mainly modification with a sugar chain in which 2 to 5 sugar chains containing a β-N-acetylglucosamine residue are bound to a trimannosyl core. is there.

<Purified cat-derived erythropoietin>
In the present invention, “purified” cat-derived erythropoietin (fEPO) means fEPO present in a state in which the concentration of fEPO per amount of protein is higher than that in the protein solution used as a raw material before purification. A crude product may be used regardless of the degree. For example, fEPO that has undergone one purification step with a cation exchanger is also included in the purified fEPO in 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 material containing fEPO, and may be a form in which fEPO is dissolved in water. As such a protein solution, a fEPO-containing protein solution derived from an fEPO production system such as a transgenic cell, a transgenic animal, a transgenic plant, a cell-free protein synthesis system or the like that expresses fEPO as described above can be used. .

A first preferred form of the protein solution used as a raw material is a protein solution prepared using eggs of transgenic birds having a foreign gene encoding fEPO. The said egg can be various forms, such as the whole egg obtained by splitting, the egg white isolate | separated from the whole egg, and an egg yolk, Preferably it is 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 egg is preferably one that has been sufficiently homogenized by a shearing treatment. More preferably, the protein solution prepared using the egg is, for example, 3 volume parts or less, preferably 2 volumes with respect to the egg itself or the egg and the egg 1 volume part subjected to shearing treatment. Part or less, more preferably a mixed solution with 1 part by volume or less of water.

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 a transgenic cell (for example, CHO cell) having a foreign gene encoding fEPO in a medium. is there. As the protein solution according to the second preferred form, preferably, the culture mixture itself, the supernatant of the culture mixture, the disrupted solution obtained by disrupting the cells in the culture mixture, the supernatant of the disrupted solution, Or the liquid which diluted the liquid which concentrated one of these liquids, water, or aqueous solution can be illustrated.

<PH>
In the present invention, the pH value refers to a value measured using a pH meter at a liquid temperature of 22.5 ± 0.5 ° C. unless otherwise specified. The pH meter is preferably used after a two-point calibration at pH 4 and pH 7 using a commercially available standard reagent.

<PH adjustment step>
The pH adjustment 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.

Surprisingly, by adjusting the pH of the protein solution containing fEPO to a range of 4 or more and 7 or less, it is surprising that some components other than fEPO are insolubilized and precipitated out of the components contained in the protein solution. Remarkably promoted. As a result, in the protein solution that has undergone the pH adjustment step, the ratio of fEPO in the dissolved component increases. For this reason, it is possible to refine | purify fEPO efficiently by using the protein solution which passed through the pH adjustment process for the refinement | purification process mentioned later. On the other hand, when the pH of the protein solution containing fEPO is in the range of less than 4 or more than 7, precipitates due to components other than fEPO are hardly formed, and the effect of increasing the purification efficiency of fEPO cannot be obtained.

From the viewpoint of promoting the formation of precipitates, the range of the pH in the pH adjustment step is more preferably 4.1 or higher, 4.2 or higher, 4.3 or higher, 4.4 or higher, 4.5 or higher, 4 or higher. .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. 2 or less, 5.1 or less. In particular, when the pH is 4.7 or more, 4.8 or more, 4.9 or more, or 5.0 or more, and / or the pH is 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, 5.2 or less, 5.1 or less, from the protein solution This is preferable because the formation of precipitates is particularly accelerated.

Furthermore, adjusting the pH of the protein solution to the above range in the pH adjustment 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. Is also preferable. From this viewpoint, the pH range in the pH adjustment step is 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. From the viewpoint of yield, the upper limit of the pH in the pH adjustment 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, It is 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 viewpoint, the pH range in the pH adjustment step is 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 preferable to set it as 4.8 or more, 4.9 or more, and 5.0 or more. From the viewpoint of activity, the upper limit of the pH in the pH adjustment 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. .

Thus, in one embodiment of the present invention, by performing the pH adjustment step, the formation of precipitates is promoted, the yield of purification of fEPO using a support containing a cation exchange group, and the activity of fEPO are increased. An advantageous effect in holding can be achieved. That is, by performing the pH adjustment step and the subsequent purification step of fEPO under a consistent pH condition, the activity of fEPO can be maintained and the purification efficiency can be improved. Such an effect is an unexpected advantageous effect which is not suggested by the prior arts such as Patent Documents 4 and 6.

The above-mentioned effect by the pH adjustment step is obtained when the protein solution prepared using the first preferred form, that is, an egg of a transgenic bird having an exogenous gene encoding fEPO is used as the raw material protein solution. This is particularly noticeable. It is known that 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 ovalbumin is 4.6, the isoelectric point of ovotransferrin is 6.1 and the isoelectric point of ovomucoid is generally 3.9 to 4.5), whereas fEPO is the amino acid sequence shown in SEQ ID NO: 3. From this, the isoelectric point is estimated to be 8.2. Therefore, by adjusting the pH of the protein solution prepared using the eggs of transgenic birds having a foreign gene encoding fEPO to 4 or more and 7 or less, fEPO whose isoelectric point is an alkaline region is dissolved, It is presumed that proteins in the acidic region at the isoelectric point are precipitated, but are not restricted by this estimation mechanism.

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-described 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 a buffer solution and a protein solution, mixing an acid or a base as necessary, and adjusting the pH of the mixture to a pH within the above range. . More specifically, the pH of the mixture obtained by mixing a sufficient amount of buffer solution and protein solution is measured, and when the pH is outside the above range, the pH of the mixture is adjusted to the above by mixing an appropriate amount of acid or base. The range can be adjusted. A sufficient amount of the buffer is, for example, 1.5 parts by volume or more, preferably 2 parts by volume 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 volume. Hereinafter, it is typically 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 used for purification using a carrier containing a cation exchange group in the purification step. Moreover, also in embodiment which removes the deposit which precipitated in the pH adjustment process, since the removal of a deposit is easy by fully reducing the viscosity of a protein liquid, it is preferable. The viscosity of the mixture of a sufficient amount of buffer solution and protein solution in the pH adjustment step is, for example, 20 cP or less when measured with a B-type viscometer (for example, TVB-10 viscometer manufactured by Toki Sangyo Co., Ltd.) at 15 ° C. More preferably, the viscosity is 10 cP or less, and still more preferably 5 cP or less. In Patent Document 6, an acidic buffer solution of about 0.5 wt% to about 5 wt% with respect to the egg white is added to the egg white pool to form a mixed egg white having a pH of about 5 to 6.5. Thus, 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 an 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 remove precipitates or perform column chromatography.

The buffer solution is not particularly limited. For example, a buffer solution containing at least one selected from glycine, phthalic acid, citric acid, succinic acid, acetic acid and phosphoric acid can be used. In addition, those having low biotoxicity suitable for the production of pharmaceuticals are preferable. For example, acetate buffer is preferable because it is easy to use. 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 the acidic region is preferably used. Can be used. The buffering action is an action in which the pH hardly fluctuates even when an acid or a base is added or the concentration changes due to evaporation or dilution. The acidic range preferably includes the above pH range. The concentration of the buffer component in the buffer 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 more preferably 90 mM or less. 80 mM or less is more preferable. Proteins may be denatured when high concentration buffer components come in contact with the protein. A buffer solution having a relatively low concentration of the buffer component as described above is preferable because the damage given to the protein containing fEPO is small 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. Such a buffer solution containing a high concentration of acidic sodium denatures egg white. The possibility is considered high. In this 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, and further preferably 20 mM or more. The concentration of the buffer component is a concentration converted as if it existed in the form of a free form regardless of whether the buffer component is contained in a salt form or a free form.

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

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

Measure the fEPO concentration (ng / mL) by measuring pre-treated egg white raw material and fEPO standard product (in-house management) using an EPO-ELISA kit (Roche). 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. Calculated as the formula: concentration (ng / mL) = (average value of absorbance−intercept) / slope × dilution ratio.

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

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 with a reduced amount of precipitate. By performing the precipitate removal step, the amount of unnecessary protein can be reduced to increase the purification efficiency, and the handling of the protein solution in the purification step is facilitated.

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

<Purification process>
In the purification step, the protein solution whose pH was adjusted in the pH adjustment step was subjected to a precipitate removal step as necessary, and then contacted with a carrier having a cation exchange group to adsorb fEPO, and then adsorbed. elution of 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). 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. For example, a styrene carrier, an agarose carrier, or a cellulose carrier can be used. Examples of the styrenic carrier include a carrier composed of a crosslinked polymer obtained by copolymerizing styrene and divinylbenzene, and commercially available products include trade names MiniBeads, MonoBeads, and SOURCE15 commercially available from GE Healthcare Japan. SOURCE 30 can be exemplified. Commercially available agarose-based carriers include trade names such as Sepharose High Performance, Sepharose Fast Flow, Sepharose 4 Fast Flow, Sepharose XL, Sepharose XL, and Sepharose BegBesBegBeseBegBeseBegBesBeBeBeBeBeBeBeBeBeBeBeBeBeBeBeBsBgBeBeBeBeBgBBeBeBeBgBB . Cellulose-based carriers include those of the trade name Cellufine MAX series commercially available from JNC Corporation.

By using a protein solution whose pH is adjusted to 4 or more and 7 or less by the pH adjusting step as a protein solution to be contacted with a carrier having a cation exchange group (hereinafter referred to as “cation exchanger”), This is preferable because the yield of fEPO in the purification with a cation exchanger can be increased. From this viewpoint, the pH of the protein solution 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, 4.6 or higher. It is preferably 7 or more, 4.8 or more, 4.9 or more, or 5.0 or more. Among these ranges, it is particularly preferably 4.4 or more or a higher pH value range. The upper limit of the pH of the protein solution brought into contact with the cation exchanger is not particularly limited from the viewpoint of the yield, but is preferably 6.5 or less, 6.4 or less, 6.3 or less, 6.2 or less, 6.1 or less. Below, it is 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.

In addition, it is also preferable from the viewpoint of maintaining the activity of fEPO to use a protein solution whose pH is adjusted to 4 or more and 7 or less by the pH adjusting step as a protein solution to be brought into contact with the cation exchanger. From this viewpoint, the pH of the protein solution 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, 4.6 or higher. It is preferable to set it as 7 or more, 4.8 or more, 4.9 or more, and 5.0 or more, and it is preferable that it is especially the range of 4.8 or more or a higher pH value among these ranges. From the viewpoint of this activity, the upper limit of the pH of the protein solution to be contacted 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.

Thus, by bringing the protein solution adjusted to the predetermined pH range in the pH adjustment step into contact with the cation exchanger, the yield of purification of fEPO using the carrier containing the cation exchange group is improved, and the fEPO An advantageous effect in maintaining the activity can be obtained. As described above, the formation of precipitates can be promoted by the pH adjustment step. Thus, 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 contacting the pH-adjusted protein solution with the cation exchanger, first, the bed formed by packing the cation exchanger in a column or the like is adsorbed at a pH lower than the isoelectric point of fEPO. It is preferable to fill and equilibrate with the working buffer. The buffer for adsorption 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 Although it is a citrate buffer solution or the like, an adsorption buffer solution having a buffering action in an acidic region can be suitably used in view of the isoelectric point of fEPO. The pH value of the acidic region can be selected from the same range as the pH value of the protein solution to be contacted with the cation exchanger, preferably substantially the same as the protein solution to be contacted with the cation exchanger. The same pH value can be obtained. The buffering action is an action in which the pH hardly fluctuates even when an acid or a base is added or the concentration changes due to evaporation or dilution. fEPO is estimated to have an isoelectric point of 8.2 from the amino acid sequence shown in SEQ ID NO: 3, and is considered to be positively charged under acidic pH conditions. For this reason, 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 the fEPO is purified. can do. This effect is particularly remarkable when the protein solution prepared using the above-described first preferred form, ie, an egg of a transgenic bird having an exogenous gene encoding fEPO, is used as the raw material protein solution. As mentioned above, many of the major proteins contained in the eggs of transgenic birds have an isoelectric point in the acidic region and are difficult to adsorb on the cation exchanger under acidic conditions, so they have a buffering effect on the acidic region. It is considered that fEPO mixed with the main protein can be selectively adsorbed to the cation exchanger in the adsorption buffer solution.

∙ Load the pH-adjusted protein solution onto the equilibrated cation exchanger bed, adsorb fEPO onto the cation exchanger, and wash the bed as necessary. Thereafter, an elution buffer whose ionic strength is increased stepwise or continuously by adding salts to the adsorption buffer is passed through a bed of a cation exchanger on which fEPO is adsorbed to elute fEPO. , Recovered as a fEPO-containing liquid. The ionic strength of the elution buffer is preferably 100 mM or more and 300 mM or less. The purification using a cation exchanger is preferably performed within a temperature range of 20 ° C. or more and 25 ° C. or less. The pH of the washing buffer used for washing and the adsorption buffer can be set to substantially the same pH value as the protein solution brought into contact with the cation exchanger.

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

In the purification step of the present invention, in addition to purification using a 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. Moreover, you may perform the other protein purification process which is not illustrated. 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 support to which a triazine dye such as Cibacron Blue 3G is linked. However, the method of the present invention preferably does not include a step of purification by dye affinity chromatography. This is because by not including the step of purification by dye affinity chromatography, it is possible to prevent the dye from being mixed into the target purified fEPO. As the ion exchange chromatography, in addition to the above cation exchange chromatography, anion exchange chromatography can also be used.

When performing a plurality of purification treatments, a buffer solution for dissolving a protein containing fEPO can be exchanged between treatments as necessary. The exchange of the buffer solution can be performed by a normal 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 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 produce reaction precursors and can be added to proteins by synthetic reactions. Among these, since PEG has no antigenicity and is nontoxic, it is effective from the viewpoint of reducing the antigenicity of the modified protein and suppressing the expression of anti-protein antibodies as a side effect.

When fEPO is administered to rodents such as mice and rats to confirm the hematopoietic effect, it has been reported that the increase in reticulocytes is greatest on the 4th day after a single administration in blood and disappears on the 7th day. However, it is known that addition of PEG inhibits metabolism in the liver and prolongs the life span in blood, thereby extending the duration of drug efficacy, and in rat hematopoietic experiments it is known that the effect lasts 14 days.

As the molecular weight of the water-soluble long-chain molecule such as PEG added increases, the blood life of the water-soluble long-chain molecule-fEPO complex increases. However, since the addition of a very 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 in vivo hematopoietic effect. Is preferably 10 kDa or more, more preferably 30 kDa or less. More preferably, it is 20 kDa.

fEPO has at least three water-soluble long-chain molecule binding sites, and 1 (mono), 2 (di), and 3 (tri) molecules can be attached to a single fEPO polypeptide. However, since the binding ability of PEG at multiple sites inhibits the receptor binding ability of EPO and reduces in vivo activity, the mono- or di-form is preferred, and the di-form is particularly preferred.

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

As a method of chemically modifying fEPO with a water-soluble long chain molecule, fEPO and the water-soluble long chain molecule are mixed at a molar ratio of about 1: 1 to 10 and then mixed at 4 to 37 ° C. while mixing at 30 to 180. For example, the reaction may be stopped by adding about 1/10 volume of a 100 mM glycine solution as a reaction terminator and mixing at 4 to 37 ° C. for 1 hour.

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

Generally, as a method for covalently bonding a water-soluble long chain molecule to a protein, there is a chemical reaction with a polyol, lactol, amine, carboxylic acid or carboxylic acid derivative which is a functional group capable of oxidizing protein or sugar chain. There are also methods using sulfonate ester activated polymers such as sulfonate ester activated PEG. Also when adding to EPO, it can be added by these methods.

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

As a PEGylation reaction precursor used for covalently bonding PEG to a protein, one obtained by methoxylation of one end of a long chain molecule can be used. Furthermore, a PEG having a non-methoxylated end modified to a group capable of forming a covalent bond by undergoing a nucleophilic reaction by a nucleophilic group such as an amino group of 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
(Where
Y represents a leaving group such as a succinimidyloxy group,
X is — (CH ₂ ) _m —C (═O) —
A group represented by
m is 1 or more and 8 or less, more preferably 4 or more, more preferably 6 or less, and most preferably an integer of 5.
n is an integer indicating the degree of polymerization)
Is preferable from the viewpoint of reactivity. It is known that when a 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 EPO has a plurality of lysine residues, the number of PEG additions increases as the reaction proceeds, resulting in a mixture of isomers with different numbers of additions. In fEPO consisting of SEQ ID NO: 2, alanine 27, lysine 71 and / or lysine 78 are preferably PEGylated, and in mature fEPO consisting of SEQ ID NO: 3, alanine 1, lysine 45 and / or lysine 52 are preferably PEGylated.

<Virus removal process>
In the method of the present invention, when the water-soluble long chain molecule addition step is performed, the virus is preferably removed by passing the water-soluble long chain molecule-added fEPO obtained in the water-soluble long chain molecule addition step through a filter. A virus removal step is further included. In the water-soluble long-chain molecule addition step, a liquid composition in which 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 nonwoven 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 viruses removed by the virus removal step include human parvovirus, mouse microvirus, porcine parvovirus, encephalomyocarditis virus, hepatitis virus, and human immunodeficiency virus.

<Formulation process>
In the method of the present invention, when the water-soluble long-chain molecule addition step is performed, the osmotic pressure of a mammalian body fluid is more preferable. And a formulation step of preparing a liquid composition having the same osmotic pressure and pH as the 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 body fluid of the subject mammal is preferred because it hardly causes pain when administered subcutaneously to the subject mammal. For example, when producing a liquid composition suitable for administration to a mammal having a body fluid with an osmotic pressure of about 280 mOsm / KgH ₂ O and a pH of about 7.4, it is prepared in a formulation process. 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 and 8.0 or less, more preferably 7.3 or more, and more preferably 7.7 or less. Further, when the osmotic pressure of the mammalian body fluid 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 mammalian body fluid is Y, the pH of the liquid composition is preferably (Y ± 0.5), more preferably (Y ± 0.1). Here, osmotic pressure and pH are values measured at 20 to 25 ° C.

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

The purified fEPO, water-soluble long-chain molecule-added fEPO, and the liquid composition produced by the method of the present invention include, in addition to purified fEPO or water-soluble long-chain molecule-added fEPO, a solvent such as water, an excipient, 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 It is a pharmacologically acceptable ingredient for non-human animals to be administered. The purified fEPO, water-soluble long-chain molecule-added fEPO and the liquid composition produced by the method of the present invention are a combination of purified fEPO or water-soluble long-chain molecule-added fEPO and other additives. Examples of such additives include lubricants, coating agents, coloring agents, anti-aggregation agents, absorption promoters, solubilizers, health food materials, nutritional supplement materials, vitamins, fragrances, sweeteners, preservatives At least one additive selected from the group consisting of agents, preservatives and antioxidants can be used. The additive is preferably an pharmacologically acceptable additive for the non-human animal to be administered.

The method of the present invention may further include a step of preparing a solution, gel, or powder containing purified fEPO or water-soluble long-chain molecule-added fEPO, and then preparing various preparations. Here, as the form of the preparation, for example, injections, drops, injections, liquids for external use, patches, coating agents (creams, ointments etc.), inhalants, sprays, suppositories, rectal capsules , Subcutaneous implantable sustained-release preparations, micelle preparations, gelled preparations, liposome preparations, and forms of preparations for parenteral administration such as pessaries for intravaginal administration, and forms of preparations for oral administration. Examples of the application of injections and drops include intravenous, subcutaneous, intradermal, intramuscular, intraorgan, intranasal, eye drop, intracerebral, intraperitoneal, and lesion. The injection may be in the form of a prefilled syringe formulation. Examples of the application of the injection include rectal and vagina. 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. It is done. Among these forms of drugs, preparations in which fEPO is dissolved in the liquid are included in the above-mentioned “fEPO-containing liquid composition”.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited to these examples. When the product name was described, the instructions in the attached manual were followed unless otherwise specified.

<Experiment 1>
Preparation of fEPO-containing egg white by a transgenic chicken was carried out by a modified method based on the method described in Japanese Patent Application Laid-Open No. 2007-89578. 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 having the amino acid sequence shown in SEQ ID NO: 3 (mature fEPO). FEPO produced in egg white is a polypeptide (mature fEPO) having the amino acid sequence shown in SEQ ID NO: 3.

1. 1. Preparation of egg white containing feline-derived EPO using transgenic chicken 1.1. Microinjection and Artificial Hatching of Retroviral Vector into Chicken Embryos A retroviral vector for fEPO expression was microinjected into chicken embryos to produce transgenic chickens that express fEPO. Microinjection and artificial hatching were performed under aseptic conditions.

The DNA base sequence encoding fEPO (full 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. The virus titer of this 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 the chicken fertilized egg (manufactured by Shiroyama Breeder) was sterilized with a disinfectant (manufactured by Showa Franchi) and ethanol. Incubator P-008 (B) (made by Showa Franchi Co., Ltd.) was set to 38 ° C and humidity 50-60%, and the power-on time was the incubation start time (0 hour). Incubation was performed while turning 90 ° every minute.

After about 55 hours from the start of incubation, the egg was 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 a stereomicroscope system SZX12 (Olympus), femtochip II (Eppendorf) was injected with about 2 μl of the virus solution prepared by the procedure described in Example 5 of JP-A-2007-89578, and femtojet (Eppendorf) And then injected into the heart of the chicken embryo from the hole.

After injecting the virus solution, the hole was closed with Scotch tape BK-15 (manufactured by Sumitomo 3M), and the egg was returned to the incubator and incubation was continued. The incubator's turning was changed to 30 ° turning every 30 minutes. One week after the start of incubation, oxygen was supplied to the incubator at 60 cc / min for incubation. On the 19th day after the start of incubation, the eggs were turned and waited for natural hatching. The chicks that hatched naturally were bred and grown to obtain a female adult chicken. As feed, young SX safety and Neosafety 17 (manufactured by Toyohashi Feed Co., Ltd.) were used. The expression of fEPO in the egg white laid by the transgenic chicken was confirmed by a 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.

The eggs of chicken individuals whose fEPO activity has been confirmed in the egg white are collected, divided using a diamond cutter type egg breaking machine (made by Mitaka Denki Co., Ltd.), and egg white separated using an egg white separation slit (made by Mitaka Denki Co., Ltd.). The egg yolk was separated and only the egg white was collected. The collected egg white was sheared by passing a 1 mm caliber egg white strainer (manufactured by Sankyo Giken Co., Ltd.). 10 to 20 L of sheared egg white was collected in a 20 L tank, and then stirred and mixed at 750 rpm for 5 minutes using a stirrer. The egg white after stirring was subdivided into containers, and stored frozen using a −80 ° C. freezer (CLN-50CD1 manufactured by Japan Freezer).

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

1.2. Measurement of the activity of cat-derived erythropoietin The activity of fEPO in egg white was determined by a cell proliferation assay using BaF / EPOR, which is an EPO-dependent cell line (JP-A-10-94393). The cell proliferation assay was performed using epogin (manufactured by Chugai Pharmaceutical Co., Ltd.) as a standard erythropoietin, and a calibration curve for the growth was drawn to measure the erythropoietin activity of unknown samples. RPMI 1640 liquid medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 5% fetal bovine serum (FBS), 50 units / ml penicillin and streptomycin was used as the medium for BaF / EPOR cells. Epodin was added to a final concentration of 1 IU / ml during normal BaF / EPOR cell culture. Cells in logarithmic growth phase were used for the cell proliferation assay.

In order to perform a cell proliferation assay with BaF / EPOR cells, first, epodine in the medium was removed. The cultured BaF / EPOR cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was removed, and 10 ml of a medium not containing epogin was added to the precipitate and suspended. The same operation was repeated three times to remove epodine from the medium. The cells were counted and diluted to a concentration of 55555 Cells / ml in medium lacking epogin. 90 μl was seeded in each well of a 96-well microtiter plate. To this, add 10 μl of epodine diluted to 25, 16, 10, 6.4, 4.0, 2.5, 1.6, 1.0 IU / ml in culture medium and suspend evenly. It became turbid (final erythropoietin concentrations were 2.5, 1.6, 1.0, 0.64, 0.4, 0.25, 0.16, 0.1 IU / ml, respectively).

Samples used in the assay were serially diluted by about 2 to 4 times with a medium so as to fall within the measurement range of the calibration curve, and 10 μl each 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 Chemical Laboratory) was added to each well. After a color reaction 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 result of the standard sample was logarithmically approximated to obtain an approximate expression. The activity of the unknown sample was converted from the obtained approximate expression. The activity of fEPO corresponding to 1 IU of Epogin 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 physical methods. The prepared sample was stored frozen at −80 ° C. until the activity was measured.

2. Pretreatment of egg white As the pretreatment for purifying fEPO from the 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 white obtained in 1 above and stored in the -80 ° C freezer was 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 processing.

2.2. Shear The egg white after thawing was sheared by the following procedure.

The collected egg white was weighed 40.8 kg into a 100 L capacity tank, and then sheared by passing it through a 1 mm caliber egg white strainer (manufactured by Sankyo Giken Co., Ltd.) and recovered into a 100 L capacity tank. 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 A 50 mM acetic acid-sodium acetate buffer solution was prepared as a pH adjusting buffer solution by the following procedure.

In a 5 L mug, about 2 L of purified water was added, and 1149.6 g of sodium acetate trihydrate (Merck) and 187.2 g of glacial acetic acid (Merck) were added to the water. Thoroughly stirred and made up to 4 L was prepared as a concentrated buffer.

136 L of purified water was added to a 500 L tank, 4 L of the concentrated buffer solution was added to the water, and the volume was increased to 158 Kg with purified water, followed by thorough stirring.

In a 500 L tank, add 158 Kg of the buffer solution and stir at 190 rpm. About 72 Kg of egg white liquid obtained by the shearing treatment 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 a pH-adjusted egg white solution). The pH-adjusted egg white liquor was clouded immediately after mixing the acetate buffer and egg white.

PH measurement was performed using a pH meter (Horiba Seisakusho) that was calibrated at two points at pH 4 and pH 7 using a calibration solution manufactured by Nacalai Tesque. The solution temperature during the measurement was 22.5 ± 0.5 ° C. Unless otherwise specified, all pH values in this specification are values measured under these conditions.

2.4. Membrane filtration Membrane filtration of the pH-adjusted egg white solution in which white turbidity occurred was performed in the following procedure.

2.3. The pH-adjusted egg white solution prepared in (1) was pumped and passed through a stainless steel wire mesh with a mesh diameter of 100 to 300 μm to remove large aggregates. Thereafter, the filtrate was filtered using a microfiltration membrane (depth filter with a pore diameter of 0.2 to 5 μm, manufactured by Merck Millipore) at a flow rate of about 20 to 30 LMH, and the filtrated liquid was collected in a tank. The liquid remaining on the microfiltration membrane was collected by flowing 160 kg of the extrusion buffer solution.

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

As the extrusion buffer solution used above, an acetic acid-sodium acetate buffer solution (pH 5.1) was prepared by the following procedure. Add 1000 g of purified water to a 1 L beaker and add 383.2 g of sodium acetate trihydrate (Merck) and 63.68 g of glacial acetic acid (Merck) to the water. Stir. After visually confirming that the powder was completely dissolved, the 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 produce a total of 160 kg of extrusion buffer solution.

The protein concentration of the egg white treatment liquid after membrane filtration (hereinafter referred to as “pretreated 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. The absorbance at wavelengths of 280 nm and 320 nm is measured using a spectrophotometer and a quartz cell having an optical path length of 1 cm, and the amount of protein is calculated from the following equation. Formula: Protein amount (mg / mL) = (Abs280-Abs320) × 0.959 × dilution ratio Measurement method of fEPO concentration: EPO-ELISA kit (Roche), pre-treated egg white raw material and PEGylated Measure mature fEPO standards (in-house management) that have not been completed. A calibration curve of 4.0 to 1.1 ng / mL is prepared from the standard product, and the erythropoietin concentration in the pretreated egg white raw material is calculated using the following formula. Formula: Concentration (ng / mL) = (Average value of absorbance−intercept) / slope × dilution rate

<Experiment 2>
The relationship between the pH and the amount of precipitate in chicken egg white solution was investigated.
1. Method The fEPO-containing chicken egg white prepared by the procedure described in “1.1. Preparation of feline-derived EPO using a transgenic chicken” in Experiment 1 was stored frozen at −20 ° C., and then incubator at 15 ° C. Thaw and incubate overnight.

The thawed egg white was sheared with a strainer in the same manner as in “2.2.

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

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

After re-adjusting the pH, D600 was measured.

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

All samples were individually transferred to glass containers and then homogenized by inversion mixing at the same time. After homogenization, the photo was taken after standing for 1 hour.

2. Results The measurement results of the turbidity (OD 600) of the egg white solution in each sample are shown in Table 1 below.

Also, a photograph of each sample after uniformization and standing for 1 hour (see Table 1 for sample numbers) is shown in FIG.

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

From the above results, it was revealed that precipitates were efficiently generated from egg white at pH 4-7. O. Since the value of D600 is maximized, pH 5.1 is optimal for removing the precipitate. It was also found that precipitates are efficiently generated within the range of pH 4.4 to 6.1, particularly within the range of pH 4.7 to 6.02.

<Experiment 3>
Chicken egg white contains ovalbumin, ovomucoid and the like as main proteins. It is known that the isoelectric point (pI) of ovalbumin is 4.6, and that 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. Individual proteins have different charge states depending on the pH of the solution, and are 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 fEPO can be separated from the main protein in egg white such as ovalbumin and ovomucoid by ion exchange chromatography.

Therefore, the conditions for fEPO binding to the cation exchanger and the conditions for removing ovalbumin were examined by the batch method.

1. Method 1.1. PH adjustment of egg white solution Prepare 50 mM sodium acetate solution of pH 3.5, 4.4, 5.0, 5.6 and mix with egg white thawed and sheared in the same manner as described in Experiment 2. , PH adjusted egg white liquids of 3.5, 4.4, 5.0, and 5.6 were prepared. The pH-adjusted egg white solution was subjected to an experiment after centrifugation to remove the precipitate.

1.2. Adsorption and recovery on cation exchange column carrier The 50 mM acetate buffer described in this column was 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 1M NaCl was added and suspended, followed by centrifugation (1000 g, 3 minutes), and the supernatant was discarded. 1 ml of 50 mM acetate buffer was added, suspended, centrifuged (1000 g, 3 minutes), and the supernatant was discarded. Again, 1 ml of 50 mM acetate buffer was added, suspended, centrifuged (1000 g, 3 minutes), and the supernatant was discarded. For precipitation, 500 μl of 50 mM acetate buffer and 1.1. After adding 250 μl of pH-adjusted egg white solution prepared in the column and suspending, the mixture was 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 shaken for 1 hour at room temperature. 1 ml of 1M NaCl was added and shaken for 1 hour at room temperature. After shaking, the mixture was centrifuged (1000 g, 3 minutes), 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 cat-derived erythropoietin” 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 adsorbed fraction indicates that fEPO was detected and that the amount of fEPO was 50% or more when the amount of fEPO in the egg white raw material after pretreatment was 100%.

Regarding ovalbumin removal, x indicates that ovalbumin has not been removed at all in the adsorbed fraction, Δ indicates that it has been removed to some extent, and ○ indicates that it has been generally removed.

It has been 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 under 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 object of removing ovalbumin.

<Experiment 4>
Using an AKTA chromatography system, the influence 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, pH 4.8, 4.9, 5.0, and 5.2 instead of 5.1 were used. An egg white raw material was prepared after pretreatment. Therefore, the pH values of the obtained pretreated egg white raw materials are adjusted to 4.8, 4.9, 5.0, and 5.2, respectively. All purifications by cation exchange column chromatography were performed at a temperature of 20 ° C.

An XK26 column (manufactured by GE Healthcare) was packed 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, and 5.2, and experiments were performed using a buffer solution having the same pH as that of the pretreated egg white raw material.
A solution: 50 mM acetic acid-sodium acetate buffer B solution: 50 mM acetic acid-sodium acetate buffer / 70 mM sodium chloride C solution: 50 mM acetic acid-sodium acetate buffer / 150 mM sodium chloride D solution: 50 mM acetic acid-sodium acetate buffer / 1 M Sodium chloride

After liquid A was passed and equilibrated, 88 mg of pretreated egg white raw material as a total protein amount was loaded per 1 ml of the carrier volume. The linear flow rate during loading was 30 cm / h. In order to wash away non-adsorbed and weakly bound protein on the carrier, the solution B was passed through 3 CV. The main fraction was eluted with 5 CV C solution, and 1 CV was collected at a time. After collection, the mixture was mixed so as not to foam and sampled. The remaining protein components remaining on the column were eluted with 3 CV solution D.

The following analysis was performed on the fraction that was not adsorbed at the time of egg white addition (flow-through), the washed fraction, the eluted fraction, and the total eluted fraction.

<Analysis>
Protein amount: The protein concentration was measured by the same procedure as described in “2.4. Membrane filtration” in Experiment 1 above. The protein concentration x the amount of recovered solution was defined as the total protein amount.

SDS-PAGE: Equivalent amounts of each sampling solution and 10 mM DTT-containing sample loading buffer are mixed and heated 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 (Invitrogen) to confirm the presence or absence of OVA.

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

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

FIG. 3 shows molecular weight marker M, pre-treated egg white raw material P before purification, non-adsorbed fraction F, washing fraction W, and elution fraction when purification by cation exchange column chromatography is performed at pH 4.8. The results of SDS-PAGE of fraction E and total elution fraction A are shown.

The fEPO yield for each pH condition during adsorption is shown in the following table.

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

<Experiment 5>
In Experiment 1, 2.1. And the egg white liquid obtained through the thawing treatment of 2.4 of the above Experiment 1. Viscosity at 15 ° C. was measured using two TVB-10 viscometers manufactured by Toki Sangyo Co., Ltd. for 2 lots of pretreated egg white raw material obtained through the membrane filtration treatment.
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 not subjected to the pH adjustment and membrane filtration steps has a very high viscosity, and the viscosity is reduced by the pH adjustment and membrane filtration steps.

<Experiment 6>
The effect of pH on fEPO activity was examined 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” in Experiment 7 below.

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

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

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

PH 6.0 buffer: 1N hydrochloric acid was added to phosphate buffered saline (PBS) to adjust pH to 6.0.

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

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

This result is because when the pH is 5 or more, the activity of fEPO is easily retained, and when the pH is around 5.0, fEPO is not easily deactivated.

From Tables 3 to 5 above, if the pH of the protein solution at the time of loading the chromatography in the purification step is 4.4 to 6.0, ovalbumin is removed and the recovery rate of fEPO is 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 that the pH of egg white is adjusted to pH 5.1, membrane filtration is performed, and then cation exchange chromatography is performed at pH 5.1, as a condition for purifying fEPO from egg white. They estimated. Therefore, in Experiment 7 below, 2.4. The pretreated egg white raw material (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 did.

1. Purification by cation exchange chromatography <Process overview>
In this step, 2.4. 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 eluted by eluting the crude fEPO. The target of the process is removal of ovalbumin (hereinafter referred to as OVA) which is a main impurity.

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

<Carrier for purification>
・ SP Sepharose FastFlow (SPFF: GE Healthcare)
Bed height 15cm, compression rate 113%

<Method of packing the carrier into the column>
Liquid passing through the column at the time of packing was performed from the top to the bottom of the column (hereinafter referred to as Down Flow). The packing operation was performed at room temperature (the room temperature range was 20.0 to 25.0 ° C. below), and column evaluation and column storage conditions were 20 to 25 ° C.
Packing liquid: water Carrier compressibility (natural sedimentation volume / filling volume): 1.13
Final bed height: 15cm

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

Each buffer solution prepared was used within 72 hours after 0.2 μm filter filtration.

PH measurement was carried out using a calibration solution manufactured by Nacalai Co., Ltd., and two-point calibration was performed at pH = 4 and pH = 7. 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 Corporation)
Sodium chloride (Merck Corporation)

<Preparation method>
Liquid A: 479.00 g of sodium acetate and 79.600 g of acetic acid were added to, dissolved in, and mixed with 100.00 kg of water, and the pH and electrical conductivity were confirmed.
Liquid 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: After dissolving 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, and after mixing, check the pH and electrical conductivity. It was.
Liquid D: After dissolving 958.00 g of sodium acetate in 200.00 kg of water, 11688.0 g of sodium chloride is dissolved, 88.480 g of acetic acid is added, and after mixing, the pH and electrical conductivity are confirmed. It was.

<Process monitoring>
The flow rate, liquid volume, electrical conductivity, absorbance (280 nm), pressure (below the filling pressure), and temperature built in the chromatographic system were monitored.

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

<Operation procedure>
The temperature during purification was 20-25 ° C. The linear flow rate was 30 cm / h when the egg white raw material was loaded after pretreatment and 60 cm / h during fractionation. In order to prevent clogging of the column due to the generation of aggregates, 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 preservation solution in the column, the following operation was performed after passing 1 column volume (hereinafter referred to as CV) of water.

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

2) Loading of egg white raw material after pretreatment 88 mg of pretreated egg white raw material was loaded as a total protein amount per 1 ml of the carrier volume (*). In order to prevent clogging of the column, the pre-treated egg white raw material was subjected to 0.2 μ filter filtration. The egg white raw material after pretreatment is stored at 4 ° C., but it was returned to 20-25 ° C. before being added to the column. The linear flow rate was 30 cm / h, and the egg white raw material was loaded after pretreatment. The linear flow rate during fractionation (washing / elution / total elution) was 60 cm / h.
* The carrier volume is the volume of the carrier after compression and packing, and the column cross-sectional area x bed height.

3) Washing After the pretreatment, after loading the egg white raw material, 3 CV was passed through the solution B 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 solution C and collected together. After collection, the mixture was sampled so as not to foam, sampled, and the following process control test was performed. The rest was stored at 4 ° C. until use.

5) Total elution The remaining protein components remaining on the column were eluted with 3 CV D solution.

<Sampling>
The non-adsorbed fraction (flow-through), the washed fraction, the eluted fraction, and the total eluted fraction at the time of egg white addition were mixed well, sampled, and subjected to the following process control test.

<Process control test>
Process control tests were conducted on the following items. The process control test was carried out after the chromatography and before carrying out the ultrafiltration as the next process.

The elution fraction obtained in this step was designated as “SPFF elution fraction”.

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

SDS-PAGE: Equivalent amounts of each sampling solution and 10 mM DTT-containing sample loading buffer are mixed and heated at about 98 ° C. for 5 minutes. This is electrophoresed on a 12.5% polyacrylamide gel, and then silver stained using a silver staining kit (manufactured by Invitrogen). As a result, there was no OVA in the loaded sample in the eluted fraction.

ELISA: Measure the fEPO concentration of the load solution and the eluted fraction in the same procedure as described in “2.4. Membrane filtration” in Experiment 1 above. These concentrations and load weight or elution fraction weight are integrated, and the relative value of the fEPO amount of the elution fraction with respect to the load amount is obtained. 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 step by ultrafiltration The cation exchange chromatography eluate (SPFF elution fraction) obtained by the above was subjected to concentration and salt exchange using an ultrafiltration membrane.

As the ultrafiltration membrane, an ultrafiltration membrane (regenerated cellulose molecular weight cut off 5 k), a Pericon ultrafiltration membrane and a V screen membrane manufactured by Merck & Co., Inc. were used. An ultrafiltration membrane of 0.4 m ² or more was used per 11.6 L of the treatment liquid.

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

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

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

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

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

FEPO concentration: The purified EPO fraction is diluted with a phosphate buffer, 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) × 0.959 × dilution ratio

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

SDS-PAGE / WB: The purified EPO fraction is mixed with a 10 mM DTT-containing sample loading buffer and heated at about 98 ° C. for 5 minutes. This is electrophoresed on a 12.5% 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 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 a 10 mM DTT-containing sample loading buffer and heated at about 98 ° C. for 5 minutes. This is electrophoresed on a 15% polyacrylamide gel and then stained with CBB. As a result, a single band was confirmed.

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

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

Bioactivity measurement: 1.2 of the above Experiment 1. 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 (1000000 times to 3375000 times) and added to the culture solution. After culturing at 37 ° C. in a 5% CO ₂ atmosphere for 2 days, Cell Counting Kit 8 (Dojindo Laboratories) was added and the number of cells was measured. A standard curve was prepared, the activity was read, and the specific activity of the preparation was calculated. As a result, it was 2.8 × 10 ⁵ U / mg.

ELISA: OVA measurement kit (manufactured by Morinaga Bioscience), ovotransferrin measurement kit (manufactured by ICL) and lysozyme measurement kit (manufactured by CUSABIO), except for using OVA (manufactured by SIGMA) as a standard product The amount of each substance in the purified EPO fraction is quantified according to the procedure of 1. 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 PEGating agent was added to the purified EPO fraction obtained in (1).
SUNBRIGHT ME-200HS (PEGylating agent for linear PEG having an average molecular weight of 20K, manufactured by NOF Corporation)

The purified EPO fraction and the PEGating agent were mixed at a molar ratio of fEPO and PEGating agent of 1: 5, and then reacted for 2 hours while mixing at 4 ° C. 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 The PEGylation reaction solution obtained in 1) was diluted with 10 volumes of 50 mM acetic acid-sodium acetate buffer (pH 4.5), filtered through a 0.2 μm filter, mono-PEG, di-PEG, oligo-PEG and unreacted In order to separate and recover PEG and unreacted EPO, separation and purification were performed using a cation exchange column.

The following operations were performed at 20-25 ° C.

The diluted solution was loaded on a cation exchange column (MacroCapSP: manufactured by GE Healthcare Japan Co., Ltd.). The column after loading was washed with 50 mM acetic acid-sodium acetate buffer (pH 4.5), and then step elution was performed with 50 mM acetic acid-sodium acetate buffer, 0.15 M NaCl (pH 4.5). It was collected. The collected elution fraction was homogeneously stirred to obtain “MCSP elution fraction”. Subsequently, the residual protein was eluted with 50 mM acetic acid-sodium acetate buffer, 1 M NaCl (pH 4.5).

“MCSP elution fraction” corresponds to the fraction from which the peaks of mono-PEG and di-PEG were collected.

<Sampling>
The PEGylation reaction solution and the eluted fraction were mixed well, sampled, and a process control test was performed.

SDS-PAGE: Equivalent amounts of each sampling solution and 10 mM DTT-containing sample loading buffer are mixed and heated 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 observed at 60 kDa and 120 kDa.

6). Preparation of drug substance A buffer solution containing the same components in water except that PEGylated fEPO and Tween 80 were not included among the components shown in Table 6 was used as the drug substance buffer solution. 5. above. The MCSP elution fraction obtained in 1 above was subjected to salt exchange with the drug substance buffer solution by ultrafiltration, and the protein concentration was adjusted to 0.27 mg / mL.

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

The above-mentioned solution after virus removal was filtered through a 0.2 μm filter and aseptically filled into a bag as the drug substance.

7). Formulation 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, the drug substance buffer was added to a total of 100 mL, and the mixture was stirred well to obtain Tween 80 solution. The drug substance 468.9 mL was accurately weighed and placed in a 2 L graduated cylinder. After accurately weighing 13 mL of Tween 80 solution and putting it in the same 2 L graduated cylinder, the drug substance buffer solution was added to make up to 1300 mL. The composition of the PEGylated fEPO solution formulation thus obtained is shown in Table 6. Filtration was performed using Millipak 20 (manufactured by Merck), and 1.1 mL each was filled into a glass vial. Sealed with a rubber stopper and an aluminum cap.

FEPO concentration: 3. The fEPO concentration measured by the same procedure as described in 1 was 0.11 mg / mL.

SDS-PAGE (CBB staining): 3. SDS-PAGE (CBB staining) was performed in the same procedure as described in 1. above. As a result, bands were confirmed at 60 kDa and 120 kDa.

HPLC-RP: 3. HPLC-RP was performed in the same procedure as described in 1. As a result of obtaining the other peak areas relative to the peak area of the PEG body, no peak of 0.5% or more was observed.

HPLC-SEC: 3. HPLC-SEC was performed in the same procedure as described in 1. As a result of obtaining a relative value from the peak areas of the aggregate, triPEG body, monoPEG body and diPEG body, the ratio of the aggregate was less than 2.0%.

Bioactivity measurement: 1.2 of the above Experiment 1. 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 preparation, it was 1.8 × 10 ⁴ U / mg.

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

Claims (9)

1. A method for producing a purified cat-derived erythropoietin,
   A pH adjusting step for adjusting the pH of the protein solution containing cat-derived erythropoietin to a range of 4 or more and 7 or less;
   a purification step comprising contacting the protein solution adjusted in the pH adjustment step with a carrier having a cation exchange group to adsorb cat-derived erythropoietin, and then eluting the adsorbed cat-derived erythropoietin from the carrier; Including methods.
2. The method according to claim 1, further comprising a precipitate removing step of removing the precipitate from the protein solution whose pH has been adjusted in the pH adjusting step before the purification step.
3. The method according to claim 1 or 2, wherein the pH of the protein solution is adjusted to a range of 4.7 or more in the pH adjustment step.
4. 4. The pH adjustment step, wherein the protein solution is mixed with a buffer solution having a buffering action in an acidic region containing a component having a buffering effect in the acidic region at a concentration of 1 M or less. The method described in 1.
5. 5. The pH adjustment step, wherein the protein solution is mixed with at least 1.5 parts by volume of a buffer solution having a buffering action in an acidic region with respect to 1 part by volume of the protein solution. The method according to item.
6. The method according to any one of claims 1 to 5, wherein the protein solution used in the pH adjustment step is a protein solution prepared using an egg of a transgenic bird having a foreign gene encoding a cat-derived erythropoietin. .
7. A step of producing a purified cat-derived erythropoietin by the method according to any one of claims 1 to 6;
   Water-soluble long-chain molecule-added cat-derived erythropoietin comprising a water-soluble long-chain molecule-added step of chemically modifying the purified cat-derived erythropoietin with a water-soluble long-chain molecule to obtain a water-soluble long-chain molecule-added cat-derived erythropoietin Production method.
8. The method according to claim 7, further comprising a virus removal step of removing the virus by passing the erythropoietin derived from the water-soluble long chain molecule-added cat obtained in the water-soluble long chain molecule addition step through a filter.
9. A formulation step of preparing a liquid composition comprising the erythropoietin derived from the 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 that of a mammalian body fluid The method according to claim 7 or 8, further comprising:

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