NZ733430A - A method for controlling glycosylation of recombinant glycoprotein - Google Patents
A method for controlling glycosylation of recombinant glycoproteinInfo
- Publication number
- NZ733430A NZ733430A NZ733430A NZ73343015A NZ733430A NZ 733430 A NZ733430 A NZ 733430A NZ 733430 A NZ733430 A NZ 733430A NZ 73343015 A NZ73343015 A NZ 73343015A NZ 733430 A NZ733430 A NZ 733430A
- Authority
- NZ
- New Zealand
- Prior art keywords
- recombinant glycoprotein
- insulin
- glycoprotein
- glycosylation
- culture medium
- Prior art date
Links
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- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000977 initiatory Effects 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- SZQUEWJRBJDHSM-UHFFFAOYSA-N iron(3+);trinitrate;nonahydrate Chemical compound O.O.O.O.O.O.O.O.O.[Fe+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O SZQUEWJRBJDHSM-UHFFFAOYSA-N 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 230000001404 mediated Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 150000004682 monohydrates Chemical class 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L na2so4 Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Polymers 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 230000004526 pharmaceutical effect Effects 0.000 description 1
- 230000013595 protein glycosylation Effects 0.000 description 1
- 201000004681 psoriasis Diseases 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 108091007521 restriction endonucleases Proteins 0.000 description 1
- 235000019192 riboflavin Nutrition 0.000 description 1
- 239000002151 riboflavin Substances 0.000 description 1
- 125000005629 sialic acid group Chemical group 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 230000001225 therapeutic Effects 0.000 description 1
- MYVIATVLJGTBFV-UHFFFAOYSA-M thiamine(1+) chloride Chemical compound [Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N MYVIATVLJGTBFV-UHFFFAOYSA-M 0.000 description 1
- 230000001131 transforming Effects 0.000 description 1
- 229960001322 trypsin Drugs 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000002452 tumor necrosis factor alpha inhibitor Substances 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N α-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- NBSCHQHZLSJFNQ-RWOPYEJCSA-N β-D-mannose 6-phosphate Chemical compound O[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@@H]1O NBSCHQHZLSJFNQ-RWOPYEJCSA-N 0.000 description 1
Abstract
The controlling the glycoprotein structures is very important in the field of development of recombinant glycoprotein products for medicines and development of mass production technology. The present invention relates to a method for controlling a glycosylation pattern of a recombinant glycoprotein, comprising culturing a cell comprising polynucleotide encoding a recombinant glycoprotein in a culture medium comprising insulin. The method for controlling the glycosylation of the recombinant glycoprotein according to the present invention can control an activity, folding, secretion, stability, a half-life in plasma, and an immune response of the recombinant glycoprotein. comprising culturing a cell comprising polynucleotide encoding a recombinant glycoprotein in a culture medium comprising insulin. The method for controlling the glycosylation of the recombinant glycoprotein according to the present invention can control an activity, folding, secretion, stability, a half-life in plasma, and an immune response of the recombinant glycoprotein.
Description
DECLARATION
I, Hyoiinsa SA,
of HANOL lntelectual Property & Law at 6th Floor, 163, YangjaeCheon-ro, Gangnamgu
, Seoul, 06302, Republic of Korea,
do solemnly and sincerely declare that l have a ent knowledge of h and
Korean languages and that the attached English translation is a true and accurate
translation of the specification as filed in respect of International Patent Applicant No.
filed on December 30, 2015.
Dated at Seoul, Republic of Korea this Zith day of June, 2017.
jogs/17511.01 £2 H70 I; V]
Signature name of translator
[Description of Invention]
[Title of Invention]
A METHOD FOR CONTROLLING GLYCOSYLATION OF INANT
ROTEIN
[Technical Field]
The t invention relates to a method for controlling glycosylation of a
recombinant glycoprotein.
[Background Art]
As a TNFR-Fc fusion n in which a ligand binding part of a human p75 TNF-α
receptor (TNFR, TNF-α or) is linked to an Fc fragment of human IgG1, Etanercept was
released by Amgen under the trade name of Enbrel in 2002. Etanercept competitively
inhibits in vivo binding between TNF-α receptors on the surface of a cell, thereby inhibiting a
TNF-α-related immune response. ingly, as a TNF-α inhibitor, Etanercept is used for
rheumatoid arthritis, psoriasis, ankylosing spondylitis, etc ., and clinical studies for its
application to vasculitis, Alzheimer’s disease, and Crohn’s disease are in progress.
Meanwhile, a gene recombinant pharmaceutical product is a pharmaceutical product
containing a peptide, a protein, etc ., produced by using a genetic manipulation technique as
an active ingredient. Use of biotechnology is advantageous in that it is possible to obtain a
large number of highly pure recombinant proteins which are difficult to obtain in a l
state, but an expression structure itself may be unstable since a gene of a target n is
inserted into a host cell from outside. Besides, proteins are produced by expressing the gene
in a rganism or a cell of an animal or plant, but not in the human body, the
inant proteins may be different from native proteins in terms of structural,
physicochemical, immunochemical, and biological properties or features (Kwon, et al., FDC
Legislation Research V, vol.1, 2, 13-21, 2010).
In particular, in the case of a glycoprotein, glycosylation and a structure or form of a
glycoform (sugar chain) may differ according to a culture condition. In other words, in the
process of glycoprotein production, difference in glycoform structures or the amounts of
rides constituting the glycoform structure lead to various types of glycoforms, thereby
causing heterogeneity ing to differences in production conditions. In the case of
glycoproteins with different glycoform structures, they are different from native forms in
terms of in vivo movement or tissue distribution, or are antagonistic to the native forms,
causing an e reaction. When administered continuously for a long period of time,
they act as antigens and may cause an immunological problem.
As described above, as the glycoforms may become an important factor that may
affect a pharmaceutical effect and in vivo movement, controlling the glycoprotein structures
is very important in the field of development of recombinant glycoprotein products for
medicines and pment of mass production technology.
In this , Korean Patent Publication No. 2011-0139292, as a prior art, ses
control of protein glycosylation and compositions and methods related thereto, and Korean
Patent Publication No. 2012-0134116 discloses a method for increasing N-glycosylation site
ncy on therapeutic glycoproteins.
[Disclosure of Invention]
[Technical Problem ]
With the above background, the present inventors have made extensive efforts to find
a method for controlling ylation of a recombinant glycoprotein, and as a result, have
confirmed that the glycosylation of the recombinant glycoprotein can be controlled when a
culture medium containing insulin is used, thereby ting the present invention.
[Technical Solution]
A main object of the present invention is to provide a method for controlling a
glycosylation n of a recombinant glycoprotein, comprising culturing a microorganism
comprising a polynucleotide encoding the recombinant rotein in a culture medium
comprising insulin.
Another object of the present invention is to provide a method for controlling a
glycosylation pattern of a recombinant glycoprotein, sing (a) culturing a
microorganism comprising a polynucleotide encoding a recombinant glycoprotein in a culture
medium to grow the rganism; and (b) adding insulin in the culture medium and
ing the same to produce a glycoprotein.
[Advantageous Effect]
The method for lling the glycosylation of the recombinant glycoprotein
according to the present invention can control an activity, g, secretion, stability, a fe
in plasma, and an immune response of the recombinant glycoprotein.
[Description of Drawings]
shows a cleavage map of pCUCBin-mSig-TNFcept.
shows the number of viable cells (unit: 105 cells/mL) and viability (%)
according to cell culture time (unit: day) in an exemplary embodiment of the present
invention.
[Best Mode]
As an aspect to achieve the above s, the present ion provides a method
for controlling a glycosylation pattern of a recombinant glycoprotein, comprising culturing a
microorganism comprising a polynucleotide encoding the inant glycoprotein in a
culture medium comprising insulin.
The glycoprotein refers to a protein in which a saccharide binds to a specific amino
acid of a polypeptide, and the saccharide may refer to a glycoform, e.g. , one in which at least
one or two monosaccharides are linked. As an example, the glycoform, as an
oligosaccharide in which various monosaccharides are linked to a glycoprotein, may include
a monosaccharide such as fucose, N-acetylglucosamine, N-acetylgalactosamine, galactose,
mannose, sialic acid, e, xyloses, mannosephosphate, etc .; a branched form thereof;
etc .
As an example, the recombinant glycoprotein may be an globulin fusion
protein. The immunoglobulin fusion protein may e the Fc region which is a part of
the immunoglobulin, including the heavy chain constant domain 2 (CH2), the heavy chain
constant domain 3 (CH3), and the hinge region, excluding the variable domains of the heavy
and light chains, the heavy chain nt domain 1 (CH1), and the light chain constant
domain (CL1) of the immunoglobulin (Ig).
As another example, the recombinant glycoprotein may be a TNFR-Fc fusion
protein.
The tumor necrosis factor receptor (TNFR) refers to a or protein which binds
to a TNF-α. The TNFR protein may be a TNFRI (p55) or TNFRII (p75) protein, preferably
TNFRII protein, but is not limited thereto. Additionally, the TNFRII may be atively
used with a tumor necrosis factor receptor superfamily member 1B (TNFRSF1B). The
TNFRII protein is divided into 4 domains and transmembrane regions, e.g. , a TNFRII protein
consisting of 235 amino acids including 4 domains and transmembrane, but is not limited
thereto. Information regarding the TNFRI and TNFRII proteins can be obtained from
known databases such as National Institutes of Health GenBank. For example, the TNFRI
and TNFRII ns may be the proteins of which the accession number is NP_001056 or
P20333, but are not d thereto.
For having an activity of binding to TNF-α, which is known to cause s diseases
when overexpressed in vivo, the TNFR protein can be used for treatment of diseases mediated
by TNF-α. In order to be used for said purpose, the TNFR p rotein can be produced and used
in a form of a fusion protein with a half-life increased by fusion of the Fc region of an
immunoglobulin and the TNFR protein.
The tumor necrosis factor receptor -Fc fusion protein refers to a fusion
protein in which all or a portion of the TNFR protein is linked to the Fc region of the
immunoglobulin by an enzymatic reaction or a product in which the two polypeptides are
expressed in one polypeptide through genetic manipulation. The TNFR-Fc fusion protein
may have TNFR protein and the Fc region of the immunoglobulin directly linked via a
peptide linker, but is not limited o. A non-limiting example of the TNFR-Fc fusion
protein may be Etanercept (US patent 7,915,225; 5,605,690; Re. 36,755).
The TNFR-Fc fusion n may be produced by fusion of all or a portion of a
TNFR protein with the Fc region of an immunoglobulin, e.g. , 232 amino acids of the Fc
region of an immunoglobulin including the hinge region and the 1st to 235th amino acid sites
of the TNFRII, but is not limited thereto. Additionally, the c fusion protein may be
codon-optimized according to a host cell to be expressed and may be, for example, a TNFRFc
fusion protein codon-optimized specifically to a CHO cell, but is not limited thereto.
The TNFR-Fc fusion protein is not only an amino acid sequence, but also an amino acid
sequence which is 70% or more, preferably 80% or more, more ably 90% or more, still
more preferably 95% or more, most preferably 98% similar to the amino acid sequence, and
includes all ns which have the activity of substantially binding to TNF-α. It is s
that as long as the sequence having such similarity is an amino acid sequence identical to
TNFR-Fc fusion protein or an amino acid sequence having a corresponding ical
activity, a protein mutant having amino acid sequences of which a part is deleted, modified,
substituted, or added falls within the scope of the present invention.
The Fc refers to a part of the immunoglobulin, including the heavy chain nt
domain 2 (CH2), the heavy chain constant domain 3 (CH3), and the hinge region, excluding
the variable domains of the heavy and light chains, the heavy chain constant domain 1 (CH1),
and the light chain constant domain (CL1) of the immunoglobulin (Ig). Additionally, the Fc
region of the present invention includes not only a native form of an amino acid sequence but
also an amino acid sequence derivative thereof. The amino acid sequence tive means
that one or more amino acid residues of a native form of an amino acid sequence have
different sequences due to deletion, ion, vative or non-conservative substitution,
or a combination thereof. Additionally, the immunoglobulin Fc region may be an Fc region
derived from IgG, IgM, IgE, IgA, IgD, or a combination or hybrid thereof. Additionally, the
immunoglobulin Fc region is preferably derived from an IgG known to improve half-life of a
binding protein, and more preferably derived from an IgG1, but is not limited to its subclass
and can be obtained from any ss of IgG (IgG1, IgG2, IgG3, and IgG4).
The Fc region can genetically produce or obtain a gene encoding the Fc region by
using a recombinant vector or cutting a purified polyclonal antigen or monoclonal antigen
with an appropriate lyase such as papain, pepsin, etc., respectively.
The TNFR-Fc fusion protein can be obtained by introducing an expression vector
ing a polynucleotide encoding the fusion protein into a host cell and expressing the
same. In an exemplary embodiment of the present invention, a pCUCBin-mSig-TNFcept
vector was used as the sion vector including a polynucleotide encoding the TNFR-Fc
fusion protein and was transduced into a CHO cell to express a TNFR-Fc fusion protein.
In the present invention, the rganism can be used to have the same meaning as
the host cell or transformant. A miting example may be an animal cell line, plant, or
yeast host cell. In an exemplary embodiment of the t invention, Chinese Hamster
Ovary cell (CHO cell) was used as the microorganism, but is not limited thereto as long as
the microorganism can be ormed by a polynucleotide encoding the recombinant
glycoprotein.
The polynucleotide, as long as it can be expressed inside the microorganism, can be
inserted into a chromosome and d therein or located outside the chromosome. The
polynucleotide includes RNA and DNA which encode the target protein. A method for
including the polynucleotide is not limited as long as the method is used in the art. As an
example, the polynucleotide can be included inside a microorganism in a form of an
expression te, a gene construct ing all essential elements required for selfexpression.
As another example, a method for modifying by an expression vector including
a sequence of the polynucleotide encoding the target protein operably ted to a suitable
regulation sequence so that the target protein can be expressed in an appropriate host cell can
be used. The regulation ce includes a promoter initiating transcription, a random
operator sequence for regulation of the transcription, a sequence encoding a suitable mRNA
ribosome-binding , and a sequence for regulation of ription and translation.
The vector, after being transformed into a suitable host cell, may be ated or function
irrespective of the host genome, or may be ated into the host genome itself. The vector
used in the present invention may not be specifically limited as long as the vector is
replicable in the host cell, and any vector known in the art may be used.
The glycosylation n of the inant glycoprotein means an expression
pattern of a glycoform, which appears through glycosylation of the glycoprotein. Examples
of the glycosylation pattern include presence of glycosylation which connects a saccharide to
a protein, type of a saccharide, type of glycosylation, content of saccharide, composition of
ccharide (saccharides), ing molar ratio, location of glycoform, structure of
glycoform including sequence, location of glycosylation, glycosylation occupancy, number of
glycoforms, and relative contents according to structure. Difference in biological activity or
in vivo stability may appear according to the glycosylation pattern of the recombinant
glycoprotein.
In the present invention, the n may control ed glycosylation of the
recombinant glycoprotein. In the present invention, the N-linked glycosylation may be used
to have the same meaning as N-glycosylation. As an example, the insulin may reduce the
content of N-glycan of the recombinant glycoprotein. In the present invention, the N-glycan
may be used to have the same meaning as N-glycoform, and may refer to a case in which a
saccharide is connected to asparagine of protein.
In the present invention, the insulin may control O-linked ylation of the
recombinant glycoprotein. In the present invention, the N-linked glycosylation may be used
to have the same meaning as O-glycosylation. As another example, the insulin may reduce
the content of O-glycan of the recombinant glycoprotein. In the present invention, the O-
glycan may be used to have the same meaning as O-glycoform, and may refer to a case in
which a saccharide is connected to serine or threonine of protein.
In the present invention, the insulin may control the N-linked glycosylation and O-
linked glycosylation of the recombinant glycoprotein.
In an exemplary ment of the present invention, the insulin addition ed
to influence the glycosylation pattern of the glycoprotein (Table 2). Specifically, it was
confirmed that the an and/or an content is controlled to be reduced by addition
of the insulin. In ular, among culturing processes of a cell capable of producing
rotein, addition of insulin during the production phase was confirmed to play an
important role in l of the ylation pattern.
The insulin concentration may be 0.0001 mg/L to 1 g/L relative to the total volume of
the culture medium. In an exemplary embodiment of the present invention, it was
confirmed that as the insulin concentration increases, the N-glycan and/or O-glycan content
could be controlled to be reduced (Table 2).
The culture medium is not limited as long as it is used for culturing a microorganism
or host cell including a polynucleotide encoding a glycoprotein in the art. For example, the
culture medium may include an amino acid such as L-glutamine, ine, alanine, ne
monohydrochloride, asparagine monohydrate, aspartic acid, cysteine, glycine, histidine,
cine, leucine, lysine monohydrochloride, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine, (disodium salt, dehydrate), and valine. As another example,
the culture medium may include glucose, sodium bicarbonate, sodium chloride, calcium
de anhydrous, cupric sulfate pentahydrate, ferric nitrate nonahydrate, ferrous sulfate
heptahydrate, potassium chloride, ium sulfate anhydrous, magnesium de
anhydrous, sodium phosphate (monobasic or c, monohydrate), zinc sulfate heptahydrate,
hypoxanthine, putrescine dihydrochloride, sodium pyruvate, biotin, D-calcium pantothenate,
choline chloride, cyanocobalamin, folic acid, i-inositol, nicotinamide, pyridoxal
monohydrochloride, pyridoxine monohydrochloride, riboflavin, thiamine monohydrochloride,
glucose anhydrous, potassium chloride, sodium phosphate O 4·H2O), sodium
hydrogen carbonate (NaHCO3), HEPES (free acid), dextran sulfate, sodium chloride, ascorbic
acid, D-biotin, Hypep 1510, or a combination of two or more. For initial seed e, MTX
may be further included for an increase in expression level.
The culturing may be a perfusion culturing method. The ing may be a
culturing method of perfusing culture fluid around a microorganism. Through the perfusion
culturing method, the insulin concentration can be easily controlled according to a target
glycosylation pattern.
As another aspect, the present invention provides a method for controlling a
glycosylation pattern of a recombinant glycoprotein, comprising (a) ing a
microorganism comprising a polynucleotide encoding a recombinant glycoprotein in a culture
medium to grow the microorganism; and (b) adding insulin in the culture medium and
culturing the same to e a glycoprotein.
As an example, the recombinant glycoprotein may be an immunoglobulin fusion
protein. As another example, the recombinant glycoprotein may be TNFR-Fc fusion protein,
which is described above.
Step (a), which is a growth phase, may further include seed culturing.
The culture medium of step (a) may not include insulin.
Step (b) may be a step of adding insulin at different concentrations according to a
target glycosylation pattern. In an exemplary embodiment of the present invention, it
appeared in the growth phase that the N-glycan and/or O-glycan ts vary according to
addition of insulin. Specifically, it was confirmed that the insulin addition can l N-
glycan and/or O-glycan contents to be reduced. In particular, among culturing processes of
a cell capable of producing glycoprotein, addition of insulin during a production phase was
med to play an important role in control of a ylation pattern.
The insulin concentration may be 0.0001 mg/L to 1 g/L relative to the total volume of
the culture medium. It was confirmed that as the insulin concentration ses, the N-
glycan and/or an content could be controlled to be reduced (Table 2).
The insulin may l N-linked glycosylation and O-linked glycosylation of a
recombinant glycoprotein. As an example, the insulin may reduce the N-glycan content of
the recombinant glycoprotein. As another example, the insulin may reduce the O-glycan
content of the recombinant glycoprotein.
As another aspect, the present invention provides a culture medium composition for
controlling the inant glycoprotein glycosylation pattern. The insulin may be
included in a concentration of 0.0001 mg/L to 1 g/L relative to the total volume of the culture
For example, the culture medium may be used only during the production phase
among microorganism culturing processes.
[Mode for Invention]
Hereinbelow, the t ion will be described in detail with accompanying
exemplary embodiments. r, the exemplary embodiments disclosed herein are only
for illustrative purposes and should not be construed as limiting the scope of the present
invention.
Example 1: Preparing cell line for glycoprotein production
1-1. Preparing vector
Methods commonly used in molecular biology such as treatment of restriction
enzyme, purification of plasmid DNA, conjugation of DNA sections, and transformation of E.
coli were conducted by applying minimum modifications to the methods introduced in
Molecular Cloning (2nd edition) of ok, et al.
A human p75 TNF receptor (TNFR) gene was cloned using a cDNA library which
uses mRNA isolated from a HUVEC cell line as a template, and the cloned gene was fused
with the Fc region of a human IgG1 to obtain a TNFR-IgG1. A pCUCBin-mSig-TNFcept
vector was prepared using a pTOP-BA-RL-pA vector (Korean Patent Publication No. 10-
2012-0059222; comprising , “CB”, and “beta-actin intron”) as a template and the
TNFR-IgG 1.
1-2. Culturing mother cell
CHO/dhfr- (CHO DXB11) was used as a mother cell. CHO/dhfr- is a cell isolated
from CHO cell and is deficient in dihydrofolate reductase (DHFR).
1-3. Transformant and selecting cell line for production
A transformant cell was prepared using CHO/dhfr- (CHO DXB11) and the
pCUCBin-mSig-TNFcept vector including p75 TNF receptor (TNFR) gene, and the gene was
amplified using MTX tration. The cells identified as the transformant cells and
monoclines were chosen as the cell line for production. The cell lines were then inserted
into a glass jar and stored in liquid nitrogen.
Example 2: Culturing cell line for glycoprotein production and harvesting
protein
Different culture media were used according to culturing phase. Insulin was added
to 5.8 g/L of media X011SB (Merck Millipore, Cat. No. 102443) to prepare the basic culture
medium. The culture medium (Media EC-SI) in which 10 g/L of glucose anhydrous (Sigma)
and 0.584 g/L of ine, glycine, and serine (Sigma) were added to the basic e
medium was used for the seed cultivation phase. The culture medium (EC-GM) in which 5
g/l of glucose anhydrous and 0.584 g/L of L-glutamine, glycine, and serine were added to the
basic culture medium for the growth phase. The culture medium (EC-PM) in which 15 g/L
of glucose anhydrous and 0.584 g/L of L-glutamine, glycine, and serine were added to the
basic culture medium was used for the production phase.
The glass jar ning the cell strain prepared in Example 1 was quickly defrosted
in a water tank, and the cells n were moved to a falcon tube containing 10 mL of the
culture medium. The resulting cells were centrifuged, and the first supernatant was
removed. The cells were then resuspended with 10 mL of Media EC-SI and were inoculated
into an Erlenmeyer flask to a final volume of 50 mL. Using a 5 L CelliGen310 cell e
ctor, the cells were cultured to obtain 2 L based on working volume. When the viable
cell number d 2 × 106 cells/mL through five times of seed ing, the culture
medium started to change to EC-GM through the perfusion culturing. As the viable cell
number increased, the exchange rate of the culture medium increased to differentiate the cells
effectively. When the viable cell number d 1.5 × 107 cells/mL (Fig. 2), the culture
medium changed to EC-PM, proceeding from the growth phase to the production phase.
The harvest was conducted a total of four times, and the harvested protein was purified. The
ing value was the average value of the four harvests.
Example 3: Analyzing glycan content
3-1. Analyzing O-glycan content
The en purified in Example 2 was diluted with 25 mM of sodium phosphate
buffer at pH 6.3 to be 100 µL at a concentration of 1.0 mg/mL. 4 µL of N-glycosidase F (1
U/ µL, Roche), 2 µL of neuraminidase (1 U/100 µL), and 2 µL of trypsin (1 mg/mL, Promega)
were added to each specimen and reacted at 37°C for 18 hours. LC-MS analysis was then
conducted.
80 µL of the specimen was inoculated, and then tryptic peptide was ed using
C18 RP (4.6 mm × 250 mm, 5 µm, 300 Å; Vydac, Cat. No. 218TP54). Mobile phase A used
0.1% TFA in water, and mobile phase B used 0.1% TFA in 80% cold CAN. The analysis
was conducted in a gradient condition for 150 minutes. Using a UV detector, a peptide was
detected at 215 nm, and the t separated through LC was connected to a mass
spectrometer (LTQ XL, Thermo) for MS analysis to calculate a relative area (%) of O-
glycopeptide.
3-2. Analyzing N-glycan content
The specimen purified in Example 2 and a reference standard rcept, )
were d with the specimen diluent (25 mM sodium phosphate (pH 6.3 buffer)) to be 3.0
mg/mL. 100 µL of each specimen and 6 µL of N-glycosidase F solution were mixed and
reacted at 37°C for 20 hours. 400 µL of ethanol was added to the solution after the reaction
and was mixed in a vortex. The resulting solution was centrifuged, and the supernatant was
then transferred to an Eppendorf tube and dried completely using a speed-vac trator.
After adding 10 µL of a 2-AA labeling agent to the dried specimen and mixing them, the
mixture was reacted at 45°C and cooled at room temperature.
A GlycoClean S cartridge was put on a disposable e tube, and then distilled
water, 30% acetate, and acetonitrile were perfused sequentially. The cooled specimen was
loaded onto the center of the cartridge membrane and perfused with acetonitrile. In order to
elute N-glycan, distilled water was added to the cartridge for collection in the Eppendorf tube.
The resulting glycan solution was lyophilized and stored until it was analyzed.
The analysis was ted with HPLC column (AsahiPak NH2P-50 4E, 4.6 × 250
mm) in a gradient ion for 130 minutes using 0.5 mM ammonium acetate (pH 6.7) and
250 mM ammonium acetate (pH 5.6) as mobile phases A and B, respectively. A
fluorometric detector was used for detection, and the sum of the area of the peaks per number
of sialic acids present at the terminal of N-glycan was calculated. In a case where there was
no sialic acid, it was marked as l. In cases of one (monosialyl) and two (disialyl), they
were marked as -1 and -2, tively.
Experimental Example 1. Culturing cell strain using culture medium not
comprising insulin added during production phase
The same culture medium as that of the production phase in Example 2, excluding
insulin, was used to culture the cell . N-Glycan contents (%) and relative surface area
ratios (%) of O-glycopeptide per temperature were analyzed and are shown in Table 1 below.
[Table 1]
Culture temperature Harvest N-Glycan-2 charge Relative e area ratio of O-
(production phase) (Nth) (%, avg) glycopeptide (%, avg)
°C H1 12.5 56.13
32°C H1 16.1 54.38
Experimental Example 2. Comparison of changes in glycosylation patterns
ing to insulin addition during the production phase
The ylation patterns were compared in accordance with the insulin addition
and are shown in Table 2 below.
[Table 2]
Culture Insulin concentration Harvest N-Glycan-2 Relative surface area ratio
temperature in e medium (Nth) charge (%, avg) of O-glycopeptide (%,
avg)
°C 0 mg/L H1 12.5 56.13
0.003 mg/L H1 10.4 54.68
0.009 mg/L H1 10.9 52.68
0.03 mg/L H1 9.7 49.5
As a result, it was shown that among culturing processes of a cell capable of
producing glycoprotein, the insulin on during the production phase affected the
glycosylation pattern. In particular, the N-glycan and/or O-glycan content was shown to
change in accordance with the insulin on. Specifically, it was confirmed that the N-
glycan and/or O-glycan content could be controlled to be reduced by the insulin addition.
While the present invention has been described with reference to the particular
illustrative embodiments, it will be understood by those skilled in the art to which the present
invention pertains that the present invention may be embodied in other specific forms without
departing from the technical spirit or essential characteristics of the present invention.
ore, the embodiments described above are ered to be illustrative in all respects
and not restrictive. Furthermore, the scope of the present invention is defined by the
ed claims rather than the ed description, and it should be understood that all
modifications or variations derived from the meanings and scope of the t invention and
lents thereof are included in the scope of the appended claims.
[Industrial Applicability]
As it is capable of changing the N-glycan and/or O-glycan content according to the
insulin addition particularly during the growth phase, the method of controlling ylation
pattern of the recombinant glycoprotein according to the present invention can be very useful
in production of a pharmaceutical recombinant glycoprotein in which uniformity of binding
of saccharide molecules plays an important role.
Claims (15)
- [Claim 1] A method for controlling a glycosylation pattern of a recombinant rotein, comprising culturing a microorganism comprising a polynucleotide encoding the recombinant glycoprotein in a culture medium comprising insulin.
- [Claim 2] The method of claim 1, wherein the recombinant glycoprotein is an globulin fusion protein.
- [Claim 3] The method of claim 1, wherein the recombinant glycoprotein is a TNFR-Fc fusion protein.
- [Claim 4] The method of claim 1, wherein a concentration of the insulin is 0.0001 mg/L to 1 g/L relative to the total volume of the culture medium.
- [Claim 5] The method of claim 1, wherein the n controls ed glycosylation and O- linked glycosylation.
- [Claim 6] The method of claim 1, wherein the insulin reduces a N-glycan content of the recombinant glycoprotein.
- [Claim 7] The method of claim 1, wherein the insulin reduces an O-glycan content of the recombinant glycoprotein.
- [Claim 8] The method of claim 1, n the culturing is a perfusion culturing method.
- [Claim 9] A method for controlling a glycosylation n of a recombinant rotein, comprising: (a) culturing a microorganism comprising a polynucleotide encoding a recombinant glycoprotein in a culture medium to grow the microorganism; and (b) adding insulin in the culture medium and ing the same to produce a glycoprotein.
- [Claim 10] The method of claim 9, wherein step (b) is a step of adding insulin at different concentrations according to a target ylation pattern.
- [Claim 11] The method of claim 10, wherein the insulin reduces a content of N-glycan or O- glycan of the recombinant glycoprotein.
- [Claim 12] The method of claim 9, wherein the recombinant glycoprotein is an immunoglobulin fusion protein.
- [Claim 13] The method of claim 9, wherein the recombinant glycoprotein is a TNFR-Fc fusion protein.
- [Claim 14] The method of claim 9, wherein a concentration of the insulin is 0.0001 mg/L to 1 g/L relative to the total volume of the culture medium.
- [Claim 15] The method of claim 9, wherein the culturing is a perfusion culturing method. ngs] [
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Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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KR10-2014-0195976 | 2014-12-31 |
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