CN108137665A - Constituent comprising long-acting erythropoietin - Google Patents
Constituent comprising long-acting erythropoietin Download PDFInfo
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- CN108137665A CN108137665A CN201680058467.9A CN201680058467A CN108137665A CN 108137665 A CN108137665 A CN 108137665A CN 201680058467 A CN201680058467 A CN 201680058467A CN 108137665 A CN108137665 A CN 108137665A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/505—Erythropoietin [EPO]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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Abstract
The present invention provides constituent containing long-acting erythropoietin and preparation method thereof.More particularly provide the EPO Fc fused protein constituents with excellent biological sustainability and high-purity, wherein the sialic acid content of EPO Fc is either more for 17mol/mol and includes protein (HCP) impurity derived from host cell with 100ng/mg or less amount.
Description
Technical field
The present invention relates to a kind of medical component for including long-acting erythropoietin (erythropoietin) and its
Preparation method.
Background technology
Erythropoietin(EPO) (EPO) including 165 amino acid is a kind of glycoprotein to promote hemocytoblast in marrow
(erythroblast) differentiation and generation red blood cell.EPO most more than kidney generate and it is known also on a small quantity in liver generate.Example
Such as, such as in shown in chronic renal failure (CRF), the loss of renal function typically causes the decline of EPO generations and therefore causes
The decline of related red blood cell.Have found that EPO other anaemias for the anaemia caused by CRF and caused by the polynary origin cause of formation are controlled
Treating has effects that different hematopoiesis functions excellent and with such as surgical operation auxiliary.
In general, as the polypeptide of EPO has low stability and easily by proteolytic enzyme, that is, the albumen in blood
Enzyme is degenerated and is decomposed, and is thus easily excluded via kidney or liver.Therefore, polypeptide (partly declines between having short biological half-life
Phase).So in order to maintain to contain the haemoconcentration and potency of protein medicine that polypeptide is formed as pharmacy, protein doctor
Medicine needs are often offerd medicine to patient.However, in the case of the protein the to be offerd medicine medicine is injection composite type, often
It injects to keep the haemoconcentration of active peptides that may usually cause patient pain.
In order to increase the half-life period of EPO polypeptides, the fusion protein prepared by using immunoglobulin (Ig) has been developed at present
Matter.Ig is the important constructivity composition of blood.Mankind Ig (hIg) may include plurality of classes such as IgG, IgM, IgA, IgD and IgE.
Immunoglobulin contains there are four polypeptide chain, and more specifically, two heavy chains and two light chains, wherein these chain warps
It is combined to form the tetramer together by cystine linkage.Each chain is made of region of variability and constant region.Constant region (the light chain constant of heavy chain
Area) three or four sections (site) such as CH1, CH2, CH3 and CH4 can be further divided into according to its homotype (isotype).Heavy chain constant region
Fc areas may include hinge (hinge), CH2, CH3 and/or CH4 functional domain (domain) according to the homotype of Ig.
About half-life period, being blended in the known display of chimeric protein in the Fc regions of immunoglobulin increases stability with prolonging
Long serum half-life.
Furthermore, it is known that stick the prevention that sialic acid can induce protein degradation to the surface of epo protein matter in liver.Saliva
Liquid acid protects second galactosyl in asialoglycoprotein receptor and therefore influences (the biology work of the activity of the EPO in live body
Property).Important angle is played the part of in extension of the sialic acid of the known sugar chain for being present in erythropoietin(EPO) in erythropoietin(EPO) half-life period
Color finally, influences its effect.Furthermore the because it have been observed that reaction that the bioactivity of erythropoietin(EPO) passes through sialidase
And inactivate, more actively carry out research of the sialic acid to the function of erythropoietin bioactivity.In most cases, it uses
Sugar in therapeutic agent manufacture has basis and indispensable role in essential effect of performance glycoprotein.Especially, for
Erythropoietin(EPO) derived from people, sugared role are absolute demands.In other words, if not including sugar, erythropoietin(EPO) is not
Display activity, is significantly affected according to the structure composition of sugar or the content of the sialic acid in end, the bioactivity of EPO.
During host cell expression, the sialic acid content in EPO-Fc can be by adding N- acetyl mannosamines to culture
Solution and increase.Obtained EPO-Fc is typically the EPO-Fc of high sialic acid content and low sialic acid content after the above process
The mixture of EPO-Fc.Here, the EPO-Fc of the relatively low sialic acid contents of the EPO-Fc of known high sialic acid content is with longer
Half-life period.Therefore, the technology in need for developing selectively EPO-Fc of the manufacture with increased sialic acid content.
It when EPO-Fc is manufactured, needs to cultivate inverted host cell, expresses a large amount of EPO-Fc, be disintegrated cell and pure
Change EPO-Fc.Therefore, it is extremely important for mass production high-quality EPO-Fc, optimization culture and purification technique.In current
Developed culture and purification technique still have big improvement space, therefore, there is an urgent need to be via developing above-mentioned technology with most
Suitableization EPO-Fc preparation process.Korean Patent Laid Reference Number 897938 (login on May 8th, 2009) discloses immunoglobulin fusion
Protein.
[existing technical literature]
Korean Patent accession number 0897938 (2009.5.8)
Invention content
Problems to be solved by the invention
It is therefore an object of the present invention to provide a kind of EPO-Fc in the living body with excellent duration (biological duration)
Fused protein constituent.
Further, it is another object of the present invention to provide a kind of EPO-Fc fused protein groups with high sialic acid content
Into object.
Furthermore it is a further object to provide a kind of EPO-Fc fused protein constituents with high-purity.
Further, it is a further object to provide impurity derived from a kind of host cell for having and reducing content
EPO-Fc fused protein constituents.
The solution to the problem
The above-mentioned purpose of the present invention is reached by following characteristics：
(1) a kind of EPO-Fc constituents, including EPO-Fc, the EPO-Fc have sialic acid content be 17mol/mol or
Person is more and host cell derived from the amounts of protein impurities be 100ng/mg or less.
(2) such as the EPO-Fc constituents of above-mentioned (1), wherein sialic acid content range is from 20 to 28mol/mol.
(3) such as the EPO-Fc constituents of above-mentioned (1), wherein EPO-Fc is with pI≤6.0.
(4) such as the EPO-Fc constituents of above-mentioned (1), wherein EPO-F is with 4.5≤pI≤5.3.
(5) such as the EPO-Fc constituents of above-mentioned (1), wherein protein impurities derived from host cell are the aggegations of EPO-Fc
Object or segment.
(6) such as the EPO-Fc constituents of above-mentioned (1), the amount of wherein protein impurities derived from host cell is 60ng/mg
It is or less.
(7) such as the EPO-Fc constituents of above-mentioned (1), further comprise derived from 0.5ng/mg or less host cell
DNA impurity.
(8) a kind of method for preparing EPO-Fc constituents, including：In 35 DEG C to 39 DEG C of temperature range and 6.5≤pH≤
With 4vvd or lower flow rate flowing EPO-Fc cell culture solutions under conditions of 7.5, to prepare perfusion culture solution；From
Perfusion culture solution obtains the pure solution of EPO-Fc；And the pure solution of EPO-Fc is adsorbed to anion exchange resin to prepare Q effluents,
It is 17mol/mol or more EPO-Fc that it, which is included with sialic acid content,.
(9) such as the method for above-mentioned (8), the preparation of wherein Q effluents include：EPO-Fc is adsorbed to anion exchange resin；
And the sodium chloride of 0.1M is arrived using the sodium phosphate containing 0.005 to 0.02M, 0.05 to 0.2M L-arginine and 0.02, and
Buffer solution with 6.7≤pH≤7.1 elutes the EPO-Fc through absorption.
(10) such as the method for above-mentioned (8), wherein the step of obtaining EPO-Fc pure solution includes culture solution will be perfused and pass through
POD filters.
(11) such as the method for above-mentioned (8), wherein the amount of protein derived from host cell (HCP) impurity is in EPO-Fc compositions
For 100ng/mg or less in object.
(12) such as method of above-mentioned (8), wherein EPO-Fc cell culture solutions, with 2.0 × 105Cell/mL's or more
Amount is seeded to incubator.
(13) such as the method for above-mentioned (10), further comprise, it, will be above-mentioned after perfusion culture solution is by POD filters
Solution is by being filled through the tubing string of a-protein-binding resin.
(14) such as the method for above-mentioned (8), it is 6.0 or lower that contained EPO-Fc, which has pI, wherein in Q effluents.
(15) such as the method for above-mentioned (8), further comprise using POD filters, ultrafiltration membrane and nanofilter respectively
Filter Q effluents.
The effect of invention
EPO-Fc constituents according to the present invention are characterized in that the part that often occurs of sugar-chain end is to use saliva acid blocked, therefore
Increase half-life period and enhance the convenience of patient.
EPO-Fc constituents according to the present invention are miscellaneous with protein impurities and DNA derived from the host cell for reducing content
Matter, therefore with high EPO-Fc purity.
The method according to the present invention for being used to prepare EPO-Fc constituents can be suitable for EPO-Fc as characterized above
Selective purification.
What the method according to the present invention for being used to prepare EPO-Fc constituents can significantly improve sialic acid is attached to EPO-
The survival of Fc and enhancing host cell and enhancing process efficiency.
Description of the drawings
The above-mentioned and other purpose of the present invention, feature are made by the schema of following detailed description and accompanying and other are excellent
Point is more clearly appreciated that.
Fig. 1 is to show the testing result figure that EPO-Fc isoelectric focusings (isoelectric focusing) check.
Specific embodiment
The present inventor based on for improveing the host cell culture technique of manufacturing and sialic acid content, high sialic acid contains
It measures EPO-Fc selection techniques and completes the present invention for removing impurity derived from host cell.
The invention discloses a kind of EPO-Fc constituents, contain tool 17mol/mol or more sialic acids and 100ng/
It the EPO-Fc of protein (host cell proteins matter, HCP) impurity derived from mg or less host cell and is prepared for it
Method.
Hereinafter, the present invention will be described in further detail.
EPO-Fc fused proteins are given birth to by the way that the Fc areas of human immunoglobulin heavy's constant region are fused to red blood cell
Cheng Su (EPO) and formed, and with compared with EPO be longer half-life period.The increased half-life period refers to increase the biology of effect
Sustainability.
Sialic acid is present in the sugar chain of EPO, protects second galactolipin group of asialoglycoprotein receptor, so big
Influence to width the bioactivity of EPO.Further, sialic acid can be induced in liver EPO degradation prevention thus increase partly decline
Phase.
Therefore, in addition to result from Fc fusions half-life period increase effect other than, contained saliva in EPO-Fc fused proteins
The attachment of liquid acid can be provided the effect of increasing half-life period.
During the culture of EPO-Fc host cells, N- acetyl mannosamines (NAM) can be made an addition to culture solution with
Increase the sialic acid content of EPO-Fc.The EPO-Fc of gained generally includes the EPO-Fc of high sialic acid content and low after the above process
The mixture of the EPO-Fc of sialic acid content, it is known that the EPO- of the relatively low sialic acid contents of the EPO-Fc of high sialic acid content
Fc has longer half-life period.
A concrete example according to the present invention, being attached to the sialic acid number of EPO-Fc increases, and pI values reduce.Based on this side
Face optionally purifies the EPO-Fc of high sialic acid content using anion exchange resin.It preferably uses containing crosslinking
The anion exchange resin of agarose.According to a concrete example, GE medical companies (GE Healthcare Co.) can be used made
The Q-Sepharose resins made.
By using the purification process of anion exchange resin, optionally purifying has the EPO-Fc of pI≤6.Especially
Ground, it is that 17mol/mol or more EPO-Fc can be obtained through thus process to have sialic acid content.According to a concrete example,
The situation of 4.5≤pI≤6.0, it is 17 constituents containing EPO-Fc for arriving 28mol/mol that can obtain sialic acid content.According to
Another concrete example, the situation in 4.5≤pI≤5.3, it is 20 to 28mol/mol to contain EPO-Fc that can obtain sialic acid content
Constituent.
During EPO-Fc is manufactured, protein derived from host cell (HCP) impurity may be mixed with.HCP impurity may include,
For example, the protein of the abnormal peptide containing the different agglutinators and segment that may be such as derived from host cell and other materials is miscellaneous
Matter, DNA impurity, intrinsic or external viral and other particle or the like.
Eliminate the quality that these impurity may be an important process and directly affect EPO-Fc fused proteins.When this
A constituent is when being prepared via a series of processes according to the present invention, and HCP is controllable in 100ng/mg or less amount,
And preferably 60ng/mg or less.DNA impurity derived from host cell can be contained with 0.5ng/mg or less amount.
The method of the EPO-Fc constituents of the present invention is used to prepare, it can be according to following completions：
Being used to prepare the EPO-Fc constituents of the present invention may include：(1) in 35 DEG C to 39 DEG C of temperature range and 6.5≤pH
Under conditions of≤7.5, EPO-Fc cell culture solutions are flowed with 4vvd or lower turnover rate to prepare perfusion culture solution；
(2) the pure solution of EPO-Fc is obtained from perfusion culture solution；(3) the pure solution of EPO-Fc is adsorbed to anion exchange resin, to prepare
With the sialic acid content 17mol/mol or more Q effluents containing EPO-Fc.
The perfusion culture solution of step (1) can be such as following preparations.
First, EPO-Fc master cell banks (master cell bank, MCB) can be from the cell bank studied for EPO-Fc
(RCB) it establishes and EPO-Fc working cardial cells library (working cell bank, WCB) can be from EPO-Fc master cell banks
(MCB) it establishes.
Culture for establishing MCB and WCB may include under the conditions of 3 to 7%CO2 and 37 DEG C, in including L-Glutamine
And in the fluid nutrient medium of the EX-cell CHO DHFR (-) of methotrexate (MTX), then cultured cell line, product distribution is existed
For the cryogenic vial for freezing and storing.
After chilled and storage WCB cell strains thaw, the cell strain through defrosting is provided and is suspended in the medium
With the proliferative cell in culture medium.For example, shaking flask can be used to be trained in the CO2 for being set to 3 to 7%CO2 and 37 DEG C for secondary culture
Case is supported, with 1:2 to 1:6 ratio is spaced 50 to 90 hours and carries out.It can make the cell number of culture in the culture using several shaking flasks
Reach and be enough to be inoculated into the enough of cell incubator.
In this case, EPO-Fc culture mediums can be used.EPO-Fc culture mediums may include, for example, EX-cell CHO
Powder culture medium, L-Glutamine, methotrexate (MTX) and the sodium bicarbonate of DHFR (-).
When ensuring for enough host cells of inoculation via shaking flask culture, the cell can be inoculated into main culture
Case, to be irrigated culture.Main incubator used herein can be 30 liters of bio-incubators.
After main incubator is inoculated into, it is 2.0 × 10 that can obtain cell concentration5Cell/mL or higher.
The cultivation temperature range of main incubator can be from 35 to 39 DEG C or from 36 to 38 DEG C etc..
The acidity of main incubator can maintain range 6.5≤pH≤7.5 (after pH regulation and control), 6.8≤pH≤7.2 (
After pH regulation and control) etc..
During perfusion culture, if necessary, culture solution can sample and with micro- sem observation, to monitor cell
Situation, process detection can be by analyzing pH, cell number, cell activity, concentration of glucose, glutamine concentration, ammonia density, oozing
Thoroughly pressure etc. and implement.
Controllable perfusing rate is 4vvd or lower.Perfusing rate range can from 0 to 4vvd, 0 to 2vvd, 1 to 3vvd, 1 to
2vvd etc..
It may include for perfusion culture medium herein made by adding N- acetyl mannosamines in EPO-Fc culture mediums
Standby culture medium (being the culture medium of added N- acetyl mannosamines) adds N- acetylmannosamines in EPO-Fc culture mediums
Culture medium (manufacture culture medium) or both culture mediums prepared by osamine and glucose can be used sequentially.
For example, according to daily 40 liters of perfusion cultures 4 days, it can obtain 140 to 180 kilograms in total of the EPO-Fc that is used for and manufacture
Cell culture solution.It is once to ten times or secondary to five times to repeat above-mentioned collection cell culture solution, it is possible to provide more increment
For the cell culture solution of EPO-Fc manufactures.
The cell culture solution obtained as described above arrived can become the perfusion culture solution of the step (1) of the present invention.
The step of preparing perfusion culture solution according to the present invention, 17mol/mol or more, 17 to 28mol/mol or 20
Sialic acid to 28mol/mol can be adhered to EPO-Fc.Further, the survival of cell can improved thus enhancing EPO-Fc groups
Into the productivity of object.
The method for being used to prepare the EPO-Fc constituents of the present invention can further comprise that (2) are obtained from perfusion culture solution
The pure solution of EPO-Fc.
There is above-mentioned step main purpose to be to remove in addition to EPO- contained in the perfusion culture solution of step (1)
Other compositions other than Fc.This removal may include any conventional method for cell culture.This removing method can with such as
The upper filtering and at least one combination of purification process.
After the perfusion culture solution of recycling step (1), multiple steps that this solution is acceptable for filtering and purify,
Thus remove impurity derived from host cell.Impurity derived from the host cell may include, for example, containing such as different aggegations
The protein impurities of object and the aberrant polypeptide of segment, DNA impurity, intrinsic viral, external virus and other particles.
Host cell refers in step (1) for all cells of EPO-Fc expression.
According to a concrete example, in order to which the host for removing contained in the host cell culture solution manufactured for EPO-Fc is thin
(HCP) impurity of protein derived from born of the same parents or DNA impurity can use POD depth filters.POD depth filters are commonly used in removal
Cell, however, the present invention is used to remove protein.
Using POD depth filters and filter, can be removed such as the host of protein aggregates, segment and other particles
Cell-derived protein (HCP) impurity.
For example, such as POD filters, the Millistak+ (MA1HC10FS1) that Merck Millipore can be used to manufacture.Into
One step, filter used herein can be the Sartobran P levels of sterility capsules of Sartorius Co. manufactures.
It is Protein A purified according to another concrete example, low pH is virally inactivated and/or hydroxyapatite purifies etc., it is applicable to
EPO-Fc is purified.
According to another concrete example, ultrafiltration apparatus, that is, tangential flow filtration (tangential flow filtration,
TFF) membranous system is replaced available for concentration and buffer solution.If conductivity and acidity via buffering fluid exchange and in term of reference,
Then complete the process (reference of concentration and buffer solution replacement：9.0 ± 1.0mS/cm of conductivity, acidity pH 6.9 ± 0.1).
The pure solution of EPO-Fc in step (2) can be prepared as described above.
The method for being used to prepare the EPO-Fc constituents of the present invention may include that the pure solution of (3) EPO-Fc is adsorbed onto anion friendship
Resin is changed, to prepare Q effluents, which includes having sialic acid content 17mol/mol or more EPO-Fc.
Above-mentioned steps are to select EPO-Fc contained in constituent with sialic acid content with main purpose
17mol/mol or higher special person.
This step can be by by the pure solution of EPO-Fc in step (2), by being filled through containing Sepharose
Anion exchange resin tubing string carry out.
According to a concrete example, the Q Sepharose resins of GE medical companies can be used, for example, can be used shown in the following table 1
Q Sepharose Fast Flow resins.
Table 1
Preparing the process of Q effluents can include：By using the equilibration buffer containing 0.005 to 0.02M sodium phosphates
Balance Q-Sepharose resins；The pure solution of EPO-Fc is made to pass through Q-Sepharose resins to adsorb EPO-F；By using above-mentioned
Equilibration buffer rebalances Q-Sepharose resins；With will contain 0.005 to 0.02M sodium phosphates, 0.05 to 0.2M L- essence
Propylhomoserin and 0.02 to 0.1M sodium chloride and with acidity for 6.7≤pH≤7.1 efflux by the tubing string to collect EPO-Fc
Q effluents.
It can suitably repeat to elute.For example, when elution repeats 4 times, obtained in being purified first to third time
Q effluents No.1 to 3 be stored in superfreeze refrigerator, and complete the 4th time elution after, all Q effluents
No.1 to 4 can be collected.
Optionally, it after elution, can be filtered using POD filters.POD filters used herein can include,
For example, the Millistak+ (MA1HC10FS1) manufactured by Merck Millipore Co..
Other than the above process, optionally, it may further include and the process of filter is installed in tubing string and is passed through
Resin is made to contact the process to sterilize and wash Q-Sepharose resins with CIP.According to a concrete example, filtering used herein
Device may include, for example, the Sartobran P levels of sterility capsules manufactured by Sartorius Co..
Contained EPO-Fc can have pI≤6.0 in the Q effluxes obtained in step (3) and sialic acid content is
17mol/mol is more.For example, it can be range 17 to 28mol/ that EPO-Fc, which can be 4.5≤pI≤6.0 and sialic acid content,
It can be range 20 to 28mol/mol that mol or EPO-Fc, which can be 4.5≤pI≤5.3 and sialic acid content,.
The method for being used to prepare the EPO-Fc constituents of the present invention can also include (4) respectively via POD filters, ultrafiltration membrane
And/or nanofilter filtering Q effluents.
According to above-mentioned steps, EPO-Fc can have increased concentration, can replace buffer solution, and can eliminate disease
Poison.
POD filters used herein can be the Millistak+ (MA1HC manufactured by Merck Milipore Co.
10FS1)。
Ultrafiltration membrane used herein can include, for example, tangential flow filtration (TFF) membranous system.For example, using injection slow
Fliud flushing washing film (retention：After 30K), Formulation Buffer can be used to make membrane equilibrium.Formulation Buffer can contain 0.01M lemons
Sour sodium, 0.1M glycine and 0.1M sodium chloride, and the acidity with pH 6.2 ± 0.2.
After completing balance, target protein can be concentrated into about 1.5 ± 0.3mg/mL.If the solution of recycling has about
The protein concentration of 1.5 ± 0.3mg/mL, then can be by being continuously added to be used to prepare the buffering of crude solution with same volume
Liquid carries out buffer-exchanged.If the conductivity and acidity of the solution finally recycled complete concentration in its term of reference
With (the reference of buffer solution exchange process：12.0 ± 2.0mS/cm of conductivity, acidity pH6.2 ± 0.2).
When the concentration of the solution finally recycled and buffer solution exchange process are completed, nanofilter (PALL can be used
(NT6DV20P1G)) it is filtered, to eliminate the disease of additional material that may originate from host cell or use in this process
Poison.
According to a concrete example, after the filter for virus filtration is washed, the integrity test of filter is carried out, and can
By the way that the buffer solution for being used for crude manufacture is reached balance by virus filter.After completing balance, concentration and buffer solution are more
The solution finally recycled after the completion of the process changed can pass through filter under the pressure of 30 ± 3psi.Therefore, nothing can be recycled
The viral filtrate of virus.After the completion of filtering, injection buffer solution washing virus filter can be used, then carries out integrality survey
Examination.
Protein compression can be made by adding in the polysorbate20 of a concentration of 0.12g/Kg and Formulation Buffer to viral filtrate
Degree is adjusted to 1.1 ± 0.3mg/mL, then can manufacture EPO-Fc crude solutions using sterilising filter.
According to a concrete example, Formulation Buffer can include 0.01M sodium citrates, 0.1M glycine and 0.1M sodium chloride,
And the acidity with pH 6.2 ± 0.2.According to a concrete example, sterilising filter used herein can be by Sartorius
Co. the Sartobran P300 manufactured.Prepared EPO-Fc crude solutions can distribute and be stored in superfreeze refrigerator
In.
By adding in Formulation Buffer to EPO-Fc crude solutions, then filtered by sterilising filter, by protein compression
Degree is adjusted to 0.5 ± 0.1mg/mL, obtains final EPO-Fc crude solutions.The sterilising filter used herein can include
Such as the Sartobran P midicap manufactured by Sartorius Co..
Hereinafter, the present invention is more fully described in reference implementation example.However, propose that these embodiments are merely to illustrate
The preferred embodiment of the present invention, and the scope of the present invention is not particularly limited to this.
Embodiment 1：The preparation of EPO-Fc constituents
<Process 1：The preparation of EPO-Fc master cell banks (MCB)>
To adding in L-Glutamine and methotrexate (MTX) in EX- cells CHO DHFR (-) fluid nutrient medium to form culture medium
And after the cell bank (RCB) studied for EPO-Fc is inoculated into culture medium, increase total cell number and total training via secondary culture
Volume is supported, so as to build MCB.Culture carries out in 5.0 ± 2.0%CO2 incubators, until culture solution reaches 37 DEG C of temperature
Degree.
<Process 2：The preparation in EPO-Fc working cardial cells library (WCB)>
To adding in L-Glutamine and methotrexate (MTX) in EX- cells CHO DHFR (-) fluid nutrient medium to form culture medium
And after the MCB prepared by process 1 is inoculated into culture medium, increase total cell number and total volume of culture via secondary culture, so as to
Build WCB.Culture carries out in 5.0 ± 2.0%CO2 incubators, until culture solution reaches 37 DEG C of temperature.
<Process 3：WCB is merged>
By it is chilled in process 2 and storage WCB thaw after, the WCB through defrosting be suspended in EPO-Fc culture mediums (including
EX- cells CHO DHFR (-) powder culture medium, L-Glutamine, methotrexate (MTX) and sodium bicarbonate) in, then cultivated
It is provided in 37 DEG C of the CO2 incubators of 5%CO2.
<Process 4：The culture of shaking flask cell>
The culture solution of process 3 was subjected to secondary culture with the interval of 64 to 80 hours, to ensure to be enough to be inoculated into
Cell number needed for cell incubator.Using EPO-Fc culture mediums in the CO2 incubators for being provided with 5%CO2 and 37 DEG C with 1:3
To 1:4 ratio carries out secondary culture.
<Process 5：Manufacture and culture in main incubator (30L bio-incubators)>
After the culture solution for obtaining enough process 4, collect cell and be inoculated into main incubator with reach 2.0 ×
105Cell/mL.Then, it is above-mentioned molten using the EPO-Fc medium cultures for being wherein further added with N- acetyl mannosamines
Liquid.Cultivation temperature is kept to be irrigated culture for 37 ± 1 DEG C and pH7.0 ± 0.2 (after pH regulation and control) in culture.If desired,
Process detection is carried out by sampling culture solution.According to process detection as a result, by range of the filling rate regulation and control 0 to 2vvd.
In order to prepare culture solution, culture medium is manufactured by new EPO-Fc and (N- acetylmannosamines sugar is added in EPO-Fc culture mediums
Prepared by amine and sodium bicarbonate) culture medium used is replaced, then further it is irrigated culture.Continue 4 days with about daily 40L
140 are collected to 180kg culture solutions, to obtain a sub- batch.By repeating the above process 4 times in total, continue 16 days, carry out
Culture is until collecting a sub- batch in four (4) in total.It (is filled for the culture solution of the host cell of EPO-Fc manufactures as a result, obtaining
Note culture solution).
<Process 6：The recycling and filtering of culture solution>
Use POD filters (MA1HC10FS1) and filter (Sartobran P- levels of sterility capsule) filter process 5
For the host cell culture solution of EPO-Fc manufactures, filtrate and the storage of perfusion culture solution are then obtained.
<Process 7：The purifying of a-protein>
The tubing string of resin is bonded simultaneously with equilibrium state filling albumin A using equilibration buffer (0.01M sodium phosphates) making
After the filtrate of process 6 is adsorbed onto tubing string, washing buffer (0.7M L-arginines, pH 5.7 ± 0.05, conductivity are used
37.0 ± 1.0mS/cm) cleaning tubing string, then using elution buffer (0.02M sodium acetates anhydride, 0.2M L-arginines,
7.94% (v/v) glycerine, pH 3.7 ± 0.05) elution target protein (EPO-Fc), then store.
<Process 8：Low pH is virally inactivated>
If the pure solution of process 7 measures the result of its pH to be not suitable as referring to, pH is added in it and adjusts buffering
Liquid (1M sodium hydroxides (NaOH), 10% acetic acid) is to adjust pH (3.7 ± 0.05), and then carrying out this process, agitating solution is held simultaneously
It is 2 hours continuous.After the process is completed, adjust buffer solution (1M sodium hydroxides, 10% acetic acid) using pH and adjust pH to about pH
6.9±0.1。
<Process 9：The purifying of hydroxyapatite>
In the tubing string balanced filled with stationary phase (hydroxyapatite) and with equilibration buffer (0.01M sodium phosphates)
After filter (Sartobran P- levels of sterility capsule) is installed, the pure solution of process 8 is adsorbed onto tubing string, then, is removed
Filter (Sartobran P- levels of sterility capsule), then using elution buffer (0.1M sodium phosphates and 0.1M L- essence ammonia
Acid, pH 6.9 ± 0.1) elution target protein, then store.
<Process 10：It is concentrated by ultrafiltration and buffer solution replaces 1>
Using ultrafiltration apparatus (film：30K is blocked, including 0.5m2) after the pure solution of concentration process 9, by being continuously added to
Equilibration buffer (0.01M sodium phosphates) carries out buffering fluid exchange.When conductivity and pH are in its term of reference, which completes
And the delay solution of concentration and buffer solution replacement 1 is collected, obtain the pure solution of EPO-Fc.
<Process 11：The purifying of Q Sepharose>
In the tubing string installation filter (Sartobran P- levels of sterility capsule) filled with stationary phase (Q Sepharose)
And with the equilibration buffer Q Sepharose resins comprising 0.01M sodium phosphates after, the pure solution of the EPO-Fc of process 10 is inhaled
Tubing string is attached to, then, removes filter, i.e. Sartobran P levels of sterility capsule, and Q Sepharose resins are put down again
Weighing apparatus is then eluted using elution buffer (0.01M sodium phosphates and 0.1ML- arginine, 0.05M sodium chloride, pH 6.9 ± 0.1)
Target protein, to obtain and store the Q effluents of EPO-Fc.
<Process 12：The defrosting and filtering of effluent>
The Q effluents through storage of process 11 are thawed and collected.Using Formulation Buffer (0.01M sodium citrates,
0.1M glycine, 0.1M sodium chloride, pH 6.2 ± 0.2) balance POD filters (MA1HC054H1), then by leading to Q effluxes
Filter is to obtain filtrate.
<Process 13：It is concentrated by ultrafiltration and buffer solution replaces 2>
Using ultrafiltration apparatus (film：30K being retained, including 0.1M2) Q of concentration process 12 collects filtrate with the egg that achieves the goal
After white matter concentration, by continuously adding Formulation Buffer (0.01M sodium citrates, 0.1M glycine, 0.1M sodium chloride, pH 6.2
± 0.2) buffering fluid exchange is carried out.When conductivity and pH are in its term of reference, which completes, and collects concentration and buffering
The delay solution of fluid exchange 2.
<Process 14：Nanofilter filters>
Formulation Buffer (0.01M sodium citrates, 0.1M glycine, 0.1M sodium chloride, pH 6.2 ± 0.2) is flowed into nanometer
For filter (Pall (NT6DV20P1G)) so that after its balance, the delay solution of the concentration of process 13 and buffer solution replacement 2 flows into it
In, so as to eliminate the viruses being likely to be present in 2 delay solution of concentration and buffer solution replacement.
<Process 15：The preparation of EPO-Fc crude solutions>
0.12g/kg polysorbate20s are added in the viral filtrate obtained in process 14, and are adjusted using Formulation Buffer
Protein concentration.Then, it is sterilized and is filtered using sterilizing and filter (Sartobran P300) and is thick to prepare EPO-Fc
Solution processed, while meet the (reference of crude protein concentration：1.1 ± 0.3mg/mL), it is then dispensed to storage container and is incited somebody to action
The containers store is in superfreeze refrigerator.
<Process 16：The preparation of the final crude solutions of EPO-Fc>
In a water bath after the EPO-Fc crude solutions of course of defrosting 15, Formulation Buffer (0.01M citric acids are added in it
Sodium, 0.1M glycine, 0.1M sodium chloride, pH 6.2 ± 0.2, polysorbate20 0.12g/Kg) to reach 0.5 ± 0.1mg/
The protein concentration of mL is sterilized and is filtered using sterilizing and filter (Sartobran P midicap), so as to prepare most
Whole EPO-Fc crude solutions.
Embodiment 2：The purity detecting of EPO-Fc constituents
<Detection 1：The test of protein derived from host cell (peptide) (HCP) impurity content
In order to detect the HCP impurity contents in final crude solution, using CHO host cell proteins matter kit carry out with
Lower test：1) it dilutes and the standard solution provided in kit is provided；2) test solution is prepared as diluting using dilution buffer
State；3) addition test (spiked test) solution is prepared by the way that standard solution is added in above-mentioned test solution；4) make
After standard solution, test solution and addition test solution are reacted with anti-CHO (phosphatase attachment), make the mixture anti-
It is reacted in the coated microtiter plates of CHO；With 5) after plate is washed, PNPP is added in it by matter, and survey using ELISA plate reader
Measure absorbance.Sample used herein is lot number 747R0001 and 747R0002.Testing result is summarised in the following table 2.It is tied from test
The content that fruit can be seen that protein impurities derived from host cell is 60ng/mg.
Table 2
<Detection 2：The test of DNA impurity contents derived from host cell>
In order to detect the content of DNA impurity derived from host cell in the final crude solutions of EPO-Fc, following test is carried out：
1) by the colleges and universities' standard dilution agent provided in kit and used as standard solution；2) the zero school standard that will be provided in kit
Dilution agent is simultaneously used as test solution；3) addition test solution is prepared by the way that standard solution is added in test sample；4) herein
The zero school standard agent used is the zero school standard agent provided in kit；5) by the way that standard solution zero school standard agent of addition is made
Zero school standard agent of standby addition used herein；6) standard solution, test solution, addition test solution prepared by, zero school standard
Agent and zero school standard agent of addition carry out DNA marker；It is combined with 7) reference test bar (stick) is carried out using thresholding system filter module,
Then read test bar is to confirm result.Sample used herein is lot number Nos.GC1113-PUR-0901-PR, GC1113-
PUR-0902-PR, 747R0001 and 747R0002.Testing result is summarised in the following table 3.From test result as can be seen that host is thin
The content of DNA impurity derived from born of the same parents is 0.5ng/mg or lower, is up to 0.215ng/mg, average out to 0.143ng/mg.
Table 3
Embodiment 3：The sugar cloth detection of EPO-Fc in EPO-Fc constituents
<Detection 3：The analysis of EPO-Fc glycosylation positions>
EPO-Fc refers to the fusion protein of homodimer, including EPO the and Fc regions combined by IgD hinges
Two kinds of monomers combined by cystine linkage in hinge area.In EPO sequences there are three N- glycosylation positions (N24, N38 and
) and an O- glycosylation position (S126) N83.In addition, there are other N- glycosylations in the IgD CH2 structural domains of Fc sequences
Position (N261).Speculate that EPO-Fc can have total of eight N- glycosylation positions and two O- glycosylation positions.
Practical glycosylation position confirms via the Protagen companies for receiving request.
Processing DTT and iodoacetamide with thus restore/alkylated protein after, using PNGase F handle protein with
Remove the sugar in N- glycosylation positions.Contain using the protein of trypsase and GluC/ trypsin treatments without sugar and still
There are the other oroteins of sugar to form multiple peptides, the size of these peptides is then compared using MALDI-MS, so that it is determined that N- glycosyls
Change position.
In order to confirm O- glycosylation positions, after HexNAc is marked, it with PNGas F is handled and glycosylates position to remove N-
All sugar in putting.By using Arg C, trypsase/Glu C and Asp N/Tripsin processing, multiple peptides are obtained, then
Compare the size of these peptides using MALDI-MS, so that it is determined that O- glycosylation positions.
Analysis result is summarised in the following table 4.Identify be present in EPO sequences three N- glycosylation positions (N24, N38 and
N83 another N- sugar) and an O- glycosylation position (S126), and in the IgD CH2 structural domains of Fc sequences is also found
Base position (N261).
Table 4
<Detection 4：The analysis of the composition composition of EPO-Fc monosaccharide>
The sugar chain of glycoprotein is usually by monosaccharide such as fucose, N- acetyl glucosamines (GluNAc), N- acetyl galactoses
The compositions such as amine (GalNAc), galactolipin, mannose, sialic acid.Alternatively, it may also comprise glucose, xylose, Man-6-P
Or the like etc..
The Protagen companies for receiving request analyze the composition composition of monosaccharide.With 4M hydrochloric acid (ultimate density) by EPO-Fc
100 DEG C hydrolysis 4 hours after, be then dried via traditional vacuum, resulting materials be dissolved in third time distilled water and via
(high performance anion exchange chromatography method uses pulsed amperometric detection to liquid chromatography；HPAEC-PAD).By in the same terms
Area between lower standard monosaccharide substance of the analysis with known concentration and more identical and each monosaccharide estimates each monosaccharide and sugared egg
White mole of molar ratio.The following table 5 is summarised in using the molar ratio of each monosaccharide of standard substance.From test result as can be seen that 1
It is respectively present about 2.2 moles of fucose in mole EPO-Fc, about 42.1 moles of GluNAc, about 9.3 moles of galactolipin, about
7.0 moles of mannose.
Table 5
Embodiment 4：The detection of the sialic acid content of EPO-Fc in EPO-Fc constituents
<Detection 5：The isoelectric focusing test of EPO-Fc>
In order to determine the pI values of prepared EPO-Fc, following isoelectric focusing (IEF) gel electrophoresis test is carried out：1) will
Standard solution and test solution each are mixed and are prepared with sample buffer (2X)；2) sample prepared by uses IEF pH 3-7
Gel carries out electrophoresis；3) gel for completing to be obtained after electrophoresis is fixed using solution of trichloroacetic acid；It is mixed with 4) use
Staining solution including Brilliant Blue R, methanol and acetic acid will be gel-colored, and methanol is included using what is mixed
It decolourizes with the liquid lime chloride of acetic acid, and drying.Used herein is lot number GC1113-PUR-0902-PR.Testing result is summarized
In Fig. 1.
As shown in the test result of Fig. 1, it can be seen that the main band of manufactured EPO-Fc is distributed in pI's 4.5 to 6.0
Range.
<Detection 6：The test of sialic acid content>
In order to detect the sialic acid content of manufactured EPO-Fc, following test is carried out：1) pass through dilution in distilled water
N- acetyl group nerve amino acids prepare standard solution；2) it is diluted using distilled water and prepares test solution in diluted state；3) between use
Benzenediol reagent heats after handling standard solution and test solution；4) it is recycled using extraction solution (n-butyl alcohol, butyl acetate) anti-
Answer product；With 5) 580nm measure recycling reaction product absorbance with estimate test solution sialic acid content, Ran Houji
Calculate the sialic acid content (mol/mol) of every 1 mole of EPO-Fc.Sample used herein is lot number Nos.GC1113-PUR-0901-
PR, GC1113-PUR-0902-PR, 747R0001 and 747R0002.Testing result is summarised in the following table 6.It can from test result
Go out, manufactured EPO-Fc has sialic acid content for 20mol/mol or more.
Table 6
Claims (15)
1. a kind of EPO-Fc constituents, it includes EPO-Fc, the EPO-Fc has sialic acid content for 17mol/mol or more
Protein impurities derived from more and 100ng/mg or less host cell.
2. EPO-Fc constituents according to claim 1, wherein, the sialic acid content ranging from 20 to 28mol/mol.
3. EPO-Fc constituents according to claim 1, wherein, the EPO-Fc has pI≤6.0.
4. EPO-Fc constituents according to claim 1, wherein, the EPO-Fc has 4.5≤pI≤5.3.
5. EPO-Fc constituents according to claim 1, wherein, protein impurities derived from the host cell are EPO-
The agglutinator or segment of Fc.
6. EPO-Fc constituents according to claim 1, wherein, the amount of protein impurities is derived from the host cell
Containing 60ng/mg or less.
7. EPO-Fc constituents according to claim 1 also derive comprising 0.5ng/mg or less host cell
DNA impurity.
8. a kind of method for preparing EPO-Fc constituents, including：
Under conditions of 35 DEG C to 39 DEG C of temperature range and 6.5≤pH≤7.5, with 4vvd or lower turnover rate flowing EPO-Fc
Cell culture solution is to prepare perfusion culture solution；
The pure solution of EPO-Fc is obtained from the perfusion culture solution；With
The pure solution of the EPO-Fc is adsorbed onto anion exchange resin to prepare Q effluents, the Q effluents include having saliva
Liquid acid content is 17mol/mol or more EPO-Fc.
9. according to the method described in claim 8, the preparation of wherein described Q effluents includes：The EPO-Fc is adsorbed onto institute
State anion exchange resin；And it uses containing 0.005 to 0.02M sodium phosphates, 0.05 to 0.2M L-arginines and 0.02 to 0.1M
Sodium chloride, and the buffer solution with 6.7≤pH≤7.1 elutes adsorbed EPO-Fc.
10. according to the method described in claim 8, the step of wherein obtaining EPO-Fc pure solution includes making the perfusion culture molten
Liquid passes through POD filters.
11. according to the method described in claim 8, wherein, the host cell derived protein in the EPO-Fc constituents
(HCP) amount of impurity is 100ng/mg or less.
12. according to the method described in claim 8, wherein, by the EPO-Fc cell culture solutions with 2.0 × 105Cell/mL
Or more amounts are inoculated into incubator.
13. according to the method described in claim 10, further include, make after the perfusion culture solution passes through the POD filters,
The solution is made to pass through the tubing string for being filled through albumin A bonding resin.
14. according to the method described in claim 8, wherein, in the Q effluents contained EPO-Fc have pI for 6.0 or
It is lower.
15. it according to the method described in claim 8, further includes respectively using described in POD filters, ultrafiltration membrane and nanofilter filtering
Q effluents.
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| PCT/KR2016/011169 WO2017061780A1 (en) | 2015-10-07 | 2016-10-06 | Composition comprising long-acting erythropoietin |
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| KR20170041384A (en) | 2017-04-17 |
| KR101847169B1 (en) | 2018-04-09 |
| TW201731523A (en) | 2017-09-16 |
| AU2016334954A1 (en) | 2018-05-24 |
| TWI654991B (en) | 2019-04-01 |
| WO2017061780A1 (en) | 2017-04-13 |
| AU2016334954B2 (en) | 2020-02-27 |
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