KR102600616B1 - A method for preparing vaccine for COVID-19 - Google Patents

Abstract

The present invention relates to a method for manufacturing a COVID-19 vaccine.

Description

COVID-19 vaccine manufacturing method {A method for preparing vaccine for COVID-19}

The present invention relates to a method for manufacturing a COVID-19 vaccine.

The new coronavirus (2019-nCoV), which occurred in Wuhan, China in 2019, was named COVID-19 in the sense that human infection was first confirmed in the second half of 2019. It is a virus that causes respiratory disease in humans or animals and causes 2 to 3 deaths. It is known that various symptoms appear after an incubation period of up to two weeks. Main symptoms include lethargy, high fever over 37.5 degrees, cough, sore throat, phlegm, muscle pain, headache, difficulty breathing and pneumonia, and respiratory failure due to lung damage. In severe cases, it can lead to death. Similar diseases include MERS and SARS. Although it has a lower fatality rate than MERS or SARS, it is known to be much more contagious. There is an increasing demand from the medical industry for efficient vaccine production to prevent COVID-19 as described above and production of antigen proteins for vaccines.

Accordingly, the present inventors developed a method for producing and purifying proteins used in coronavirus vaccines and completed the present invention.

US 2021-0338807 A1 KR 2021-0117808 A

The object of the present invention is to provide a method for producing multimeric protein assemblies.

Another object of the present invention is to provide a method for producing an immunogenic composition.

Another object of the present invention is to provide a method for producing a vaccine composition for preventing COVID-19, including the step of preparing a multimeric protein assembly.

One aspect of the invention is a method for making multimeric protein assemblies.

In one embodiment, the production method is (a) (i) a fusion process wherein SEQ ID NO: 4 or a protein having at least 75% sequence identity therewith and SEQ ID NO: 5 or a protein having at least 75% sequence identity therewith are linked through a linker. Culturing a cell line expressing protein A; (ii) recovering and purifying the fusion protein A from the culture medium of (i); (b) (i) cultivating a cell line expressing protein B, having SEQ ID NO: 6 or at least 75% sequence identity thereto; (ii) recovering and purifying protein B from the culture medium of (i); and (c) mixing the fusion protein A obtained in (a) and the protein B obtained in (b) to form a self-assembled multimeric protein assembly.

In the manufacturing method according to the preceding embodiment, (a) and (b) are performed simultaneously, at time intervals, sequentially, or in reverse order.

In the production method according to any one of the preceding embodiments, the production method further includes the step of (d) filtering the reactant of (c).

The production method according to any one of the preceding embodiments, wherein (a) (i) includes seed culture and main culture,

In the manufacturing method according to any one of the preceding embodiments, (a) (ii) includes centrifuging the culture medium to recover the supernatant.

The production method according to any one of the preceding embodiments, wherein (a) (ii) includes filtering the recovered culture medium.

The preparation method according to any one of the preceding embodiments, wherein (a) (ii) includes treatment with a nuclease.

In the production method according to any one of the preceding embodiments, (a) (ii) includes performing first chromatography, second chromatography, and third chromatography.

The preparation method according to any one of the preceding embodiments, wherein (a) (ii) includes virus filtration.

The manufacturing method according to any one of the preceding embodiments, wherein (a) (ii) includes ultrafiltration.

In the manufacturing method according to any one of the preceding embodiments, (b) (i) includes seed culture and main culture steps.

In the manufacturing method according to any one of the preceding embodiments, (b) (ii) includes disrupting cells.

The manufacturing method according to any one of the preceding embodiments, wherein (b) (ii) includes treating with a nuclease.

The preparation method according to any one of the preceding embodiments, wherein (b) (ii) includes performing first chromatography and second chromatography.

In the production method according to any one of the preceding embodiments, the filtration in (d) includes ultrafiltration and diafiltration.

In the production method according to any one of the preceding embodiments, the cell line of (a) (i) is derived from Chinese Hamster Ovary (CHO) cells.

In the manufacturing method according to any one of the preceding embodiments, (a) (i) includes adjusting the pH of the culture medium.

In the manufacturing method according to any one of the preceding embodiments, (a) (i) includes maintaining the pH of the culture medium at 6.75 to 7.15.

In the production method according to any one of the preceding embodiments, the content of sodium bicarbonate introduced into the culture medium at the late stage of the culture in (a) (i) is lower than the content of sodium bicarbonate introduced into the culture medium before the culture.

In the production method according to any one of the preceding embodiments, the air sparging rate introduced into the culture medium in the late stage of the culture in (a) (i) is less than half of the air sparging rate before the culture.

In the production method according to any one of the preceding embodiments, the gas injection rate at the late stage of the culture in (a) (i) is 0.0005 to 0.001 vvm.

In the manufacturing method according to any one of the preceding embodiments, the oxygen flow rate to the bioreactor where the culture is performed during the culture period of (a) (i) is maintained at 20 LPM or less.

In the manufacturing method according to any one of the preceding embodiments, the carbon dioxide flow rate to the bioreactor in which the late stage culture of (a) (i) is performed is maintained at 5 LPM or less.

In the production method according to any one of the preceding embodiments, the culture temperature at the latter stage of the culture in (a) (i) is lower than the culture temperature at the beginning of the culture.

In the production method according to any one of the preceding embodiments, the culture temperature at the latter stage of the culture in (a) (i) is 1°C to 10°C lower than the culture temperature at the beginning of the culture.

In the production method according to any one of the preceding embodiments, the culture period of (a) (i) is 6 to 15 days.

In the manufacturing method according to any one of the preceding embodiments, (a) (i) includes adding a culture additive to the culture medium.

In the manufacturing method according to any one of the preceding embodiments, (a) (i) includes periodically monitoring the glucose concentration in the culture medium.

In the production method according to any one of the preceding embodiments, the culture additive of (a) (i) includes a sugar source and a protein expression promoter.

In the production method according to any one of the preceding embodiments, the glucose concentration in the culture medium of (a) (i) is maintained at 5.0 to 7.0 g/L.

In the production method according to any one of the preceding embodiments, (a) (i) includes alternately adding a sugar source and a protein expression promoter to the cell culture medium at intervals of one or more days from the second day of culture or later.

In the production method according to any one of the preceding embodiments, the protein expression promoter included in the culture additive of (a) (i) is a substance that promotes an increase in the yield of protein produced by CHO cells.

In the production method according to any one of the preceding embodiments, the sugar source included in the culture additive of (a) (i) is a glucose solution.

In the production method according to any one of the preceding embodiments, the culture period of (a) (i) is 10 to 15 days.

In the manufacturing method according to any one of the preceding embodiments, (a) (i) includes adding a sugar source to the cell culture medium on the third day of culture.

In the manufacturing method according to any one of the preceding embodiments, (a) (i) includes adding a sugar source to the cell culture medium every other day from the third day of culture until the end of culture.

In the production method according to any one of the preceding embodiments, the sugar source is added to the cell culture medium at least once for 2 or 3 consecutive days from the 3rd day of culture until the end of culture.

In the manufacturing method according to any one of the preceding embodiments, the sugar source is added four or more times during the cultivation period.

In the manufacturing method according to any one of the preceding embodiments, (a) (i) includes adding a protein expression promoter to the cell culture medium on the second day of culture.

In the manufacturing method according to any one of the preceding embodiments, (a) (i) includes adding a protein expression promoter to the cell culture medium every other day from the second day of culture until the end of culture.

In the production method according to any one of the preceding embodiments, the protein expression promoter is added to the cell culture medium at least once at intervals of 2 or 3 days or more until the end of the culture.

In the production method according to any one of the preceding embodiments, the protein expression promoter is added to the cell culture medium four or more times.

In the manufacturing method according to any one of the preceding embodiments, (a) (ii) includes i) centrifuging the cell culture medium to recover the supernatant; and ii) filtering the recovered culture medium of i).

In the manufacturing method according to any one of the preceding embodiments, the step i) centrifuging the cell culture medium and recovering the supernatant is performed using a continuous centrifuge.

In the manufacturing method according to any one of the preceding embodiments, in the step i) centrifuging the cell culture medium to recover the supernatant, the feed flow during centrifugation is 200 L/h to 400 L/h.

In the manufacturing method according to any one of the preceding embodiments, in the step i) centrifuging the cell culture medium to recover the supernatant, the feed flow during centrifugation is 200 L/h to 300 L/h.

In the manufacturing method according to any one of the preceding embodiments, in the step i) centrifuging the cell culture medium to recover the supernatant, the feed flow during centrifugation is about 250 L/h.

In the manufacturing method according to any one of the preceding embodiments, in the step i) centrifuging the cell culture medium to recover the supernatant, the ejection interval during centrifugation is 100 to 250 seconds.

In the manufacturing method according to any one of the preceding embodiments, in the step i) centrifuging the cell culture medium to recover the supernatant, the discharge interval during centrifugation is 190 to 210 seconds.

In the manufacturing method according to any one of the preceding embodiments, the step of filtering the recovered culture medium of ii) i) is performed using a microfiltration filter.

In the manufacturing method according to any one of the preceding embodiments, the microfiltration filter of ii) is a depth filter.

The preparation method according to any one of the preceding embodiments, the method comprising further treating the filtrate of ii) with a surfactant.

In the manufacturing method according to any one of the preceding embodiments, (a) (ii) adjusts one or more factors selected from the group consisting of the concentration of nuclease, treatment time, and concentration of Mg ²⁺ ions in the culture medium. It includes doing.

In the manufacturing method according to any one of the preceding embodiments, (a) (ii) includes the steps of i) recovering a cell line culture medium; ii) filtering the recovered culture medium; iii) treating the filtered culture medium with nuclease and controlling one or more factors selected from the group consisting of nuclease concentration, treatment time, and concentration of Mg ²⁺ ions in the culture medium; and iv) purifying the culture medium.

In the production method according to any one of the preceding embodiments, the nuclease is benzonase.

In the production method according to any one of the preceding embodiments, iii) includes adjusting the concentration of nuclease to 1 unit/ml to 20 unit/ml.

In the production method according to any one of the preceding embodiments, iii) includes adjusting the concentration of nuclease to 2 unit/ml to 14 unit/ml.

In the production method according to any one of the preceding embodiments, iii) includes adjusting the concentration of nuclease to 2 unit/ml to 4 unit/ml.

In the production method according to any one of the preceding embodiments, iii) includes adjusting the concentration of nuclease to about 2 unit/ml.

In the production method according to any one of the preceding embodiments, iii) includes adjusting the nuclease treatment time from 2 hours to 7 days.

In the production method according to any one of the preceding embodiments, iii) includes adjusting the nuclease treatment time to 2 to 6 hours.

In the production method according to any one of the preceding embodiments, iii) includes adjusting the concentration of Mg ²⁺ ions in the culture medium to 2 mM or less.

In the production method according to any one of the preceding embodiments, iii) includes the nuclease concentration of 2 unit/ml to 14 unit/ml, the nuclease treatment time of 2 hours to 7 days, and the culture medium. It includes adjusting the concentration of Mg ²⁺ ions to 2 mM or less.

In the production method according to any one of the preceding embodiments, iii) the concentration of the nuclease is 2 unit/ml to 4 unit/ml; The nuclease treatment time is 4 hours or more, or 2 to 6 hours; And/or it includes adjusting the concentration of Mg ²⁺ ions in the culture medium to 2 mM or less.

The preparation method according to any one of the preceding embodiments, wherein iv) comprises one or more chromatography steps.

The preparation method according to any one of the preceding embodiments, wherein iv) includes first chromatography, second chromatography and third chromatography steps.

In the production method according to any one of the preceding embodiments, the first chromatography step of (a) (ii) is hydrophobic interaction chromatography.

In the production method according to any one of the preceding embodiments, the second chromatography step of (a) (ii) is mixed mode chromatography.

In the production method according to any one of the preceding embodiments, the third chromatography step of (a) (ii) is anion exchange chromatography.

In the manufacturing method according to any one of the preceding embodiments, (a) (ii) includes 'ultrafiltration and diafiltration (buffer exchange: Diafiltration).

In the production method according to any one of the preceding embodiments, the ultrafiltration step of (a) (ii) uses an ultrafiltration membrane filter having a molecular cutoff value of 30 kilodaltons (kDa) to 100 kDa.

In the manufacturing method according to any one of the preceding embodiments, the ultrafiltration step of (a) (ii) uses a conductivity of 25 mSm/cm or less.

In the production method according to any one of the preceding embodiments, a buffer solution of pH 6.0 to 9.0 is used in the ultrafiltration step (a) (ii).

In the production method according to any one of the preceding embodiments, the cell line of (b) (i) is a microorganism of the genus Escherichia.

In the production method according to any one of the preceding embodiments, the cell line of (b) (i) is E. coli.

In the production method according to any one of the preceding embodiments, the culture medium of (b) (i) contains glycerol in a content of more than 0 and less than 4% (v/v).

In the production method according to any one of the preceding embodiments, the microorganism of (b) (i) includes SEQ ID NO: 6 or a nucleic acid sequence encoding a protein having at least 75% identity thereto; or an expression vector containing the same.

In the production method according to any one of the preceding embodiments, the expression vector contained in the microorganism of (b) (i) includes the lac operon.

In the production method according to any one of the preceding embodiments, the expression vector contained in the microorganism of (b) (i) has a cleavage map shown in FIG. 13.

In the manufacturing method according to any one of the preceding embodiments, the culturing step containing glycerol in a content of more than 0 and less than 4% (v/v) in step (b) (i) is performed after the seed fermentation step. do.

In the production method according to any one of the preceding embodiments, the glycerol-containing medium in step (b) (i) is a growth phase medium of E. coli.

In the production method according to any one of the preceding embodiments, the microorganism of (b) (i) is Escherichia coli BL21 strain.

The manufacturing method according to any one of the preceding embodiments, wherein (b) (i) includes adding IPTG to the culture medium.

In the production method according to any one of the preceding embodiments, (b) (i) includes adding IPTG to the culture medium and culturing the cell line in the presence of IPTG exceeding 0 and not exceeding 2.0mM.

In the manufacturing method according to any one of the preceding embodiments, the addition of IPTG occurs when microbial growth enters the late logarithmic phase.

In the manufacturing method according to any one of the preceding embodiments, (b) (i) includes adding IPTG and culturing for 1 hour to 30 hours.

A manufacturing method according to any one of the preceding embodiments, wherein (b) (ii) includes suspending microorganisms into a suspension; and crushing the suspension.

In the production method according to any one of the preceding embodiments, the volume of the suspension is 8 ml to 14 ml per gram of microorganism.

In the manufacturing method according to any one of the preceding embodiments, the crushing pressure is 14000 psi to 21000 psi.

In the manufacturing method according to any one of the preceding embodiments, the microorganisms are pelleted or frozen through centrifugation.

In the manufacturing method according to any one of the preceding embodiments, (b) (ii) includes the step of treating with nuclease.

In the production method according to any one of the preceding embodiments, the nuclease is benzonase.

In the manufacturing method according to any one of the preceding embodiments, the nuclease treatment in (b) (ii) is performed so that the content is 10 units/mL.

In the production method according to any one of the preceding embodiments, the nuclease treatment step of (b) (ii) is carried out for 300 minutes or less after the nuclease treatment.

In the production method according to any one of the preceding embodiments, the nuclease treatment step (b) (ii) is performed under pH 7 to 9 conditions.

In the production method according to any one of the preceding embodiments, (b) (ii) further includes the step of separating the supernatant and the inclusion body after the nuclease treatment step.

In the production method according to any one of the preceding embodiments, the supernatant and inclusion bodies of (b) (ii) above are separated through centrifugation and/or filtration.

In the production method according to any one of the preceding embodiments, the disrupted suspension of (b) (ii) includes an inclusion body form of protein B.

The preparation method according to any one of the preceding embodiments, wherein (b) (ii) includes the step of solubilizing the inclusion body.

The preparation method according to any one of the preceding embodiments, wherein (b) (ii) includes performing i) first anion exchange chromatography and ii) second anion exchange chromatography.

In the production method according to any one of the preceding embodiments, i) first and ii) second anion exchange chromatography of (b) (ii) is an anion exchange filled with a resin having a secondary or tertiary amine as an exchange group. It includes loading into a resin column.

In the manufacturing method according to any one of the preceding embodiments, the anion exchange resin column of i) first and ii) second anion exchange chromatography of (b) (ii) is PEI (Polyethyleneimine), PAB (Para-aminobenzyl) , AE (amino ethyl), DEAE (diethyl aminoethyl), or DMAE (dimethyl aminoethyl) column.

In the production method according to any one of the preceding embodiments, i) first anion exchange chromatography of (b) (ii) is performed in negative mode.

In the production method according to any one of the preceding embodiments, ii) secondary anion exchange chromatography of (b) (ii) is performed in positive mode.

In the preparation method according to any one of the preceding embodiments, the buffer of ii) secondary anion exchange chromatography of (b) (ii) includes detergent.

In the production method according to any one of the preceding embodiments, the detergent content of the buffer solution for ii) secondary anion exchange chromatography of (b) (ii) is 0.01 to 5% (v/v).

In the manufacturing method according to any one of the preceding embodiments, the detergent is Triton X-100.

In the manufacturing method according to any one of the preceding embodiments, the second anion exchange chromatography of (b) (ii) includes first washing the resin using a phosphate buffer solution containing detergent and salt. ; and secondary washing of the resin using a phosphate buffer containing detergent and salt.

In the manufacturing method according to any one of the preceding embodiments, the salt contained in the buffer of the second washing step is contained at a concentration of 150mM or more and less than 250mM.

In the manufacturing method according to any one of the preceding embodiments, the salt contained in the buffer in the second washing step is sodium chloride (NaCl).

In the production method according to any one of the preceding embodiments, the purification of (b) (ii) includes filtering the solution recovered from secondary chromatography.

In the manufacturing method according to any one of the preceding embodiments, (b) (ii) includes ultrafiltration and diafiltration (buffer exchange: Diafiltration).

In the manufacturing method according to any one of the preceding embodiments, the filtration process is carried out in the presence of a buffer containing Tris, sodium chloride, and detergent. In the manufacturing method according to any one of the preceding embodiments, the detergent contained in the buffer of the filtration process is CHAPS.

In the manufacturing method according to any one of the preceding embodiments, the content of detergent contained in the buffer of the filtration process is 0.01% to 5% (v/v).

The production method according to any one of the preceding embodiments, wherein (b) (ii) above is characterized in that no additional chromatography process is performed.

In the production method according to any one of the preceding embodiments, the molar ratio of fusion protein A and protein B in (c) is 1:10 to 2:1.

In the manufacturing method according to any one of the preceding embodiments, the mixing reaction time in (c) exceeds 1 hour.

In the production method according to any one of the preceding embodiments, the mixing reaction of (c) is performed in the presence of a buffer solution containing a stabilizer.

In the production method according to any one of the preceding embodiments, the stabilizer contained in the buffer solution of (c) is polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorb Bate-80, Polysorbate-85, Poloxamer-188, Sorbitan Monolaurate, Sorbitan Monopalmitate, Sorbitan Monostearate, Sorbitan Monooleate, Sorbitan Trilaurate, Sorbitan Tristearate , sorbitan trioleate, arginine, sucrose, or combinations thereof.

In the manufacturing method according to any one of the preceding embodiments, the content of the stabilizer contained in the buffer solution of (c) is greater than 0 and less than 0.050% (v/v).

In the manufacturing method according to any one of the preceding embodiments, the filtration step of (d) includes filtration with a 0.01 μm to 0.5 μm filter.

In the manufacturing method according to any one of the preceding embodiments, the filtration in (d) is performed by ultrafiltration followed by diafiltration.

In the manufacturing method according to any one of the preceding embodiments, the filtration membrane of (d) has a molecular weight cut-off (MWCO) of 100 kDa or more and less than 500 kDa.

In the manufacturing method according to any one of the preceding embodiments, the filtration step of (d) uses tangential flow filtration.

In the production method according to any one of the preceding embodiments, the ultrafiltration in (d) is performed so that the protein concentration is less than 4 mg/mL.

In the production method according to any one of the preceding embodiments, the initial concentration of protein during diafiltration in (d) is less than 4 mg/mL.

In the production method according to any one of the preceding embodiments, the protein load per filtration membrane area of (d) is 300 mg/0.1m ² or more and 750 mg/0.1m ^{2 or} less.

In the production method according to any one of the preceding embodiments, the linker of the fusion protein A includes a Gly-Ser linker.

In the preparation method according to any one of the preceding embodiments, the linker is a (GGGGS)n, (GSSGSS)n, (GSSSSSS)n, (SSSSSS)n, (GSGGSGSGSGGSGSG)n, (GGSGGSGS)n or (GSGGSGSG)n linker. am.

In the manufacturing method according to any one of the preceding embodiments, the linker includes a helical linker EKAAKAEEAAR sequence.

In the production method according to any one of the preceding embodiments, the fusion protein A has SEQ ID NO: 1 or at least 75% sequence identity thereto.

In the method according to any one of the preceding embodiments, the multimeric protein assembly includes a trimer of fusion protein A and a pentamer of protein B.

In the production method according to any one of the preceding embodiments, the multimeric protein assembly is composed of 20 trimers of fusion protein A and 12 pentamers of protein B.

In the production method according to any one of the preceding embodiments, the multimeric protein assembly has an average diameter of 20 nm to 80 nm.

In the production method according to any one of the preceding embodiments, the multimeric protein assembly has an icosahedral structure.

In the production method according to any one of the preceding embodiments, the multimeric protein assembly is used to produce a vaccine composition.

Another aspect of the present invention is a method for producing an immunogenic composition comprising preparing the multimeric protein assembly.

Another aspect of the present invention is an immunogenic composition comprising the multimeric protein assembly prepared above.

Another aspect of the present invention is a vaccine composition comprising the multimeric protein assembly prepared above.

In one embodiment, the vaccine composition is for preventing COVID-19.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered limited by the specific description described below. Additionally, numerous papers and patent documents are referenced and citations are indicated throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the content of the present invention and the level of technical field to which the present invention pertains.

Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Additionally, such equivalents are intended to be encompassed by this invention.

As used herein, the term “about” may appear before a specific numeric value. As used in this application, the term “about” includes not only the exact number written after the term, but also a range that is approximately that number or close to that number. By considering the context in which the number is presented, one can determine whether it is close to or close to the specific number mentioned. As an example, the term “about” may refer to a range of -10% to +10% of a numeric value. As another example, the term “about” may refer to a range of -5% to +5% of a given numeric value. However, it is not limited to this.

As used herein, the term “and/or” should be understood as specifically disclosing each of two specified features or elements, one in conjunction with the other or without the other. Accordingly, as used herein in phrases such as “A and/or B,” the term “and/or” means “A and B,” “A or B,” “A” (alone), and “B” (alone). It is intended to include. Likewise, the term "and/or" used in phrases such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (sole); B (sole); and C (exclusive).

As used herein, the term “consisting essentially of” means that an unspecified ingredient may be present if the characteristics of the claimed subject matter are not substantially affected by the presence of the unspecified ingredient.

As used herein, the term “consisting of” means that the proportion of a particular ingredient(s) totals 100%. Ingredients or features that follow the term “consisting of” may be essential or obligatory. In some embodiments, in addition to the components or features listed below as “consisting of” any other optional or non-essential components may be excluded.

As used herein, the term “comprising” means the presence of a feature, step, or component described below the term, and does not exclude the presence or addition of one or more features, steps, or components. Components or features described herein below as “comprising” may be essential or mandatory, but in some embodiments, they may further include other optional or non-essential components or features.

As used herein, the term “comprising” may, in some embodiments, be modified to refer to “consisting essentially of” or “consisting of.”

With respect to amino acid sequences herein, a polypeptide "comprising" an amino acid sequence set forth in a particular SEQ ID NO, a polypeptide "consisting of" an amino acid sequence set forth in a particular SEQ ID NO, or a polypeptide or protein "having" an amino acid sequence set forth in a particular SEQ ID NO. Even if it is written that, if it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the corresponding sequence number, a protein having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added may also be used in the present application. It is obvious that it can be done. For example, if the amino acid sequence has a sequence added to the N-terminus and/or C-terminus that does not change the function of the protein, a naturally occurring mutation, a silent mutation, or a conservative substitution. However, it is not limited to this.

As used herein, the terms “first, second, third…” “(i), (ii), (iii)…” “i), ii), iii)…” or “(a), (b), Descriptions such as "(c), (d)..." are used to distinguish similar configurations, and these terms are not limited to being performed sequentially or in sequence. For example, when the term is used in connection with steps in a method, use, or analysis, the steps may be performed simultaneously, with no time interval, or may be performed in seconds, minutes, hours, days, or Alternatively, it may be performed sequentially or in reverse order at intervals of several months.

The multimeric protein assembly of the present invention may be a protein assembly disclosed in WO2019-169120, US 9630994 B2, WO2021-163481, WO2021-163438, PCT/US2021/037341. In addition, regarding fusion protein A and protein B, which are components of the multimeric protein assembly of the present invention, the contents disclosed in WO2019-169120, US 9630994 B2, WO2021-163481, WO2021-163438, PCT/US2021/037341 can be referred to. .

“Fusion protein A,” which is a component of the multimeric protein assembly of the present invention, is a protein having SEQ ID NO: 4 or at least 75% sequence identity thereto and SEQ ID NO: 5 or a protein with at least 75% sequence identity thereto linked through a linker. It can be used to prepare multimeric protein assemblies that display antigens or antigen fragments as fusion proteins.

In the above fusion protein, the protein of SEQ ID NO: 4 is a fragment of the SARS-CoV-2 Spike protein and may exhibit immunogenicity. As used herein, the term "immunogenicity" refers to the ability of a specific protein or a specific region thereof to induce an immune response against a specific protein or a protein containing an amino acid sequence with a high degree of identity with the specific protein. do. Therefore, the immunogenic protein that can be included in the multimeric protein assembly is SEQ ID NO: 4 or at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Sequences may have greater than 97%, 98%, or 99% identity.

The protein of SEQ ID NO: 5 of the present invention is linked to an immunogenic protein to form a multimeric protein assembly structure and display an antigen. The protein of SEQ ID NO: 5 may include any modification that does not disturb assembly into a multimeric protein assembly, for example, addition, deletion, insertion, substitution or modification of amino acids. In one embodiment, SEQ ID NO: 5 or at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof It can have sequences with identity. In any one of the above-described embodiments, the protein that is linked to the immunogenic protein to form the structure of a multimeric protein assembly and displays the antigen has the amino acid sequence of SEQ ID NO: 5, 17, 18, 19, 20 or 21. It may comprise, consist essentially of, or consist essentially of the above sequence. Examples of proteins with functions corresponding to the protein of SEQ ID NO: 5 include proteins represented by the amino acid sequences of SEQ ID NOs: 7, 29, 30, 31, and 39 of WO2019-169120.

In any one of the above-described embodiments, the protein of SEQ ID NO: 5 of the present invention includes an amino acid sequence identical to SEQ ID NO: 5 at at least 1, 2, 3, 4, or 5 identified interface positions. Includes. For the above protein, the interface residues identified as being present at the interface to assemble to form a nanostructure may be 25, 29, 33, 54, and 57 from the N-terminus of SEQ ID NO: 5.

The linker may be of any suitable length. For example, the linker may be a peptide linker, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 It may be composed of more than one amino acid. In one embodiment, it may be a Gly-Ser linker containing glycine and/or serine residues. In any one of the preceding embodiments, the linker is a (GGGGS)n, (GSSGSS)n, (GSSSSSS)n, (SSSSSS)n, (GSGGSGSGSGGSGSG)n, (GGSGGSGS)n, or (GSGGSGSG)n linker. It can be. In any one of the preceding embodiments, the linker may include a helical linker sequence. Specifically, the helical linker may be represented by the EKAAKAEEAAR sequence. A specific example of the linker may include, but is not limited to, amino acids 237 to 263 from the N-terminus of SEQ ID NO: 1.

The linkage between the antigen protein and the nanostructure can be achieved by expressing a fusion protein fused to the N- or C-terminus, such as the fusion protein of SEQ ID NO: 1, but is not limited to this, and is not limited to this, and can be achieved by post-translational covalent attachment. It can be achieved by, or can be achieved by non-covalent attachment. Additionally, depending on how the antigen protein is attached, the antigen protein can be displayed in an arbitrary orientation.

In any one of the above-described embodiments, the fusion protein A is SEQ ID NO: 1 or at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, Sequences may have at least 96%, 97%, 98%, or 99% identity. Herein, the protein of SEQ ID NO: 1 may be referred to as “Component A”. A plurality of isolated protein monomers of SEQ ID NO: 1 may self-assemble through interaction to form a trimer. This assembly can be achieved through non-covalent protein-protein interaction reactions.

In the multimeric protein assembly of the present invention, the “protein B” can be used to prepare a multimeric protein assembly displaying an antigen or antigen fragment, which is used in the preparation of an immunogenic composition. Specifically, it may be the protein represented by SEQ ID NO: 6, but those having the same function are included without limitation.

In one embodiment, the protein B is SEQ ID NO: 6 or at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof. Alternatively, it may have a sequence having more than 99% identity. In any one of the preceding embodiments, the protein B comprises or consists essentially of the amino acid sequence of SEQ ID NO: 6, 9, 10, 11, 12, 13, 14, 15 or 16 ( It may consist essentially of) or consist of. Examples of proteins with functions corresponding to the protein of SEQ ID NO: 6 include proteins represented by the amino acid sequences of SEQ ID NOs: 6, 8, 10, 27, 28, 32, 33, and 38 of WO2019-169120.

In any one of the preceding embodiments, the protein B is present at at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 identified interface positions. contains the same amino acid sequence. In the above protein, the interface residues identified as being present at the interface that allow assembly to form a nanostructure are 24, 28, 36, 124, 125, 127, 128, 129, 131 from the N-terminus of SEQ ID NO: 6. It may be 132, 133, 135, and 139. Herein, the protein of SEQ ID NO: 6 may be referred to as “Component B”. A plurality of isolated protein monomers of SEQ ID NO: 6 may self-assemble through interaction to form a pentamer. This assembly can be achieved through non-covalent protein-protein interaction reactions.

Component B and Component A can be self-assembled to form an immunogenic composition, and for example, can be a component of a vaccine against coronavirus.

Fusion proteins A and protein B of the invention may contain any modification that does not perturb assembly into a multimeric protein assembly, for example, additions, deletions, insertions, substitutions or modifications of amino acids. Examples include cases involving conservative sequence modifications.

In the present invention, “conservative sequence modification” may mean an amino acid modification that does not significantly affect or change the structural characteristics of the target protein. These conservative modifications include amino acid substitutions, additions, and deletions. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue that has a similar side chain. These substitutions are generally based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Families of amino acid residues with similar side chains are defined in the art. These families include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), and uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. Examples include amino acids such as tyrosine, phenylalanine, tryptophan, and histidine.

The multimeric protein assembly of the present invention can be formed by self-assembly of a trimer of fusion protein A and a pentamer of protein B. The multimeric protein assembly formed by self-assembly of Component A and Component B can also be referred to as “Nanoparticle,” “nanoparticle,” or “nanostructure.”

Specifically, the multimeric protein assembly of the present invention may have a structure comprising 20 trimers of the fusion protein A and 12 pentamers of the protein B. Accordingly, the multimeric protein assembly of the invention comprises 60 copies of fusion protein A and 60 copies of protein B. Meanwhile, the multimeric protein assembly may be formed through non-covalent interactions between fusion protein A and protein B. In other words, a covalent bond may not be formed between fusion protein A and protein B.

The multimeric protein assembly of the present invention may have an icosahedral symmetrical structure. The multimeric protein assembly of the present invention may have a diameter of about 20 nm to 80 nm, specifically 30 nm to about 70 nm.

In the manufacturing method of the present invention, (a) and (b) may be performed simultaneously, performed at time intervals, sequentially, or in reverse order.

In the manufacturing method of the present invention, (i) and (ii) may be performed simultaneously, at time intervals, or sequentially.

In the production method of the present invention, cell lines expressing fusion protein A and protein B can be manufactured individually and then stored in a cell bank.

In the production method of the present invention, fusion protein A and protein B may be prepared and stored separately and then step (c) may be performed, but is not limited thereto.

The polynucleotide encoding fusion protein A or protein B, which is the target protein provided by the present invention, has the amino acid sequence of the protein due to codon degeneracy or in consideration of the preferred codon in the organism in which the protein is to be expressed. Various modifications can be made to the coding area within the scope of not changing the.

In addition, the polynucleotide of the present invention is hybridized under strict conditions with a probe that can be prepared from a known gene sequence, for example, a complementary sequence for all or part of the base sequence, and encodes the target protein of the present invention. Any sequence may be included without limitation.

The “stringent condition” refers to conditions that enable specific hybridization between polynucleotides. These conditions are described in, e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology. , John Wiley & Sons, Inc., New York).

The polynucleotide encoding the target protein provided by the present invention can be manipulated in various ways to enable expression of the target protein. Depending on the expression vector, it may be necessary to manipulate the polynucleotide prior to inserting it into the vector. Such manipulation may use methods known in the art.

The "vector" of the present invention is a DNA preparation containing the base sequence of a polynucleotide encoding the target protein operably linked to a suitable expression control region (or expression control sequence) so that the target protein can be expressed in a suitable host. it means. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating termination of transcription and translation. After transformation into a suitable host cell, the vector can replicate or function independently of the host genome and can be integrated into the genome itself. Vectors that can be used in the present invention are not particularly limited, and any vector known in the art can be used.

Culture of cells expressing fusion protein A

The host cell of the cell line expressing fusion protein A according to one embodiment of the present invention is a eukaryotic cell. The eukaryotic cells include fungus cells, plant cells, or animal cells. Examples of fungal cells include yeast, for example, Saccharomyces sp., and for example, S. cerevisiae. In addition, examples of animal cells include insect cells or mammalian cells, and specific examples of animal cells include HEK293, 293T, NSO, Chinese hamster ovary cells (CHO), MDCK, U2-OSHela, NIH3T3, MOLT-4, Jurkat, These include PC-12, PC-3, IMR, NT2N, Sk-n-sh, CaSki, C33A, etc. Additionally, suitable cell lines well known in the art can be obtained from cell line depositories such as ATCC (American Type Culture Collection).

In the production method of the present invention, in order to introduce an expression vector having a genetic sequence encoding a target protein into a cell line, any method for introducing nucleic acid into a cell can be used, and depending on the host cell, techniques known in the art may be used. can be used. For example, heat shock, electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran. method, cationic liposome method, and lithium acetate-DMSO method, etc., but are not limited thereto.

The host cell of the cell line expressing fusion protein A according to one embodiment of the present invention may be Chinese hamster ovary cells (CHO), and specifically, Glutamine is absorbed by recombinant Adeno-Associated Virus (rAAV). HD-BIOP3 cells in which exon 6 of the synthetase gene has been knocked out can be used.

Culturing the cell line expressing the fusion protein A may include seed-culturing the cell line and main-culturing the seed-cultured cell line.

The term “seed culture” used in the present invention refers to culture aimed at obtaining cell lines in large quantities. The seed culture can be cultured under temperature conditions that allow the cell number to increase most actively. In other words, the cell line can be cultured to secure a certain number of cells.

The term “production culture” used in the present invention refers to mass culturing a cell line to produce a target protein.

The seed culturing step may be culturing the cell line expressing the fusion protein A by any method selected from the group consisting of subculture, suspension culture, and combinations thereof. Specifically, the cell line expressing the fusion protein A can be cultured by suspension culture and subculture.

The term "suspension culture" used in the present invention refers to a culture method in which cells are suspended in a culture medium, and cells that proliferate in suspension in vivo, such as blood cells or multiple cancer cells, are shaken or rotated. Suspension culture can be carried out even without use, but in most cases, culture can be performed by rotating the gaiter (gaiter culture), shaking each culture bottle (shaking culture), or rotating the incubator (rotating culture).

In one embodiment of the present invention, suspension culture may be performed under conditions of a stirring speed of 120 RPM to 160 RPM, but is not limited to these conditions. The number of cells inoculated in the suspension culture may be 2x10 ⁵ cells/mL to 5x10 ⁵ cells/mL, 3x10 ⁵ cells/mL to 4x10 ⁵ cells/mL, or 4x10 ⁵ cells/mL to 5x10 ⁵ cells/mL. Not limited.

The term “subculture” used in the present invention refers to a method of culturing a cell line by transferring it to the same or different fresh medium according to the culture cycle.

In one embodiment of the present invention, the subculture is performed by transferring the cell line expressing the target protein to the same fresh medium at intervals of 1 to 7 days, 2 to 5 days, 3 to 4 days, or 2 to 3 days. It can be inoculated and cultured. At this time, the number of cells inoculated may be 2x10 ⁵ cells/mL to 5x10 ⁵ cells/mL, 3x10 ⁵ cells/mL to 4x10 ⁵ cells/mL, or 4.5x10 ⁵ cells/mL to 5.5x10 ⁵ cells/mL. It is not limited to conditions. Additionally, the CO ₂ concentration during subculture may be, but is not limited to, 4.0% to 6.0%, 4.5% to 5.5%, or 5.0%.

Specifically, the seed culturing step may involve culturing the cell line expressing the fusion protein A at a temperature of 34°C to 38°C. More specifically, the cell line can be cultured at a temperature of about 36.5°C.

In one embodiment of the present invention, the seed culturing step may be performed by culturing a cell line expressing fusion protein A in a medium known to be suitable for cell growth in the art to which the present invention pertains, but is not limited to these conditions. . The medium may be CDOptiCHO medium.

The main culturing step may be culturing the seed-cultured cell line by any one method selected from the group consisting of batch culture, fed-batch culture, continuous culture, and combinations thereof. Specifically, cell lines expressing fusion protein A can be cultured using fed-batch culture.

In one embodiment of the present invention, the step of main culturing the cell line expressing the fusion protein A may be produced by suspension culture. At this time, the suspension culture is 60 RPM to 100 RPM, 65 RPM to 95 RPM, 70 RPM to 95 RPM, 75 RPM to 95 RPM, 80 RPM to 95 RPM, 85 RPM to 95 RPM, 90 RPM to 95 RPM, and 92 to 95 RPM. It may be carried out under stirring speed conditions of 94 RPM or 93 RPM, but is not limited to these conditions.

In another embodiment of the present invention, when culturing a cell line expressing fusion protein A, the number of cells inoculated is 5x10 ⁵ cells/mL to 10x10 ⁵ cells/mL, 6x10 ⁵ cells/mL to 10x10 ⁵ cells/mL, 7x10 ⁵ cells /mL to 10x10 ⁵ cells/mL, 7x10 ⁵ cells/mL to 9x10 ⁵ cells/mL, or 7.2x10 ⁵ cells/mL to 8.8x10 ⁵ cells/mL, but is not limited thereto.

In another embodiment of the present invention, in the step of culturing a cell line expressing fusion protein A, the pH of the cell line culture medium can be adjusted using a base solution such as carbon dioxide or sodium bicarbonate. Specifically, the pH of the culture medium can be adjusted using carbon dioxide and sodium bicarbonate. More specifically, the pH of the culture medium can be adjusted using carbon dioxide and 8% sodium bicarbonate. In addition, the basic solution introduced to adjust the pH of the culture medium during culture can maintain the pH of the culture medium at an appropriate level by being present in the culture medium in an appropriate ratio with lactic acid secreted by cells in the culture medium. At this time, the pH of the culture medium (deadband error range is 0.05) can be adjusted to 6.8 to 7.5, 6.9 to 7.4, 7.0 to 7.3, 7.0 to 7.4, 6.8 to 7.2, or 6.8 to 7.1. More specifically, the pH of the culture medium can be adjusted to pH 6.8 to 7.1 (deadband, the error range, is 0.05).

In this way, when adjusting the culture medium to maintain pH within a certain numerical range, the lower the upper limit of the pH numerical range (hereinafter, "pH upper value"), the lower the amount of carbon dioxide injected into the culture medium in the latter half of the culture and preventing the formation of bubbles in the culture medium. The effect appears. As the culture progresses, the lactic acid content in the culture medium decreases, causing the pH of the culture medium to rise, and this pH rise causes the injection of a large amount of carbon dioxide through a sparger located at the bottom of the bioreactor. In addition, since the injector serves to introduce carbon dioxide and oxygen into the culture medium, as the pH rises, the oxygen delivery power to the culture medium decreases due to an increase in the carbon dioxide/oxygen ratio in the air introduced into the culture medium. Reduced oxygen delivery to the culture medium reduces the amount of dissolved oxygen (DO) in the culture medium, thereby increasing the frequency of oxygen injection through the injector. However, direct injection of gas into the culture medium can be a factor in increasing the pH of the culture medium.

Meanwhile, during the culture process, when the pH is high, the lactic acid production of cells temporarily increases and the pH decreases. However, as the cells use the produced lactic acid as an energy source, the pH increases significantly again, so the amount of carbon dioxide used in the latter half of the culture is increased. There are aspects where an increase is inevitable. Therefore, when adjusting the pH in the culture medium, if the upper pH value is set low to 7.15 or less, 7.10 or less, 7.05 or less, 7.00 or less, 6.95 or less, 6.90 or less, 6.85 or less, or 6.80 or less, carbon dioxide and It can reduce oxygen usage.

In one embodiment of the present invention, compared to the case where the pH in the culture medium of the present invention is 7.25, if the pH in the culture medium is 6.8 to 7.1 (the deadband, which is the error range, is 0.05), carbon dioxide, oxygen and hydrogen carbonate introduced into the culture medium during the culture period Production costs can be reduced by reducing the amount of basic substances such as sodium.

In one embodiment of the present invention, in addition to maintaining the pH in the culture medium at 6.8 to 7.1 (the deadband, which is the error range, is 0.05), the content of sodium bicarbonate introduced into the late culture medium through a series of processes is adjusted to the pre-culture medium. It can be reduced from the amount of sodium bicarbonate introduced. At this time, the reference point for distinguishing the early and late stages of culture may be the 4th day of culture or the 5th day of culture.

In one embodiment of the present invention, the air sparging rate of the gas (meaning general air constituting the earth's atmosphere) sprayed through the injector during the cultivation period can be changed.

In another embodiment of the present invention, the gas injection rate at the beginning of the culture and the gas injection rate at the end of the culture may be different. Specifically, the gas injection speed at the late stage of culture may be less than half of the gas injection speed at the beginning of culture. More specifically, the gas injection rate after the 4th or 5th day of culture may be less than half of the gas injection rate before the 4th or 5th day of culture.

In another embodiment of the invention, at a later stage of the culture, for example after the fourth or fifth day of culture, the gas injection rate is 0.0005 to 0.01 vvm (volume air/volume media/minute), 0.0005 to 0.009 vvm, 0.0005 to 0.008 vvm. , 0.0005 to 0.007 vvm, 0.0005 to 0.006 vvm, 0.0005 to 0.005 vvm, 0.0005 to 0.004 vvm, 0.0005 to 0.003 vvm, 0.0005 to 0.002 vvm, 0.0005 to 0.0 It may be 01 vvm, or 0.001 vvm.

In one embodiment of the present invention, the DO of the culture medium of the cell line expressing fusion protein A can be maintained within a certain range. At this time, the DO of the cell line culture may be maintained at 10% to 90%, 20% to 80%, 30% to 70%, 40% to 60%, 40% to 50%, or 40%, but is not limited to these conditions. No.

In another embodiment of the present invention, when the DO of the cell line culture medium is located at the lower end of the preferred DO value range of the culture medium, the DO level of the culture medium can be maintained by supplying oxygen to the culture medium.

In one embodiment of the present invention, the oxygen flow rate to the bioreactor during the culture period may be maintained at about 20 LPM or less.

In another embodiment of the invention, later in the culture, for example, after day 4 or day 5 of culture, the carbon dioxide flow rate to the bioreactor may be maintained below about 5 LPM.

In one embodiment of the present invention, a cell line expressing the target protein may be cultured at a temperature of 30°C to 38°C.

In another embodiment of the present invention, the culture temperature at the later stage of culture, for example after the 4th or 5th day of culture, may be lower than the culture temperature at the beginning of the culture, for example before the 4th or 5th day of culture.

In another embodiment of the present invention, a cell line expressing fusion protein A can be cultured at a temperature of 37°C for a certain period of time at the beginning of the culture, and then cultured at a lower temperature. Specifically, the cell line is cultured at a temperature of 37°C until the 4th or 5th day of culture, and after the 5th or 6th day of culture, the temperature is 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C above the above temperature. It can be cultured at temperatures as low as ℃, 8℃, 9℃ or 10℃. More specifically, the cell line can be cultured at a temperature of 37°C from the 4th to 5th day of culture, and then at a temperature of 31°C during the subsequent culture period.

In another embodiment of the present invention, a cell line expressing fusion protein A may be cultured in a medium known to be suitable for cell growth in the art, but is not limited to these conditions. As an example, the medium may be Dynamis medium.

In another embodiment of the present invention, the culture medium may be treated with an antifoaming agent for the purpose of removing bubbles generated. The antifoaming agent can be used by selecting a suitable material considering the components of the culture medium, and is used when bubbles reach the bottom of the gas filter (air filter) in the bioreactor. As an example, the antifoaming agent may be ADCF defoaming agent.

In one embodiment of the present invention, a cell line expressing fusion protein A can be cultured in a flask or bioreactor. Unlike flasks, culture using a bioreactor can control and maintain culture conditions such as DO, glucose content, and pH, so cell culture can be performed under favorable conditions and mass culture is possible.

The types and culture conditions of the flask and bioreactor can be changed within a range that can be normally controlled by a person skilled in the art.

For example, a cell line expressing the target protein is inoculated into an Elenmeyer Flask, cultured in suspension, subcultured to secure the minimum number of cells to be inoculated into the bioreactor, and the subcultured cell line is placed in a disposable cell culture bag. The target protein can be produced by inoculating a bioreactor and performing fed-batch culture.

In another embodiment of the invention, the culture period may be 6 days or more. Specifically, the incubation period is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. You can. More specifically, the culture period may be about 10 days.

As used in the present invention, the term “culture additive” refers to a chemical component or biological substance added for the purpose of promoting cell growth and/or increasing the production of substances expressed by cells during cell culture, for example. Growth factors (e.g. insulin, etc.), iron sources (e.g. transferrin, etc.), polyamines (e.g. putrescine, etc.), minerals (e.g. sodium selenate, etc.), sugar sources (e.g. glucose etc.), organic acids (e.g. pyruvic acid, lactic acid, etc.), amino acids (e.g. L-glutamine, etc.), reducing agents (e.g. 2-mercaptoethanol, etc.), vitamins (e.g. ascorbic acid, d- Biotin, etc.), steroids (e.g. β-estradiol, progesterone, etc.), antibiotics (e.g. streptomycin, penicillin, gentamicin, etc.), buffers (e.g. HEPES, etc.), protein expression promoters (e.g. , brand name Hyclone CellBoost 1, brand name Hyclone CellBoost 2, brand name Hyclone CellBoost 3, brand name Hyclone CellBoost 4, brand name Hyclone CellBoost 5, brand name Hyclone CellBoost 6, brand name Hyclone Cell Boost 7A and Brand name Hyclone Cellboost 7B), etc.

In one embodiment of the present invention, the protein expression promoter and sugar source may be alternately added to the cell culture medium at intervals of one day or more from the second day of culture or later. At this time, the protein expression promoter may be a substance that promotes an increase in the yield of the protein produced by CHO cells, and the substance that promotes an increase in the yield of the protein produced by the CHO cell is a substance produced by CHO cells in the technical field to which the present invention pertains. Substances known to promote increased yield of proteins include CHO CD Efficient FeedA (Invitrogen), CHO CD Efficient FeedB (Invitrogen), CHO CD Efficient FeedC (Invitrogen), Sheff-CHO PLUS PF ACF (FM012) ) (Kerry), CHO CD Efficient Feed A+ (Invitrogen), CHO CD Efficient Feed B+ (Invitrogen), CHO CD Efficient Feed C+ (Invitrogen), FAA01A (Hyclone, Hyclone), brand name Hyclone Cellboost 1, Includes brand name Hyclone CellBoost 2, brand name Hyclone CellBoost 3, brand name Hyclone CellBoost 4, brand name Hyclone CellBoost 5, brand name Hyclone CellBoost 6, brand name Hyclone CellBoost 7A and brand name Hyclone CellBoost 7B. However, it is not limited to this. Specifically, the protein expression promoter may be the brand name Hyclone Cell Boost 7A and the brand name Hyclone Cell Boost 7B. At this time, the brand name Hyclone Cellboost 7A is a composition with a pH close to neutral containing amino acids, vitamins, salts and glucose, and the brand name Hyclone Cellboost 7B is an amino acid concentrate and is alkaline. In addition, when the brand name Hyclone CellBoost 7A is used by adding it to the culture medium, the volume used may be less than 20% of the total volume of the culture medium at the start of the culture or during the culture process, and the brand name Hyclone CellBoost 7B can be used by adding it to the culture medium. In this case, the volume used may be less than 2% of the total volume of the culture medium at the start of the culture or during the culture process. Furthermore, the substance that promotes increased yield of protein produced by the CHO cells may be a mixture of Hyclone Cell Boost 7A and Hyclone Cell Boost 7B at a volume ratio of 10:1. The mixture of Hyclone CellBoost may be obtained by dissolving Hyclone CellBoost 7A and Hyclone CellBoost 7B in a solvent and mixing them.

Additionally, the sugar source is a substance that contains glucose but does not inhibit cell proliferation. Meanwhile, a D-glucose solution may be used as the sugar source, but is not limited thereto.

In one embodiment of the present invention, the protein expression promoter may be used for the purpose of supplying components necessary for or assisting cell growth and/or expression of a target protein.

In another embodiment of the present invention, the sugar concentration of the sugar source that can be added to the cell line culture medium during the culture period is 1% (w/v) or more, or 1 g/L to 500 g/L, 100 g/L. to 500 g/L, 200 g/L to 500 g/L, 300 g/L to 500 g/L, or 400 g/L to 500 g/L, or may have a concentration of 450 g/L. However, it is not limited to this. Additionally, the sugar source may be a glucose solution or a D-glucose solution at a concentration of 45% (w/v), but is not limited thereto.

In another embodiment of the present invention, the glucose concentration in the cell culture medium can be maintained at 5.0 to 7.0 g/L during the culture period.

In another embodiment of the present invention, the sugar source and the protein expression promoter may be alternately added to the cell culture medium at intervals of one day or more from the second day of culture or later.

In one embodiment of the present invention, the sugar source may be added to the cell culture medium on the third day of culture.

In another embodiment of the present invention, the sugar source may be added to the cell culture medium every other day from the third day of culture until the end of culture.

In one embodiment of the present invention, the protein expression promoter and the sugar source are alternately added to the cell culture medium, and the sugar source may be added to the cell culture medium at least once for two consecutive days from the third day of culture until the end of culture.

In another embodiment of the present invention, the protein expression promoter and the sugar source are alternately added to the cell culture medium, and the sugar source may be added to the cell culture medium at least once for 3 consecutive days from the 3rd day of culture until the end of the culture.

In one embodiment of the present invention, the sugar source may be added four or more times during the culturing period. Additionally, the sugar source may be added 4 to 10 times during the culturing period, but is not limited thereto.

In another embodiment of the present invention, a protein expression promoter may be added to the cell culture medium on the second day of culture.

In one embodiment of the present invention, a protein expression promoter can be added to the cell culture medium every other day from the second day of culture until the end of culture.

In another embodiment of the present invention, the protein expression promoter may be added to the cell culture medium at least once at intervals of 2 days or more until the end of the culture.

In another embodiment of the present invention, the protein expression promoter may be added to the cell culture medium at least once at intervals of 2 or 3 days until the end of the culture.

In another embodiment of the present invention, the protein expression promoter may be administered four or more times during the culture period. In another embodiment of the present invention, the protein expression promoter may be administered 4 or 5 times during the culture period, but is not limited thereto.

Recovery and purification of fusion protein A

In one embodiment of the present invention, the step of recovering the supernatant by centrifuging the cell culture medium of (a) (ii) may use a centrifuge commonly used in the technical field to which the present invention pertains. For example, a centrifuge using a fixed angle angle rotor, a centrifuge using a swing rotor, a continuous centrifuge, etc. can be used. Specifically, the steps (a) (ii) of centrifuging the cell culture medium and recovering the supernatant may be performed using a continuous centrifuge.

In another embodiment of the present invention, in the step of recovering the supernatant by centrifuging the cell culture medium of (a) (ii), the feed flow, which is the feed rate at which the culture medium is supplied to the centrifuge, is 200 L/ h to 400 L/h, 200 L/h to 300 L/h, 210 L/h to 300 L/h, 220 L/h to 300 L/h, 230 L/h to 300 L/h, or 240 L /h to 300 L/h. Specifically, the supply flow rate may be about 250 L/h, but is not limited thereto.

In another embodiment of the present invention, in the step of centrifuging the cell culture medium of (a) (ii) and recovering the supernatant, the ejection interval during centrifugation is 100 to 250 seconds, 120 to 240 seconds, It may be 120 to 230 seconds, 140 to 230 seconds, 160 to 220 seconds, 180 to 210 seconds, or 190 to 210 seconds. Additionally, specifically, the discharge interval during centrifugation may be 190 to 210 seconds, for example, about 201 seconds, but is not limited to this value.

In another embodiment of the present invention, the step (a) (ii) of filtering the recovered culture medium may be performed using a microfiltration filter. The function of the microfiltration filter can be evaluated based on, for example, pressure limit, feed load of <20 liters (l)/m2, or turbidity reduction. Additionally, non-limiting examples of the microfiltration filter include microfiltration membrane, Millipore, COHC, A1HC, BIHC, XOHC depth filter, CUNO 60ZA, and CUNO 90ZA. Specifically, the microfiltration filter may be a depth filter, and more specifically, it may be an A1HC depth filter, but is not limited to these filters.

In one embodiment of the present invention, the culture medium recovered by centrifugation may be filtered using a filter to remove impurities. At this time, the pore diameter of the filter used for filtration may be 1 ㎛ or less, 0.5 ㎛ or less, 0.45 ㎛ or less, 0.3 ㎛ or less, 0.25 ㎛ or less, 0.22 ㎛ or less, or 0.2 ㎛ or less, and filters with various pore diameters may be combined. You can also use it. Additionally, the conductivity during filtration may be 10 to 20 mS/cm, 10 to 19 mS/cm, 10 to 18 mS/cm, 10 to 17 mS/cm, or 10 to 16 mS/cm, but is limited to these conditions. It doesn't work.

In another embodiment of the present invention, the culture solution recovered by centrifugation may be filtered using a filter and then further treated with a surfactant for reaction. The surfactants include sodium dodecyl sulfate (SDS), sodium deoxycholate, Triton , CHAPS, Chapso, digitonin, urea, and mixtures thereof, but are not limited to these ingredients. Specifically, the surfactant may be Triton X-100. In addition, the time for treating and reacting the surfactant may be 10 minutes or more, 20 minutes or more, or 30 minutes or more, and/or may be 40 minutes or less, 50 minutes or less, or 60 minutes or less. However, it is not limited to these conditions.

In one embodiment of the present invention, the filter filtrate treated with the surfactant may be refiltered. The pore diameter of the filter used for re-filtration may be 1 ㎛ or less, 0.5 ㎛ or less, 0.45 ㎛ or less, 0.3 ㎛ or less, 0.25 ㎛ or less, 0.22 ㎛ or less, or 0.2 ㎛ or less, and filters with various pore diameters may be combined. You can also use it. A sterility test, mycoplasma detection test, adventitious agent identification test through cell culture inoculation method, Mouse Minute virus detection test, and Mycobacterium tuberculosis detection test can be performed on the recovered culture medium. There are no bacteria in the recovered culture, and no mycoplasma, adventitious agents, micromouse virus, or tuberculosis bacillus are detected.

In one embodiment of the present invention, iii) the filtered culture medium is treated with nuclease, and at least one factor selected from the group consisting of nuclease concentration, treatment time, and concentration of Mg ²⁺ ions in the culture medium is added. In the regulating step, any enzyme that cleaves nucleic acids can be used without limitation, for example, a DNA cleavage enzyme can be used.

Nucleases of the present invention include exonuclease and endonuclease. Exonuclease is an enzyme that produces mononucleotides by sequentially decomposing 3' and 5'-phosphodiester bonds at one end of a polypeptide chain. Endonuclease is an enzyme that produces oligoribonucleotides by cleaving the 3', 5'-phosphodiester bond inside the polynucleotide chain, and depending on the substrate specificity, it is divided into two types: deoxyribonuclease I (DNase I) and deoxyribonuclease. II (DNase II), etc. It also includes restriction enzymes that cleave DNA with a specific base sequence, and also includes nucleases that degrade both DNA and RNA, such as the nuclease of a coccus called micrococcus. The nuclease may be benzonase.

In one embodiment of the present invention, in step (a)(ii)iii), the concentration of nuclease is 1 unit/ml to 20 unit/ml, 2 unit/ml to 14 unit/ml, 2 unit/ml It can be adjusted to 4 units/ml, or about 2 units/ml, but is not limited to these values.

In another embodiment of the present invention, in step (a)(ii)iii), the nuclease treatment time is 2 hours to 7 days, 2 hours to 5 days, 2 hours to 3 days, or 2 hours to 24 days. The time can be adjusted from 2 hours to 12 hours, 2 hours to 6 hours, 3 hours to 6 hours, or 4 hours to 6 hours, but is not limited to these values.

In another embodiment of the present invention, in step (a)(ii)iii), the concentration of Mg ²⁺ ions in the culture medium may be adjusted to 3 mM or less, or 2 mM or less, but is not limited to these values. No.

In one embodiment of the present invention, the culture solution purification step (a)(ii) may be performed using a general protein purification method. The step of purifying the culture medium may include one or more chromatography steps. Specifically, the step of purifying the culture solution may include first chromatography, second chromatography, and third chromatography steps.

The chromatography step used in the step of purifying the culture medium of (a) (ii) above functions as a protein capture step, for example, affinity chromatography, cation exchange chromatography, and anion exchange chromatography (AEX). , mixed mode chromatography, hydrophobic interaction chromatography or hydrophobic charge induction chromatography, chromatography using a membrane or bead-type resin, etc. may be used, and may be selected considering the characteristics of the protein to be purified.

In one embodiment of the present invention, the first chromatography in the step of purifying the culture medium in (a)(ii) may be Hydrophobic Interaction Chromatography (HIC).

In another embodiment of the present invention, the secondary chromatography in the step of purifying the culture solution in (a)(ii) may be mixed mode chromatography.

The mixed mode chromatography involves the use of solid phase chromatography supports in the form of resins, monoliths or membranes that use multiple chemical mechanisms to adsorb proteins or other solutes. Useful examples of the invention include, but are not limited to, chromatographic supports that utilize a combination of two or more of the following mechanisms: anion exchange, cation exchange, hydrophobic interaction, hydrophilic interaction, thiophilic interaction, hydrogen Bonding, pi-pi bonding and metal affinity.

In one embodiment of the invention, the mixed-mode chromatography process includes (1) anion exchange and hydrophobic interaction techniques; (2) cation exchange and hydrophobic interaction techniques; and/or (3) combine electrostatic and hydrophobic interaction techniques.

In one embodiment of the invention, the mixed-mode chromatography step can be accomplished by using one or more of the following systems: Capto® MMC (available from GE Healthcare Life Sciences), HEA HyperCel™ (available from GE Healthcare Life Sciences), (available from Pall Corporation), PPA HyperCel™ (available from Pall Corporation), MBI HyperCel™ (available from Pall Corporation), MEP HyperCel™ (available from Pall Corporation), Blue Trisacryl M (available from Pall Corporation) available), CFT™ Ceramic Fluoroapatite (available from Bio-Rad Laboratories, Inc.), CHT™ Ceramic Hydroxyapatite (available from Bio-Rad Laboratories, Inc.) and/or ABx (available from J.T. Baker). Specifically, the second chromatography in the purification step (a)(ii) may be Ceramic Hydroxyapatite chromatography.

In one embodiment of the present invention, the purification step (a)(ii) may include ultrafiltration and diafiltration for the purpose of sample concentration and buffer solution filtration after performing primary chromatography and virus inactivation. The filtration may use a tangential flow filtration system.

The term "tangential flow filtration" in the present invention, also referred to as "TFF", is a method of filtration also defined as "cross-flow filtration". This TFF mode filtration can be useful for concentrating and/or purifying biological materials, such as proteins and vaccines. In TFF mode filtration, fluid is pumped tangentially along the surface of the membrane. The applied pressure serves to force a portion of the sample to move through the membrane to the filtrate (permeate) side. Biological material and particulates (retentates) that are too large to pass through the membrane pores are retained on the upstream side. However, unlike normal filtration mode (Normal Flow Filtration, NFF), retained substances do not accumulate on the surface of the membrane. Instead, the retained material is swept along the surface of the membrane by the tangential flow of fluid. That is, in TFF mode filtration, the feed stream is parallel to the membrane plane, a portion passes through the membrane, and the remainder may be retained and recycled to the feed reservoir.

In another embodiment of the present invention, the third chromatography in the purification step (a)(ii) may be cation exchange chromatography or anion exchange chromatography.

In one embodiment of the present invention, the first chromatography may use a buffer containing 20mM sodium phosphate and 3M NaCl for column equilibration (EQ). Specifically, the buffer solution may have a pH of 6.4 to 6.6 and a conductivity of 170 to 220 mS/cm, but is not limited to these values.

In another embodiment of the present invention, the first chromatography may use a buffer solution containing 20mM sodium phosphate and 0.6M NaCl to elute the protein, and more specifically, the buffer solution has a concentration of 6.4 to 6.6. It may have a pH and a conductivity of 50 to 70 mS/cm, but is not limited to these values.

In another embodiment of the present invention, the first chromatography may use a buffer solution containing 20mM sodium phosphate to remove the remaining protein after eluting the target protein. More specifically, the buffer solution has a concentration of 6.4 to 6.6. It may have a pH of and a conductivity of 1.8 to 2.4 mS/cm, but is not limited to these values.

In one embodiment of the present invention, the secondary chromatography may use a buffer solution containing 2mM sodium phosphate and 50mM NaCl for column equilibration. More specifically, the buffer solution has a pH of 6.7 to 6.9 and a pH of 5 to 7. It may have a conductivity of mS/cm, but is not limited to this value.

In another embodiment of the present invention, the secondary chromatography may use a buffer solution containing 25mM sodium phosphate and 50mM NaCl in the wash process, and more specifically, the buffer solution has a pH of 6.7 to 6.9 and a pH of 7.5. It may have a conductivity of from 8.5 mS/cm, but is not limited to this value.

In another embodiment of the present invention, the secondary chromatography may use a buffer solution containing 240mM sodium phosphate and 50mM NaCl to elute the protein. More specifically, the buffer solution has a concentration of 8.1 to 8.3. It may have a pH and a conductivity of 28 to 32 mS/cm, but is not limited to these values.

In another embodiment of the present invention, the secondary chromatography may be performed by removing the remaining protein after eluting the target protein. The process is called strip, and a buffer solution containing 400mM sodium phosphate and 50mM NaCl can be used in the strip process. More specifically, the buffer solution has a pH of 8.1 to 8.3 and a conductivity of 38 to 44 mS/cm. may have, but is not limited to these numbers.

In one embodiment of the present invention, the third chromatography may use a buffer solution containing 2mM sodium phosphate for column equilibration. More specifically, the buffer solution has a pH of 8.1 to 8.3 and 0.3 to 0.5 mS/cm. It may have a conductivity of, but is not limited to this value.

In another embodiment of the present invention, the third chromatography may use a buffer solution containing 20mM sodium phosphate and 3M NaCl in a strip process to remove the remaining protein after passing the target protein, and more specifically, the buffer solution containing 20mM sodium phosphate and 3M NaCl. The solution may have a pH of 6.4 to 6.6 and a conductivity of 180 to 200 mS/cm, but is not limited to these values.

In one embodiment of the present invention, the purification step (a)(ii) may include the step of inactivating the virus. The virus inactivation step can be performed by adjusting the pH of the eluate after the chromatography process to acidic. Specifically, the pH of the eluate can be lowered to 4.0 or less, and more specifically, it can be lowered to pH 3.3 to 3.7, but is not limited to these values. Additionally, an acid such as HCl can be used to adjust the pH.

In one embodiment of the present invention, the purification step (a)(ii) may include one or more filtration steps. Specifically, the purification step (a)(ii) may include an ultrafiltration step and a virus filtration step. More preferably, the purification step (a)(ii) may include a first ultrafiltration step and a second ultrafiltration step. More specifically, the first ultrafiltration step may be performed after the first chromatography and virus inactivation step, and the second ultrafiltration step may be performed after the third chromatography and virus filtration step.

As used herein, the term "ultrafiltration" refers to any technique of applying a solution or suspension to a membrane (e.g., a semipermeable membrane) to separate a product (e.g., a protein) from other substances in the solution or suspension. . For example, ultrafiltration membranes retain molecules larger than the pores of the membrane, while smaller molecules such as salts, solvents, and water freely pass through the membrane. The solution retained by the membrane is referred to as the “concentrate” or “retentate”, while the solution passing through the membrane is referred to as the “filtrate” or “permeate.”

The ultrafiltration membrane filter of step (a) (ii) of the present invention may have a molecular cutoff value of 30 kilodaltons (kDa) to 100 kDa, or greater. Additionally, the ultrafiltration membrane filter of the present invention may have a molecular cutoff value of 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, or 100 kDa, or any intermediate value. In some embodiments, the molecular weight cutoff of the ultrafiltration membrane may be 100 kDa, but is not limited to this value.

The conductivity used during ultrafiltration in steps (a) (ii) of the present invention is 25 mSm/cm or less, 10 mSm/cm or less, 9 mSm/cm or less, 8 mSm/cm or less, 7 mSm/cm or less, 6 mSm. /cm or less, or 5 mSm/cm or less, but is not limited to these values. The pH of the buffer solution used during ultrafiltration may be pH 6.0 to 9.0, 6.0 to 7.0, 7.0 to 8.0, or 7.6 to 8.4, but is not limited to these values.

In the virus filtration step, viruses may be filtered using filters known in the art to be capable of filtering viruses, such as depth filters and virus filters.

In one embodiment of the present invention, the filtrate that has undergone virus filtration and ultrafiltration may be filtered through an additional filtration step. The filter used in the additional filtration step is a filter known in the art to be suitable for protein filtration, and may have a pore diameter of 0.3 μm or less.

Cell culture expressing protein B

The cell line expressing protein B of the present invention may be a microorganism, for example, a microorganism of the genus Escherichia. Specifically, the cell line expressing protein B may be E. coli. The E. coli may be, for example, E. coli BL21, JM109, K-12, LE392, RR1, DH5α or W3110 strain. Specifically, the E. coli may be an E.coli BL21 strain.

In one embodiment of the present invention, the E. coli may contain a polynucleotide encoding protein B of the present invention, or a vector containing the same.

The vector is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pBR, pUC, and pBluescriptII series can be used as plasmid vectors. , pGEM-based, pTZ-based, pCL-based, pET-based, etc. can be used. Specifically, pET28, pET29, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used. For example, a polynucleotide encoding the target protein of the present invention can be inserted into a chromosome through a vector for intracellular chromosome insertion. Insertion of the polynucleotide into the chromosome may be accomplished by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker may be additionally included to confirm whether the chromosome has been inserted. A selection marker is used to select cells transformed with a vector, that is, to confirm the insertion of a target nucleic acid molecule, and to impart selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides. Markers that do so may be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other expression traits, so transformed cells can be selected.

In any one of the above-described embodiments, the expression vector may include the lac operon. In the present invention, “operon” is a functional unit of DNA containing a group of genes whose expression is regulated by one expression control sequence, and lac operon refers to an operon in which transcription occurs in the presence of lactose or an analog thereof. .

In any one of the above-described embodiments, the lac operator in the expression vector of the present invention may be operably linked to the gene encoding protein B. As used herein, “operably linked” means a configuration in which the regulatory sequence is placed at an appropriate position so that the regulatory sequence directs the expression of the coding sequence. Therefore, when the lac operator in the expression vector of the present invention and the gene encoding protein B are operably linked, the expression of protein B of the present invention can be induced in the presence of lactose or its analogue.

As an example, the expression vector may include an antibiotic resistance gene, Lac operon, T7 promoter region, and multiple cloning site. More specifically, the expression vector may have the cleavage map of Figure 13, but is not limited thereto.

In one embodiment of the present invention, the step of culturing in the glycerol-containing medium of (b)(i) may be performed after the seed fermentation step.

In one embodiment of the above-described embodiments, the glycerol-containing medium may be a growth phase medium of E. coli. The growth phase of microorganisms is also called the exponential phase (log phase). The number of cells can be increased through culturing in a glycerol-containing medium, and cell growth can be increased. Accordingly, the expression level of the target protein can be increased.

In any one of the above-described embodiments, the glycerol may be included in the culture medium in an amount greater than 0 and less than 4% (v/v). The glycerol content is, for example, greater than 0 and less than about 4% (v/v), less than 3.9% (v/v), less than 3.9% (v/v), less than 3.8% (v/v), 3.8% Less than (v/v), less than 3.7% (v/v), less than 3.7% (v/v), less than 3.6% (v/v), less than 3.6% (v/v), 3.5% (v/v) or less, less than 3.5% (v/v), less than 3.4% (v/v), less than 3.4% (v/v), less than 3.3% (v/v), less than 3.3% (v/v), 3.2% ( v/v) or less, less than 3.2% (v/v), less than 3.1% (v/v), less than 3.1% (v/v), less than 3.0% (v/v), less than 3.0% (v/v) , 2.9% (v/v) or less, less than 2.9% (v/v), 2.8% (v/v) or less, less than 2.8% (v/v), 2.7% (v/v) or less, 2.7% (v) /v), less than 2.6% (v/v), less than 2.6% (v/v), less than 2.5% (v/v), less than 2.5% (v/v), less than 2.4% (v/v), Less than 2.4% (v/v), less than 2.3% (v/v), less than 2.3% (v/v), less than 2.2% (v/v), less than 2.2% (v/v), 2.1% (v/ v) or less, or less than 2.1% (v/v), but is not limited thereto. For example, when glycerol is included in the growth medium, cell growth increases and protein expression levels also increase compared to medium without glycerol, allowing mass production of the target protein.

In any one of the above-described embodiments, step (b)(i) may further include adding an expression inducer to the medium after culturing in a medium containing glycerol. The expression inducer may be isopropyl β-D-1-thiogalactopyranoside (IPTG). IPTG has a structure similar to lactose and can act as an inducer of the lac operon, causing transcription of the operon.

In any one of the above-described embodiments, the addition of the expression inducer may be performed when the growth of E. coli enters the late logarithmic phase.

In any one of the above-described embodiments, the addition of the expression inducer may be made at or before or after the absorbance (OD600) value of the culture medium of the strain is about 20 to 40, but is not limited thereto. When the absorbance (OD600) value of the culture medium of the above strain reaches about 20 to 40, it can be determined that the growth of E. coli has entered the late logarithmic phase.

OD600 refers to the optical density at a wavelength of 600 nm and can be used to measure the concentration of the strain. For example, in the case of E. coli , absorbance 1.0 at OD600 is converted to approximately 1X10 ⁹ CFU/mL, so the absolute amount of the strain can be confirmed by measuring the OD600 value.

The expression inducer may be decomposed by microorganisms or may not be used. Therefore, the amount of the expression inducer added may be the same as the content in the medium. In any one of the above-described embodiments, the content of the expression inducer in the medium is greater than 0mM and less than about 5.0mM, specifically less than 4.5mM, less than 4.0mM, less than 3.9mM, less than 3.8mM, less than 3.7mM. , 3.6mM or less, 3.5mM or less, 3.0mM or less, 2.9mM or less, 2.8mM or less, 2.7mM or less, 2.6mM or less, 2.5mM or less, 2.4mM or less, 2.3mM or less, 2.2mM or less, 2.1mM or less, or It may be 2.0mM or less. For example, it may be about 1.9mM or less, 1.8mM or less, 1.7mM or less, 1.6mM or less, 1.5mM or less, 1.4mM or less, 1.3mM or less, 1.2mM or less, 1.1mM or less, and 1.0mM or less.

In any one of the above-described embodiments, the time for culturing the microorganism after addition of the expression inducer may be about 1 hour to 30 hours, for example, about 2 hours to 25 hours, 2 hours to 20 hours. , 2 hours to 16 hours, 2 hours to 15 hours, 3 hours to 25 hours, 3 hours to 20 hours, 3 hours to 16 hours, 3 hours to 15 hours, 4 hours to 25 hours, 4 hours to 20 hours, 4 hours to 16 hours, 5 hours to 25 hours, 5 hours to 20 hours, 5 hours to 16 hours, 5 hours to 15 hours, 4 hours to 15 hours, 4 hours to 14 hours, 4 hours to 13 hours, 4 hours to It may be 12 hours, 4 hours to 11 hours, or 4 hours to 10 hours. As an example, it may be about 10 hours, but is not limited thereto.

Recovery and purification of protein B

In one embodiment of the present invention, the protein B can be recovered from a cell line. The cell line may be a strain.

The recovery method may include the step of crushing the strain. Cell lysis methods include the use of alkali, surfactants, organic solvents or enzymes (lysozyme, etc.), sonication or high pressure grinder (French Press), and periodic high temperature, pressure, freezing or thawing cycles. It can be performed by applying.

In any one of the preceding embodiments, the strain may be in an inactivated form. The term inactivated form includes inactivated strains that are unable to form single colonies on plates specific to the strain. As an example, the strain may be in pelleted or frozen form. The inactivated strain can be grown again when applied to an appropriate medium under conditions known in the art. As an example, the strain of the present invention can be easily stored and handled in an inactivated form while still containing protein B. However, it is not necessarily limited to this.

In any one of the above-described embodiments, the suspension in (b)(ii) may be suspending cells recovered through centrifugation.

In any one of the above-described embodiments, in the crushing step, the volume of the suspension may be about 4 ml to 18 ml, 5 ml to 15 ml, 6 ml to 15 ml, 7 ml to 14 ml, or 8 ml to 14 ml per gram of strain, For example, about 8ml to 13ml, 8ml to 12.5ml, 8ml to 12ml, 8ml to 11.5ml, 8ml to 11ml, 8ml to 10.5ml, 8.5ml to 13ml, 8.5ml to 12.5ml, 8.5ml to 12ml, 8.5ml to 11.5ml. ml, 8.5ml to 11ml, 9ml to 13ml, 9ml to 12.5ml, 9ml to 12ml, 9ml to 11.5ml, or about 9ml to 11ml. As an example, it may be about 10ml.

In any one of the above-described embodiments, in the step of disrupting the strain, the cell lysis method may include the use of an alkali, a surfactant, an organic solvent, or an enzyme (lysozyme, etc.), sonication, or high pressure. This can be accomplished through the use of a French press, applying periodic high temperature, pressure, freezing or thawing cycles. In any one of the above-described embodiments, the crushing step may be crushing by pressure. The pressure is about 14000 psi to 21000 psi, 14000 psi to 20000 psi, 14000 psi to 19000 psi, 14000 to 18000 psi, 15000 psi to 21000 psi, 15000 psi to 20000 psi, 15000 psi to 19 000 psi, 15000 psi to 18000 psi , 15500 psi to 18000 psi, 15000 to 17500 psi, 16000 psi to 18000 psi, 16000 psi to 17500 psi, 16500 psi to 18000 psi, or about 16500 psi to 17500 psi. In any one of the above-described embodiments, the number of disruptions may be appropriately repeated at a level that can obtain the target protein at a desired yield. For example, it may be performed more than once, for example, 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4 times, 3 times, or 2 times, but is not limited thereto.

In any one of the above-described embodiments, impurities may be removed after crushing the strain. For example, it can be treated with nuclease, etc.

In the recovery and purification of proteins, nucleic acids contained in cell cultures, cell lysates, etc. are recognized as impurities, so the impurities can be removed by treatment with nuclease. The nuclease may be an endonuclease, for example, Benzonase (Benzonasae: Serratia marcescens endonuclease), Omnileave (Epicentre), or DNase I.

In any one of the above-described embodiments, the strain may be treated with nuclease after the step of disrupting the strain. The nuclease may be an endonuclease, for example, Benzonase (Benzonasae: Serratia marcescens endonuclease), Omnileave (Epicentre), or DNase I.

In any one of the above-described embodiments, the nuclease may be benzonase. By adding the benzonase, the final content of benzonase is greater than 0 and less than or equal to about 10 unit/mL, specifically about 0.5 to 10 unit/mL, specifically 0.5 to 9 Unit/mL, 0.5 to 8 Unit/mL, 0.5 to 7 Unit/mL, 0.5 to 6 Unit/mL, 1 to 10 Unit/mL, 1 to 9 Unit/mL, 1 to 8 Unit/mL, 1 to 7 Unit/mL, 1 to 6 Unit/mL, 2 to 10 Unit/mL, 2 to 9 Unit/mL, 2 to 8 Unit/mL, 2 to 7 Unit/mL, 2 to 6 Unit/mL, 3 to 10 Unit/mL, 3 to 9 Unit/mL, 3 to 8 Unit/mL, 3 to 7 Unit/mL, 3 to 6 Unit/mL, 4 to 10 Unit/mL, 4 to 9 Unit/mL, 4 to 8 Unit/mL, 4 to 7 Unit/mL, or about 4 It may be from 6 Unit/mL. As an example, it may be about 5 Unit/mL.

In any one of the above-described embodiments, the nuclease treatment step may be performed in an environment with a pH of about 7 to 9.

In any one of the above-described embodiments, the reaction time after the nuclease treatment may be about 300 minutes or less, specifically about 60 minutes to 300 minutes, 90 minutes to 270 minutes, or 120 minutes to 240 minutes. And, as an example, it may be about 180 minutes.

In any one of the above-described embodiments, step (b)(ii) may further include a process for removing impurities, and for example, methods such as centrifugation, filtration, and dialysis may be used. You can.

In any one of the above-described embodiments, the disrupted suspension may include an inclusion body form of the target protein. In any one of the above-described embodiments, the target protein may be recovered from the microorganism in the form of an inclusion body.

After transcription, proteins form tertiary structures through a process called protein folding, and during this process, the unique characteristics and functions of the protein appear. During this process, if protein folding does not occur properly, the proteins clump together. An inclusion body refers to an aggregate state in which polypeptides are gathered together at high density due to misfolding. The inclusion body form has the advantage of high purity of the target protein and ease of cell destruction and purification by centrifugation. Protein solubilization and refolding are the process of releasing the inclusion body form and returning it to its normal protein form.

In any one of the above-described embodiments, the purification method may further include a step of solubilizing the inclusion body. A solubilization buffer may be used to return the target protein recovered in the form of an inclusion body to its normal protein form.

The “solubilization buffer” of the present invention is a buffer for resolving polypeptide aggregation in proteins expressed in the form of inclusion bodies.

In one embodiment, the solubilization buffer may include Na-P, NaCl, Urea, and Triton .

In any one of the above-described embodiments, the purification method (b)(ii) may further include the step of separating the supernatant and the inclusion body after the nuclease treatment step. The separation method may use any method known in the art, and is not particularly limited, but may include centrifugation or filtration, for example. The filtration may be carried out by conventionally known processes such as microfiltration, ultrafiltration, diafiltration, microfiltration, and depth filtration, and may be microfiltration as an example. The filtration may include passing the recovered liquid through a filter having a pore size of about 0.01 μm to 0.5 μm, for example about 0.1 μm to 0.3 μm, specifically about 0.2 μm. However, it is not limited to this.

In any one of the above-described embodiments, the protein recovered from the supernatant after the nuclease treatment step may exist in a liquid soluble form rather than in the form of aggregates. However, it is not limited to this.

In one embodiment of the present invention, the purification step (b)(ii) is characterized by performing first anion exchange chromatography and second anion exchange chromatography.

In the present invention, "anion exchange chromatography (AEX)" is capable of separating molecules according to their charges by binding negatively charged (or acidic) molecules to a positively charged support, Congeners of molecules (acidic, basic and neutral) can be easily separated by this technique.

“Anion exchange resin” or “anion exchange membrane” used in anion exchange chromatography refers to a solid phase that is positively charged and therefore has one or more positively charged ligands attached thereto. Any positively charged ligand that attaches to a solid phase suitable for forming an anion exchange resin can be used.

Examples of commercially available anion exchange resins include Poros HQ, Poros XQ, Q Sepharose Fast Flow, DEAE Sepharose Fast Flow, SARTOBIND® Q, ANX Sepharose 4 Fast Flow (High Sub), Q Sepharose XL, Q Sepharose Big Bead, DEAE Sepharose A-25, DEAE Sephadex A-50, QAE Sepharose A-25, QAE Sephadex A-50, Q Sepharose High Performance, Q Sepharose XL, Source 15Q, Source 30Q, Resource Q, Kapto Q, Kapto DEAE, Mono Q, Toyo Pearl Super Q, Toyo Pearl DEAE, Toyo Pearl QAE, Toyo Pearl Q, Toyo Pearl Gigacap Q, TS Gel Super Q, TS Gel DEAE, Fructogel EMD TMAE, Fructogel EMD TMAE HiCap, Fructogel EMD DEAE, Fructogel EMD DMAE, MacroPrep High Q, Macro-Prep-DEAE, Unosphere Q, Nubia Q, PORGS PI, DEAE Ceramic HyperD, Includes Q Ceramic Hyper D. Other examples include DEAE Cellulose, Poros® PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 (Applied Biosystems), Satobind® Q (Satorius), MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX Sepharose® Fast Flow, Q Sepharose® High Performance, QAE SEPHADEX® and FAST Q Sepharose® (GE Healthcare), WP PEI, WP DEAM, WP QUAT (J. T. Baker), Hydrocell DEAE and Hydrocell QA (Biochrome Labs Inc.), Unosphere Q, Macro-Prep® DEAE and Macro-Prep® High Q (BioRad), Ceramic HyperD Q, Ceramic HyperD DEAE, Trisacryl® M and LS DEAE, Spherodex LS DEAE, QMA SPHEROSIL® LS, QMA SPHEROSIL® M and MUSTANG® Q (Pall Technologies), DowX® Fine Mesh Strong Base Type I and Type II Anion Resin and DowX® Monosphere 77 Weak Base Anion (Dow Liquid Separations), INTERCEPT® Q Membrane, Matrex Cellufine® A200, A500, Q500 and Q800 (Millipore), Fructogel. ® EMD TMAE, Fructogel® EMD DEAE and Fructogel® EMD DMAE (EMD), Amberlite® Weak and Strong Anion Exchangers Types I and II, Dowex® Weak and Strong Anion Exchangers Types I and II, Die Aion® weak and strong anion exchangers types I and II, DUOLITE® (Sigma-Aldrich), TSK Gel Q and DEAE 5PW and 5PW-HR, Toyopearl® SuperQ-650S, 650M and 650C, QAE- 550C and 650S, DEAE-650M and 650C (Tosoh), QA52, DE23, DE32, DE51, DE52, DE53, Express-Ion D or Express-Ion Q (Whatman), Sartobind® Q (Satorius, New York, USA) Corporation), but is not limited to this.

As an example, in the purification step (b)(ii) of the present invention, the first anion exchange chromatography may use a weak anion exchanger.

In any one of the above-described embodiments, the weak anion exchange resin may have a weak anion exchange group. For example, an anion exchange resin filled with a primary, secondary or tertiary amine, specifically a resin having a secondary or tertiary amine as an exchange group, can be used. Positively charged forms of the above amines are also included.

In any one of the above-described embodiments, the first anion exchange chromatography is performed using a polyethyleneimine (PEI), para-aminobenzyl (PAB), amino ethyl (AE), diethyl aminoethyl (DEAE), or dimethyl aminoethyl (DMAE) column. It may be, and specifically, it may be a DEAE column. An example may be DEAE Sepharose Fast Flow (FF), but is not limited thereto.

In one embodiment, the secondary anion exchange chromatography in the purification step (b)(ii) of the present invention may use a weak anion exchanger.

In any one of the above-described embodiments, the weak anion exchange resin may have a weak anion exchange group. For example, an anion exchange resin filled with a primary, secondary or tertiary amine, specifically a resin having a secondary or tertiary amine as an exchange group, can be used. Positively charged forms of the above amines are also included.

In any one of the above-described embodiments, the secondary anion exchange chromatography is performed using a polyethyleneimine (PEI), para-aminobenzyl (PAB), amino ethyl (AE), diethyl aminoethyl (DEAE), or dimethyl aminoethyl (DMAE) column. It may be, and specifically, it may be a DEAE column. An example may be DEAE Sepharose Fast Flow (FF), but is not limited thereto.

In the purification step (b)(ii) of the present invention, the “loading” step loads the column by allowing the fluid (feed) containing the target protein to flow through the inlet, so that the feed is an adsorbent or resin. It involves a step of allowing a specific substance to bind by contact with it. Unbound substances and other molecules flow through the outlet with the fluid.

In one embodiment, the purification step of (b)(ii) of the present invention includes the steps of washing the column, eluting the target material, regenerating the adsorbent, and/or equilibrating the column before and after the loading step. It can be included.

As used herein, “buffer” refers to a substance that, by virtue of its presence in solution, increases the amount of acid or alkali that must be added to cause a unit change in pH. Buffered solutions resist changes in pH by the action of acid-base conjugate components. A buffered solution can maintain a constant concentration of hydrogen ions such that the pH of the solution is within the physiological range. Buffer components may include, but are not limited to, organic and inorganic salts, acids, and bases.

For example, phosphate buffer, citric acid buffer, or acetic acid buffer can be used as the column buffer. Specifically, the column buffer may be a phosphate buffer, for example, sodium phosphate, but is not limited thereto. As an example, the column buffer may contain salt. For example, the column buffer may include, but is not limited to, salts such as NaCl, KCl, NaOAc, (NH ₄ ) ₂ SO ₄ , Na ₂ SO ₄ and CH ₃ COONa.

In any one of the above-described embodiments, the first anion exchange chromatography may include the step of regenerating the column using a buffer solution.

In any one of the above-described embodiments, the buffer used in the column regeneration step of the first anion exchange chromatography may be a phosphate buffer containing a salt. In any one of the above-described embodiments, the buffer used in the column regeneration step of the first anion exchange chromatography may include sodium phosphate and sodium chloride. In any one of the above-described embodiments, the concentration of sodium phosphate may be about 100mM to 300mM. In any one of the above-described embodiments, the concentration of sodium chloride may be about 400mM to 600mM. However, it is not limited to this.

In any one of the above-described embodiments, the first anion exchange chromatography may include the step of equilibrating the column using a buffer solution.

In any one of the above-described embodiments, the buffer used in the column equilibration step of the first anion exchange chromatography may be a phosphate buffer. In any one of the above-described embodiments, the buffer used in the column equilibration step of the first anion exchange chromatography may include sodium phosphate and sodium chloride. In any one of the above-described embodiments, the concentration of sodium phosphate may be about 1 to 100mM, specifically about 2 to 50mM, or about 5 to 35mM, but is not limited thereto. In any one of the preceding embodiments, the concentration of sodium chloride may be about 100mM to 600mM, for example about 100mM to 500mM, 100mM to 450mM, 100mM to 400mM, 150mM to 500mM, 150mM to 450mM, 150mM It may be from 400mM to 400mM, 200mM to 500mM, 200mM to 450mM, 200mM to 400mM, 250mM to 500mM, 250mM to 450mM, 250mM to 400mM, or 300mM to 400mM. For example, it may be about 350mM. However, it is not limited to this.

The regeneration and equilibration steps may be performed prior to the loading step.

In any one of the above-described embodiments, the first anion exchange chromatography may include loading a solution containing the target protein. The target protein in the solution may be included in the form of an inclusion body, but is not limited thereto. The flow rate of the loading step may be selected appropriately, for example, may be at least about 50 cm/hr, 60 cm/hr, 70 cm/hr or more, but is not limited thereto. The amount of solution containing the loaded target protein can be appropriately adjusted. For example, the amount of sample loaded into a unit resin may be about 1 to 20 v/v, but is not limited thereto.

In one embodiment of the above-described embodiments, the first anion exchange chromatography may include performing in negative mode.

In the present invention, the term "negative chromatography" is also called "negative chromatography" or "perfusion chromatography" and refers to a chromatography method in which the target protein does not attach to the column, but impurities do attach to the column. By performing the negative mode chromatography, the target protein can be separated from impurities by allowing the target protein to exist in the flow-through. On the contrary, “positive chromatography” refers to a chromatography method in which the target protein binds to the column and impurities do not bind. After performing positive chromatography, the target protein can be obtained by eluting the target protein bound to the column.

By including the step of performing the first anion exchange chromatography in negative mode, the content of impurities can be reduced.

In any one of the above-described embodiments, the target protein of the present invention may be included in the permeate of the first anion exchange chromatography.

In any one of the above-described embodiments, the purification step (b)(ii) of the present invention involves loading the permeate of the first anion exchange chromatography back into the first anion exchange column and circulating it. Additional steps may be included. The circulation can be performed until the impurity content is lowered to a desired level. For example, the circulation volume may be about 2 times or more, 3 times or more, 4 times or more, 5 times or more, and for example, about 10 times the resin volume, but is not limited thereto.

In any one of the above-described embodiments, the purification step of (b)(ii) of the present invention may include filtering the solution obtained in the first anion exchange chromatography step. The filtration step may include passing the permeate through a filter having a pore size of about 0.01 μm to 0.5 μm, for example, about 0.1 μm to 0.3 μm, specifically about 0.2 μm. However, it is not limited to this.

In any one of the above-described embodiments, the solution obtained through the first chromatography may be loaded on a second anion exchange column.

In any one of the above-described embodiments, the secondary anion exchange chromatography may be performed in positive mode.

In any one of the above-described embodiments, the buffer solution for the secondary anion exchange chromatography may include detergent. The buffer solution includes a buffer solution for an equilibration step, a washing step, and/or an elution step.

Examples of detergents included in the buffer for the secondary anion exchange chromatography include Triton , polyoxyethylene sorbitan monooleate, Brij, NP40, CHAPS, CHAPSO or octaglucoside, and specifically selected from the group consisting of Triton X-100, Tween 80, and Tween 20. You can use any detergent that works. As an example, Triton X-100 can be used. The content of the detergent may be about 0.01% to 1%, or 0.5% to 0.05%, for example, about 0.1% (v/v).

In any one of the above-described embodiments, the secondary anion exchange chromatography may include the step of regenerating the column using a buffer solution.

In any one of the above-described embodiments, the buffer used in the column regeneration step of the secondary anion exchange chromatography may be a phosphate buffer containing a salt. In any one of the above-described embodiments, the buffer used in the column regeneration step of the secondary anion exchange chromatography may include sodium phosphate and sodium chloride. In any one of the above-described embodiments, the concentration of sodium phosphate may be about 100 to 300mM. In any one of the above-described embodiments, the concentration of sodium chloride may be about 400mM to 600mM. However, it is not limited to this.

In any one of the above-described embodiments, the secondary anion exchange chromatography may include the step of equilibrating the column using a buffer solution.

In any one of the above-described embodiments, the buffer used in the column equilibration step of the secondary anion exchange chromatography may be a phosphate buffer. In any one of the above-described embodiments, the buffer used in the column equilibration step of the secondary anion exchange chromatography may include sodium phosphate and sodium chloride. In any one of the above-described embodiments, the concentration of sodium phosphate may be about 1 to 100mM, specifically about 2 to 50mM, or 5 to 35mM, but is not limited thereto. In any one of the above-described embodiments, the concentration of sodium chloride may be about 500mM or less, for example, about 400mM or less, about 300mM or less, or about 250mM or less, for example, about 50mM to 400mM, about 50mM. to 300mM, or about 50mM to 250mM. As an example, it may be about 150mM. However, it is not limited to this.

The regeneration and equilibration steps may be performed before loading the solution containing the target protein obtained in the first chromatography onto the second chromatography column.

In any one of the above-described embodiments, in the purification step (b)(ii) of the present invention, the secondary anion exchange chromatography is performed using a phosphate buffer solution containing a detergent and a salt to prepare the resin as 1. washing the car; And it may include secondary washing of the resin using a phosphate buffer containing detergent and salt.

The washing step may be performed after loading the solution obtained through primary chromatography and discarding the passed fraction.

In any one of the above-described embodiments, the buffer solution in the first and second washing steps may be a phosphate buffer solution, specifically sodium phosphate. In any one of the above-described embodiments, the concentration of sodium phosphate may be about 1 to 100mM, specifically about 2 to 50mM and 5 to 35mM, but is not limited thereto.

In any one of the above-described embodiments, the buffer solution in the first and second washing steps may include sodium chloride. In any one of the above-described embodiments, the concentration of sodium chloride in the first washing step buffer may be about 100mM to 200mM.

In any one of the above-described embodiments, the concentration of sodium chloride in the secondary washing step buffer may be less than about 250mM, specifically about 10mM or more and less than 250mM, 25mM or more and less than 250mM, 50mM or more and less than 250mM, or 75mM or more and 250mM. It may be less than, 100mM or more and less than 250mM, or about 150mM or more and less than 250mM.

The secondary chromatography of the present invention may include recovering the eluate by adding a buffer solution after the washing step.

The elution buffer may be a phosphate buffer, specifically sodium phosphate. The buffer solution may include sodium chloride. The concentration of the sodium phosphate may be about 1 to 100mM, specifically about 2 to 50mM, or 5 to 35mM, but is not limited thereto. The concentration of sodium chloride may be about 30mM to 500mM. For example, it may be less than 500mM.

In any one of the above-described embodiments, the purification step of (b)(ii) of the present invention may include filtering the recovery solution of the secondary anion exchange chromatography. The filtration step may include passing the recovered liquid through a filter having a pore size of about 0.01 μm to 0.5 μm, for example, about 0.1 μm to 0.3 μm, specifically about 0.2 μm. However, it is not limited to this.

In the purification step (b)(ii) of the present invention, the conductivity, flow rate, or flow rate may be appropriately adjusted in the step of performing the chromatography described above.

In one specific example, the purification step (b)(ii) of the present invention may further include a filtration step after performing chromatography.

The filtration may be performed using conventionally known processes such as microfiltration, ultrafiltration, diafiltration, microfiltration, and deep filtration.

In any one of the above-described embodiments, the purification step of (b)(ii) of the present invention may include ultrafiltration and diafiltration processes after the chromatography step.

“Ultrafiltration” in the present invention refers to a membrane-based separation process that separates molecules in solution based on size. Ultrafiltration processes are known in the art and are commonly used in protein purification processes (e.g., Zeman et al., Microfiltration and Ultrafiltration: Principles and Applications, Marcel Dekker, Inc., pp. 299-301 (1996) ). By ultrafiltration, solvents and small solute molecules can pass through while macromolecules can be retained, and thus it can be used to purify and concentrate fine molecules such as proteins.

“Diafiltration” of the present invention is a type of ultrafiltration in which an aqueous buffer is added to the filtration retentate and may also be referred to as buffer exchange in the ultrafiltration process. The diafiltration is a method of removing, replacing, or lowering the concentration of salts or solvents from solutions containing proteins, peptides, nucleic acids, and other biomolecules using an ultrafiltration membrane.

In any one of the above-described embodiments, the buffer used in the filtration step is a salt selected from TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, Cacodylate, and MES; and salts selected from NaCl, KCl, NaOAc, (NH4)2SO4, Na2SO4 and CH3COONa; and Triton , CHAPS, CHAPSO, and octaglucoside. In any one of the above-described embodiments, the buffer solution may include Tris and sodium chloride. In any one of the above-described embodiments, the detergent contained in the buffer solution may be CHAPS. CHAPS, also known as 3-cholamidopropyl dimethylammonio 1-propanesulfonate, is a non-denaturing zwitterionic detergent.

In any one of the above-described embodiments, the Tris may be included at a concentration of about 10mM to 100mM, specifically about 20mM to 80mM, 30mM to 70mM, or 40mM to 60mM, and for example, may be included at a concentration of about 50mM. there is.

In any one of the above-described embodiments, the sodium chloride may be included at a concentration of about 100 to 1000mM, specifically about 200mM to 800mM, 300mM to 700mM, or 400mM to 600mM, and for example, may be included at a concentration of about 500mM. there is.

In any one of the preceding embodiments, the CHAPS is about 0.01% to 5%, specifically 0.01% to 3%, 0.01% to 2%, 0.01% to 1%, 0.01% to 1.5%, 0.05%. to 3%, 0.05% to 2%, 0.05% to 1.5%, 0.05% to 1%, 0.1% to 3%, 0.1% to 2%, 0.1% to 1.5%, 0.1% to 1%, 0.5% to 3 %, 0.5% to 2%, 0.5% to 1.5%, or 0.5% to 1% (v/v). For example, it may be included at a concentration of about 0.75%.

Assembly and purification of fusion protein A and protein B

In one embodiment, the method for producing a multimeric protein assembly of the present invention may include mixing the fusion protein A and protein B at a molar ratio of 1:10 to 2:1.

For example, in the mixing step (c), the molar ratio of fusion protein A and protein B is about 1:9, 1:8, 1:7, 1:6, 1:5.5, 1:5, 1:4.5, 1 :4, 1:3.5, 1:3, 1:2.5, 1:2.4, 1:2.3, 1:2.2, 1:2.1, 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6 , 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, 1:1, 1.05:1, 1.1:1, 1.15:1, 1.2:1, 1.25:1 or approximately 1.3:1 day. You can. In another aspect, the molar ratio of fusion protein A and protein B may be about 1:0.9 to 1.1, or 1:1.0 to 1.1.

The time for performing the mixing reaction is not particularly limited, but may be performed for more than 1 hour. For example, it may be performed for about 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, or about 4 hours. For example, from about 0.5 hours to 4 hours, from 0.5 hours to 3.5 hours, from about 1 hour to 3.5 hours, from 1.5 hours to 3 hours, from 1.5 hours to 2.5 hours, from 1.8 hours to 2.2 hours, or 1.9 hours. It can be performed for a period of time of 2.1 hours or less.

In any one of the above-described embodiments, the mixing step may be performed in the presence of a buffer solution containing a stabilizer.

The stabilizers include polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitan monolaurate, selected from sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, arginine, sucrose, or combinations thereof. You can. As an example, it may be polysorbate-80.

The content of the stabilizer may be greater than 0 and less than 0.050 v/v%. Specifically, about 0.001% or more and less than 0.050%, 0.005% or more and less than 0.050%, 0.01% or more and less than 0.050%, 0.001% or more and 0.045% or less, 0.005% or more and 0.045% or less, 0.01% or more and 0.045% or less, 0.001% or more and 0.040%. % or less, 0.005% or more 0.040% or less, 0.01% or more 0.040% or less, 0.001% or more 0.030% or less, 0.005% or more 0.030% or less, 0.01% or more 0.030% or less, 0.001% or more 0.035% or less, 0.005% or more 0.035% or less, 0.01% or more and 0.035% or less, 0.01% or more and 0.025% or less, 0.015% or more but less than 0.050%, 0.02% or more but less than 0.050%, 0.015% or more and 0.045% or less, 0.02% or more and 0.045% or less, 0.015% or more and 0.040% or less. , 0.02% or more and 0.040% or less, 0.015% or more and 0.030% or less, 0.02% or more and 0.030% or less, 0.015% or more and 0.035% or less, 0.02% or more and 0.035% or less, or about 0.02% or more and 0.03% or less. In one aspect, the content of the stabilizer may be about 0.01% or more and 0.025% or less.

In any one of the above-described embodiments, the step (d) of filtering the composition containing the multimeric protein assembly of the present invention may be further included after the mixing step (c). The filtration step (d) may include passing the permeate through a filter having a pore size of about 0.01 μm to 0.5 μm, for example, about 0.1 μm to 0.3 μm, specifically about 0.2 μm. However, it is not limited to this.

In one embodiment, the purification step (d) of the present invention may include performing ultrafiltration and/or diafiltration.

When performing diafiltration after performing ultrafiltration, the protein may be concentrated to a specific concentration by ultrafiltration and then diafiltration may be performed. At this time, the concentration of the protein concentrated by ultrafiltration may be the same as the concentration of the protein at the beginning of diafiltration.

In any one of the preceding embodiments, the initial concentration of protein in the diafiltration is less than about 10 mg/mL, such as less than about 9 mg/mL, less than 8 mg/mL, less than 7 mg/mL, less than 6 mg/mL, It may be less than 5 mg/mL, or less than about 4 mg/mL.

In any one of the above-described embodiments, the molecular weight cut-off (MWCO) of the filtration membrane used in the filtration step may be less than about 500 kDa. For example, approximately 10 kDa to less than 500 kDa, 20 kDa to less than 500 kDa, 30 kDa to less than 500 kDa, 40 kDa to less than 500 kDa, 50 kDa to less than 500 kDa, 60 kDa to less than 500 kDa, 70 kDa to less than 500 kDa, 80 kDa to less than 500 kDa, and more than 90 kDa. Less than 500 kDa, or about It may be more than 100 kDa and less than 500 kDa. For example, it may be about 200 kDa or more and 400 kDa or less, for example, about 300 kDa, but is not limited thereto.

In one embodiment of the above-mentioned embodiments, the filtration may be performed using a tangential flow filtration method. This is the same as described above.

In any one of the above-described embodiments, the protein load per filtration membrane area in the filtration step may be about 10 mg/0.1 m ² or more and 1250 mg/0.1 m ² or less. For example, about 20 mg/0.1m ² or more and 1200 mg/0.1m ² or less, about 30 mg/0.1m ² or more and 1200 mg/0.1m ² or less, about 40 mg/0.1m ² or more and 1200 mg/0.1m ² or less, about 50 mg /0.1m ² or more 1150 mg/0.1m ² or less, about 60 mg/0.1m ² or more 1150 mg/0.1m ² or less, about 70 mg/0.1m ² or more 1150 mg/0.1m ² or less, about 80 mg/0.1m ² or more 1100 mg/0.1m ² or less, about 90 mg/0.1m ² or more 1100 mg/0.1m ² or less, about 100 mg/0.1m ² or more 1100 mg/0.1m ² or less, about 110 mg/0.1m ² or more 1050 mg/0.1m ² or less, about 120 mg/0.1m ² or more 1050 mg/0.1m ² or less, about 130 mg/0.1m ² or more 1050 mg/0.1m ² or less, about 140 mg/0.1m ² or more 1000 mg/0.1m ² or less, about 150 mg /0.1m ² or more 1000 mg/0.1m ² or less, approximately 160 mg/0.1m ² or more 1000 mg/0.1m ² or less, approximately 170 mg/0.1m ² or more 950 mg/0.1m ² or less, approximately 180 mg/0.1m ² or more 950 mg/0.1m ² or less, approximately 190 mg/0.1m ² or more 950 mg/0.1m ² or less, approximately 200 mg/0.1m ² or more 900 mg/0.1m ² or less, approximately 210 mg/0.1m ² or more 900 mg/0.1m ² or less, about 220 mg/0.1m ² or more 900 mg/0.1m ² or less, about 230 mg/0.1m ² or more 900 mg/0.1m ² or less, about 240 mg/0.1m ² or more 850 mg/0.1m ² or less, about 250 mg /0.1m ² or more 850 mg/0.1m ² or less, approximately 260 mg/0.1m ² or more 850 mg/0.1m ² or less, approximately 270 mg/0.1m ² or more 800 mg/0.1m ² or less, approximately 280 mg/0.1m ² or more It may be 800 mg/0.1m ² or less, about 290 mg/0.1m ² or more and 800 mg/0.1m ² or less, or about 300 mg/0.1m ^{2 or} more and 750 mg/0.1m ² or less.

Immunogenic compositions and vaccine compositions

The multimeric protein assembly containing the target protein of the present invention can be used as a component of an immunogenic composition. Accordingly, another aspect of the present invention provides an immunogenic composition comprising the multimeric protein assembly.

The immunogenic composition can be used to generate antibodies in mammals.

The immunogenic composition of the invention may comprise an immunologically effective amount of a multimeric protein assembly. The term “immunologically effective amount” refers to an amount that is effective in eliciting an antibody response to an antigen when administered to an individual. The immunologically effective amount will depend on the health and physical condition of the individual to be treated, the age of the individual, the capacity of the individual's immune system to synthesize antibodies, the level of protection required, the formulation of the composition, the treating physician's assessment of the medical situation, and other relevant factors. It may vary depending on. The immunologically effective amount of the immunological composition of the invention can be expressed in terms of the multimeric protein assembly or the antigen content within the assembly, and can be expressed in terms of the mass of protein per dose.

The immunogenic composition of the present invention can be administered to an individual to prevent infection in the individual. Accordingly, the immunogenic composition of the present invention may be a vaccine composition.

The vaccine composition of the present invention can be used to prevent diseases caused by SARS-CoV-2 infection. The term "prevention" in the present invention refers to any action that suppresses or delays the onset of a disease. The term "SARS-CoV-2" in the present invention refers to the virus that causes coronavirus disease 19 (COVID-19), a respiratory disease.

Multimeric protein assemblies can be efficiently produced through the method of the present invention.

Figure 1 shows the structure of a vector containing a base sequence encoding the protein (Component A) of SEQ ID NO: 1.
Figure 2 shows changes in dissolved oxygen (DO), pH, oxygen flow rate, and carbon dioxide flow rate in the culture medium when the upper pH value of the culture medium in the bioreactor is set to pH 7.25.
Figure 3 shows the changes in DO, pH, oxygen flow rate, and carbon dioxide flow rate in the culture medium when the upper pH value of the culture medium in the bioreactor was lowered to pH 7.1, and the trend of cumulative sodium bicarbonate usage during the culture period.
Figure 4 shows the change in the amount of lactic acid produced in the culture medium when the upper pH value of the culture medium in the bioreactor was changed to 6.8, 6.99, 7.0, or 7.2, respectively.
Figure 5 shows the purpose when the brand name Hyclone Cell Boost 7A/7B (Cell Boost 7A/7BTM) was supplied alone or Hyclone Cell Boost 7A/7B was alternately supplied with 45% (w/v) D-glucose solution every other day. It indicates that the protein expression level changes.
Figure 6 shows the target protein when supplied only with the brand name Hyclone Cell Boost 7A/7B (Cell Boost 7A/7BTM) or when Hyclone Cell Boost 7A/7B was supplied alternately with 45% (w/v) D-glucose solution. Cell viability and viable cell density (VCD) of the expressing cell line are shown.
Figure 7 shows the cell viability and viable cell density (VCD) of cell lines expressing the target protein for each injection cycle of Hyclone Cell Boost 7A/7B (Cell Boost 7A/7BTM) and 45% (w/v) D-glucose solution. indicates.
Figure 8 shows the 10th day of culture, culture 11 measured through SDS-PAGE when the injection cycle of the brand name Hyclone Cell Boost 7A/7B (Cell Boost 7A/7BTM) and 45% (w/v) D-glucose solution was changed. The expression level of the target protein on the first day, the 12th day of culture, the 13th day of culture, the 14th day of culture, and the 15th day of culture are shown.
Figure 9 shows culture 10 measured through Western blot (WESTERN-BLOT) when the injection cycle of brand name Hyclone Cell Boost 7A/7B (Cell Boost 7A/7BTM) and 45% (w/v) D-glucose solution was varied. The expression level of the target protein on the first day, the 11th day of culture, the 12th day of culture, the 13th day of culture, the 14th day of culture, and the 15th day of culture are shown.
Figure 10 shows the target protein content in the supernatant obtained by centrifuging 2000 L of cell culture expressing the target protein at a feed flow rate of 200 L/h, 300 L/h, or 400 L/h, and A1HC in the supernatant. This shows the results of measuring the content of the target protein in the filtrate by Western blotting when filtered using a depth filter or a combination of filters with pore diameters of 0.5 ㎛/0.2 ㎛.
Figure 11 shows the content of the target protein in the supernatant obtained by centrifuging 2000 L of a cell culture medium expressing the target protein at a feed flow rate of 200 L/h, and the filtration rate when the supernatant was filtered using a filter with a pore diameter of 0.45 ㎛. The results show the results of measuring the target protein content in the liquid and the target protein content in the filtrate by Western blotting when the supernatant was filtered using a filter with a pore diameter of 0.2 ㎛.
Figure 12 shows (1) the concentration (unit/ml) of benzonase, which is a nuclease, from 1 to 20 unit/ml, (2) the nuclease treatment time (hr) from 1 to 6 hr, and (3) This shows the results of the augmented design test design derived from the DoE program (JMP, Version: 14.0) when the concentration (mM) of Mg ²⁺ ions in the culture medium was set in the range of 0 to 2 mM.
Figure 13 shows the expression vector map of Component B.
Figure 14 confirms the strain growth level according to the amount of glycerol added to the culture medium.
Figure 15 confirms the difference in expression level depending on the amount of IPTG added.
Figure 16 shows the degree of cell growth upon entering the induction phase (and the relative expression level of the target protein according to the duration of the induction phase) measured according to the test plan. Early log phase, mid log phase, and late log phase, respectively - Marked as 1, 0, 1.
Figure 17 confirms protein yield according to cell suspension volume and disruption pressure.
Figure 18 confirms protein yield and impurity levels according to changes in benzonase treatment conditions.
Figure 19 shows protein yield and impurity levels by changing the processing conditions of the solubilization buffer.
Figure 20 shows the impurity removal rate by adjusting the loading ratio, linear velocity, and circulation time of the first chromatography process.
Figure 21 confirms the particle size and polydispersity index according to the mixing molar ratio of Components A and B.
Figure 22 confirms the particle size and polydispersity index according to reaction time when mixing Components A and B.
Figure 23 shows the results confirming that unassembled proteins were removed through SDS PAGE analysis of UF/DF permeate.
Figure 24 shows the results of confirming the yield, size average, and polydispersity index according to the change in concentration of the multimeric protein assembly when performing diafiltration.
Figure 25 shows the results of confirming the yield, size average, and polydispersity index according to changes in UF/DF filtration membrane MWCO size.
Figure 26 shows the yield, size average, and polydispersity index by changing the protein load (Protein/membrane area load ratio) per UF/DF filtration membrane area (0.1 m ² ) to 300, 750, and 1250 mg.
Figure 27 shows a schematic diagram of the overall process for manufacturing nanoparticles.
Figures 28 and 29 show Component A, a component of nanoparticles, Figure 30 shows Component B, a component of nanoparticles, and Figure 31 is a schematic diagram of the process for manufacturing and purifying nanoparticles.

Hereinafter, the present invention will be described in more detail through examples and examples. However, these examples and examples are for illustrative purposes only and the scope of the present invention is not limited to these examples and examples.

Preparation Example 1. Preparation of Component A expressing cells

To create a cell line expressing the polypeptide (“Component A”) represented by the amino acid sequence of SEQ ID NO: 1, the HD-BIOP3 cell line purchased from HORIZON Discovery, UK was used as a host cell. A recombinant expression vector plasmid (M-2560) was created by inserting cDNA synthesized by Genscript into the donor plasmid (pJV145) using SalI/NotI restriction enzyme reaction (Figure 1). The M-2560 vector has a DNA sequence represented by SEQ ID NO: 3.

The recombinant expression vector plasmid (M-2560) was transfected into the HD-BIOP3 cell line. Among the transfected cells, cells capable of stable single cell cloning were selected to prepare a research cell bank (RCB) with sufficient long-term passage stability. The RCB was transferred to SK Bioscience's Andong factory, and a working cell bank (WCB) was manufactured in compliance with GMP standards.

1 vial of WCB (Working Cell Bank) was dissolved in a water bath at 37°C, diluted with CD OptiCHO medium, centrifuged, and the pellet was resuspended in CD OptiCHO medium. 6 ^{L culture} at a concentration of ⁵ L culture, subculture was expanded to allow 1600 L culture at a concentration of 8 x 10 ⁵ cells/mL in a 2000 L bioreactor.

Preparation Example 2. Cultivation of cell lines expressing Component A in a 2000 L bioreactor

A disposable cell culture bag was placed in a 2000 L bioreactor and prepared for operation, then 1400 L of Dynamis medium was injected and WCB was inoculated and cultured at a concentration of 8.0 x 10 ⁵ cells/mL. During culture, DO control was carried out using gas and oxygen, and pH control was carried out using 8% sodium bicarbonate and carbon dioxide gas.

Immediately after inoculation, sampling was performed every day to measure the number of cells and check for microbial contamination and glucose concentration in the culture medium. When the foam in the bioreactor reached the bottom of the air filter mounted on the condenser, ADCF defoamer was applied. This process was repeated repeatedly until the end of culture.

The culture conditions used during culture are shown in Table 1 below.

Cell line culture conditions Culture conditions Setting value Temperature (days 0 to 5 of culture) 37℃ Temperature (after 5th day of incubation) 31℃ culture medium Dynamis Inoculation medium CD OptiCHO stirring speed 93RPM culture volume 1600 L Initial inoculum concentration 8.0 x 10 ⁵ cells/mL Dissolved oxygen amount 40% Dissolved oxygen amount control gas, oxygen pH control 8% sodium bicarbonate, carbon dioxide

Manufacturing Example 2.2. Culture medium recovery and DNA cutting enzyme treatment

The culture medium of the cell line expressing the target protein was centrifuged at 4,500 g for 30 minutes to collect the supernatant, passed through a depth filter made of A1HC, treated with triton X-100, and filtered through a 0.5/0.2 μm filter. To remove cell line-derived DNA from the filtered culture medium, Benzonase (Merck, Benzonase® endonuclease), a DNA cutting enzyme, was treated at a final concentration of 2 Unit/mL and reacted at room temperature for 4 hours.

Preparation Example 3. Purification of Component A

Manufacturing Example 3.1. Primary chromatography

In the first purification process, impure proteins were removed and the target protein was purified using HIC (hydrophobic interaction chromatography) resin. To prepare the sample for loading, 5 M sodium chloride was added to the main culture solution after the benzonase treatment, and the conductivity was corrected to be above 170 mS/cm, particularly in the range of about 170 to 220 mS/cm. The column was equilibrated using an equilibrated sodium phosphate buffer solution containing sodium chloride in a sufficient amount of 5 times the column capacity or more, and then the sample was added to the column. At this time, the fraction that flowed out without binding to the column was discarded and thoroughly washed by flowing more than twice the column capacity of the equilibration buffer solution until the UV values were parallel. Subsequently, elution of the target protein was performed using an elution sodium phosphate buffer solution containing sodium chloride.

Manufacturing Example 3.2. Virus inactivation

To inactivate the virus, the target protein eluate was treated with 5 M HCl so that the pH was 3.5 and stirred at room temperature for 1 hour to inactivate the virus. After reaction for 1 hour, 5 N NaOH was added to the inactivated target protein eluate to restore the pH to that before the inactivation process and stabilized by stirring at room temperature for 30 minutes.

Manufacturing Example 3.3. 1st ultrafiltration

The inactivated primary purification eluate was concentrated using a 100 kDa ultrafiltration membrane filter, and desalting and buffer exchange were performed using a balanced sodium phosphate buffer solution containing sodium chloride to be used in the secondary purification process. The conductivity of the filtrate was checked and the target protein was recovered by performing buffer exchange using a buffer solution until the conductivity was at the same level as that of the buffer solution.

Manufacturing Example 3.4. Secondary chromatography

In the second purification process, CHT™ Ceramic Hydroxyapatite chromatography and mixed mode resin were used to remove remaining impure proteins and purify the target protein. The sample to be input into the column was the first ultrafiltration recovery liquid, and no separate pH and conductivity adjustments were made. The column was equilibrated using an equilibrated sodium phosphate buffer solution containing sodium chloride in a sufficient amount of 7 times the column capacity or more, and then the sample was added to the column. After sample addition was completed, the equilibration buffer solution was flowed at twice the column capacity to remove the fraction that flowed out without binding to the column. Next, impurities were removed using a washing sodium phosphate buffer solution containing sodium chloride, and the target protein was eluted using an elution sodium phosphate buffer solution containing sodium chloride.

Manufacturing Example 3.5. Tertiary chromatography

In the third purification process, the target protein was purified through anion exchange chromatography (AEX) using a column filled with cation exchange resin, and impure proteins and nucleic acids were removed. The column filled with cation exchange resin was equilibrated with a sufficient amount of 6 times the column capacity using an equilibrium sodium phosphate buffer solution, and the secondary purification eluate was diluted 4 times using an equilibrium sodium phosphate buffer solution and then added to the column. After sample injection was completed, the equilibration buffer solution was flowed at 3 times the column capacity, and the fraction that flowed out without binding to the column was taken and the column was washed.

Manufacturing Example 3.6. virus filtration

Viruses were removed by performing a virus filtration process using the column flow from the third purification process. Since the column flow-through from the third purification process is used, the above-mentioned equilibrium buffer solution is equally present in the column flow-through liquid subject to virus filtration. There are two types of filters used in the virus filtration process. A depth filter is used as a pre-filter, and viruses are removed using a virus filter after filtration through the depth filter.

Manufacturing Example 3.7. Secondary ultrafiltration

The virus filter filtrate was concentrated and buffer exchanged by ultrafiltration using a 30 kDa filter. Concentrate the virus filter filtrate to about 1/10 to 1/3 of the starting volume of filtration and add at least 8 times the volume of assembly buffer (50 mM Tris, 100 mM arginine, 150 mM NaCl, 5 w/v% sucrose buffer/ Buffer exchange was performed at pH 7.9~8.1 / conductivity 18~22 mS/cm). Samples on which secondary ultrafiltration was completed were filtered through a 0.22 μm filter, and the target protein stock solution was stored at -70°C or lower.

Manufacturing Example 3.8. Composition, pH and conductivity of the buffer used in the purification process

The composition, pH, and conductivity of the buffer solution used in the processes of Preparation Examples 3.1 to 3.7 are summarized in Table 2 below.

step Buffer solution composition buffer solution pH conductivity Manufacturing Example 3.1.
Primary chromatography HIC EQ
Column equilibration 20mM Sodium phosphate, 3M NaCl pH 6.4~6.6 170 to 220 mS/cm Manufacturing Example 3.1.
Primary chromatography HIC Elution
elution 20mM Sodium phosphate, 0.6M NaCl pH 6.4~6.6 50~70 mS/cm Washing (a simple washing step to remove proteins remaining on the HIC column after performing the first chromatography) HIC Strip
Removal of residual proteins 20mM Sodium phosphate pH 6.4~6.6 1.8~2.4 mS/cm Manufacturing Example 3.2.
Virus inactivation inactivation 20mM Sodium phosphate, 0.6M NaCl After adjusting pH to 3.5, pH recovery 50~70 mS/cm Manufacturing Example 3.3 .
Primary ultrafiltration TFF (Tangential flow filtration) percolation 2mM Sodium phosphate, 50mM NaCl pH 6.7~6.9 5~7 mS/cm Manufacturing Example 3.4.
Secondary chromatography CHT EQ
Column equilibration 2mM Sodium phosphate, 50mM NaCl pH 6.7~6.9 5~7 mS/cm Manufacturing Example 3.4.
Secondary chromatography CHT wash
wash 25mM Sodium phosphate, 50mM NaCl pH 6.7~6.9 7.5~8.5 mS/cm Manufacturing Example 3.4.
Secondary chromatography CHT Elution
elution 240mM Sodium phosphate, 50mM NaCl pH 8.1~8.3 28~32 mS/cm Washing (this is a simple washing step to remove proteins remaining on the CHT column after performing the second chromatography) CHT Strip
Removal of residual proteins 400mM Sodium phosphate, 50mM NaCl pH 8.1~8.3 38~44 mS/cm Manufacturing Example 3.5.
Tertiary chromatography AEX EQ
Column equilibration 2mM Sodium phosphate pH 8.1~8.3 0.3~0.5 mS/cm Washing (this is a simple washing step to remove proteins remaining on the AEX column after performing the third chromatography) AEX Strip
Removal of residual proteins 20mM Sodium phosphate, 3M NaCl pH 6.4~6.6 180~200 mS/cm Manufacturing Example 3.6.
virus filtration percolation 2mM Sodium phosphate pH 8.1~8.3 0.3~0.5 mS/cm Manufacturing Example 3.7.
Secondary ultrafiltration TFF (Tangential flow filtration) percolation 50mM Tris
150mM NaCl
100mM Arginine
5 w/v% sucrose pH 7.9~8.1 18~22 mS/cm

Preparation Example 4. Preparation and culture of Component B expressing cells

The strain was transformed with a vector into which the Component B protein (SEQ ID NO: 6) expression gene was introduced into BL21 competent cells distributed by Thermofisher. pET29b+ was used as the vector for gene introduction. The prepared plasmid represented by SEQ ID NO: 8 (FIG. 13) was transformed into E. coli BL21 strain, an E. coli strain with kanamycin resistance was selected, and the selected E. coli BL21 strain was inoculated into a soy-based medium containing yeast extract. Culture was performed at 37°C with shaking at 200 rpm until the absorbance was 1.8 or higher. When culture was completed, sterilized glycerol was added to a final concentration of 25% to establish a cell bank for research.

After dissolving 1 vial of the cell bank for E. coli production at room temperature, kanamycin solution was added to a final concentration of 30 ug/mL in a 1 L flask containing 500 mL of soybean-based medium containing yeast extract, and the strain for production was added. Seed culture was prepared by inoculating 0.15 mL. At this time, the seed culture conditions were cultured at 37°C with shaking at 200 rpm and cultured for more than 8 hours so that the final absorbance exceeds 1.5.

Add kanamycin to a 75 L fermenter containing 50 L of soy-based medium containing yeast extract to a final concentration of 30 ug/mL, inoculate the entire seed culture, and culture at 200 rpm at 37°C until the absorbance reaches 25. (Growth phase). When the absorbance reached 25, the fermentor settings were changed to a culture temperature of 18°C and a stirring speed of 150 rpm, IPTG was added to a final concentration of 0.5mM, and the culture was completed for 10 hours (Induction phase).

Manufacturing Example 4.2. Culture medium recovery and DNA cutting enzyme treatment

The main culture medium of Preparation Example 4.1 was centrifuged to recover the cells, suspended in a buffer solution, and then disrupted with a high-pressure crusher. Centrifugation conditions were centrifugation speed of 15,000 g and centrifugation time of 20 minutes. Culture supernatant was discarded and cells were recovered. The recovered cells were stored in an ultra-low temperature freezer below -70°C. 10 mL of buffer per 1 g of cells was used and stirred for 1 to 2 hours until the cells were completely released. The cell suspension was crushed twice at a pressure of 17,000 psi using a high-pressure crusher to recover the cell lysate.

To remove cell line-derived DNA, benzonase, a DNA cutting enzyme, was added to the cell lysate to a final concentration of 5 Unit/ml and reacted at room temperature for 3 hours. The cell homogenate after Benzonase treatment was centrifuged at 15,000 g for 60 minutes using a high-speed centrifuge, the supernatant was discarded, and the inclusion bodies were recovered. The recovered inclusion bodies were washed using sodium phosphate buffer, the inclusion bodies were dissolved using sodium phosphate buffer containing 2 M urea (20mM Na-P, 150mM NaCl, 2M Urea, 0.1% Triton X-100), and 0.2 ㎛ filtered.

Preparation Example 5. Purification of Component B

Preparation Example 5.1 First chromatography

First chromatography was performed using a column filled with diethylaminoethyl (DEAE) resin to remove host-derived impurities such as endotoxin and nucleic acid.

Specifically, a column filled with DEAE resin (DEAE sepharose FF) was regenerated using 200mM sodium phosphate and 500mM sodium chloride buffer and equilibrated using 20mM sodium phosphate and 350mM sodium chloride buffer. The inclusion body recovered in Example 3 was added to the chromatographic column on which equilibration was completed at a flow rate of 100 cm/hr. After injection, collection of the permeate was started when UV280 increased by more than 0.005 AU, and collection was stopped when the UV280 value decreased again and reached equilibrium. The recovered permeate was filtered at 0.2 μm.

Manufacturing Example 5.2. Secondary chromatography

The second chromatography used a column filled with diethylaminoethyl (DEAE) resin to further remove endotoxin and host-derived proteins remaining after the first chromatography.

The column filled with DEAE resin (DEAE sepharose FF) was regenerated using 200mM sodium phosphate and 500mM sodium chloride buffer and equilibrated using 20mM sodium phosphate and 150mM sodium chloride buffer. The first chromatography permeate was added to the chromatography column on which equilibration was completed at a flow rate of 100 cm/hr, and the fraction that passed without being adsorbed on the resin was discarded. The resin was first washed again using a buffer containing 20mM sodium phosphate, 150mM sodium chloride, and 0.1% triton At this time, all washing liquid was discarded. When washing was completed, a buffer containing 20mM sodium phosphate and 400mM sodium chloride was added, and the eluate was recovered when the UV280 value reached 0.01 AU or higher. Recovery was terminated when the UV280 value decreased again and reached equilibrium. The recovered liquid was filtered at 0.2 μm.

Manufacturing Example 5.3. UF/DF

The secondary chromatography recovery solution was concentrated (ultrafiltration) to 5 kg using a tangential flow filtration system equipped with a 100 kDa MWCO (Molecular weight cut-off) membrane with an area of 0.7 m ² and 10 times the volume of the concentrate. It was recovered by performing buffer exchange (Diafiltration) using 50 mM Tris buffer. At this time, transmembrane pressure (TMP) was maintained below 1 bar. The recovered liquid was filtered at 0.2 μm.

Preparation Example 6. Assembly and purification of Component A and Component B

Multimeric protein assembly nanoparticles used in vaccines were manufactured by assembling Component A and Component B obtained through the above-described preparation example.

A reaction was performed by mixing Component A stock solution and Component B at room temperature, 1 hour, and a stirring speed of 80 rpm. The mixing ratio of Component A:Component B was 1:1.1 (molar concentration). When the reaction was completed, the reaction solution was filtered through a 0.2 μm filter.

The filtered assembled reaction solution was concentrated (ultrafiltration) to 4 kg using a tangential flow filtration system equipped with a 300 kDa MWCO (Molecular weight cut-off) membrane with an area of 0.7 m ² and the concentrate was 10 times the volume. It was recovered by performing buffer exchange (Diafiltration) using 50mM Tris buffer. At this time, transmembrane pressure (TMP) was maintained below 1 bar.

Optimization of Component A expression cell culture conditions

Example 1. Changes due to change in culture medium pH in bioreactor

In Preparation Example 2, on the 6th day of culture, the upper pH value of the culture medium in the bioreactor reached 7.25, so the injection of carbon dioxide into the culture medium through the sprayer increased to adjust pH, and the injection of oxygen to maintain DO of the culture medium increased. This occurred (Figure 2). Additionally, as the injection of carbon dioxide and oxygen increased, the problem of increased bubble generation occurred.

Therefore, it was verified whether the above problem could be solved by setting the upper pH value of the culture medium in the bioreactor to pH 7.1 (the deadband, which is the pH error range, is 0.05).

Specifically, culture when the upper pH value of the culture medium in the bioreactor is set to pH 7.2 (deadband is 0.05) (CTIA01503 batch) and when the upper pH value of the culture medium is set to pH 7.1 (deadband is 0.05) (E21R054 batch) Changes over the period were observed. Even after the 4th to 5th day of culture, the E21R054 batch did not increase the amount of oxygen and carbon dioxide injected into the culture medium, and as the pH was maintained at a constant level, the total amount of sodium bicarbonate accumulated and used during the culture period also increased. It was found to be low (Figure 3).

In addition, when the upper pH value of the culture medium in the bioreactor was set to pH 6.8, 6.99, 7.0, or 7.2, the amount of lactic acid generated during the culture period was found to decrease as the pH was lowered (Figure 4). Therefore, by keeping the upper pH value low during the culture period, the cells did not excessively produce by-products such as lactic acid to maintain pH. As the concentration of lactic acid in the culture medium increased, the dosage of buffering agents such as sodium bicarbonate, which were separately added to maintain the pH of the culture medium, decreased. In addition, the stress that cells experience during the culture process was reduced, culture stability was greatly improved, and protein production efficiency was increased.

Example 2. Confirmation of foam suppression effect by changing gas injection speed during culturing process

As the culture is stirred in a bioreactor, gas that forms bubbles is generated in the culture medium, which can have a negative effect on cell growth in the culture medium and protein expression of the cells. Therefore, antifoams are added to the culture medium for the purpose of inhibiting foam formation in the culture medium, but antifoams cause changes in cell membrane permeability, which can inhibit cell growth and also affect protein expression. It was verified whether foam formation occurring in the culture medium could be suppressed by changing the gas injection rate introduced into the culture medium within the actor.

Considering the upper pH value of the culture medium, pH 7.1 (deadband is 0.05), which was confirmed to increase production efficiency in Example 1, the pH of the culture medium was maintained at pH 6.8 to 7.1, and cultured under the conditions shown in Table 3 below. The effect of suppressing foam generated in the culture medium was confirmed.

process conditions Incubation period DO output § Air sparging flow (vvm) Degree of foaming (1~5) Control example Day 0 ~ Day 10 0% 0 5 10% 0.002 100% Experimental Example 1 Day 0 ~ Day 5 0% 0.002 3 10% 100% Day 5 ~ Day 10 0% 0.0005 One 10% 100% Experimental Example 2 Day 0 ~ Day 5 0% 0.002 3 10% 100% Day 5 ~ Day 10 0% 0.001 One 10% 100%

§DO output: This is an indicator of the amount of oxygen that needs to be replenished. If DO ouput is 0%, it means that dissolved oxygen is sufficient in the culture medium. If it exceeds 0%, it means that oxygen must be sprayed into the culture medium. In the case of 100%, it means that the amount of oxygen consumed by the cells is the maximum amount of oxygen that can be sprayed.

In the case of the control example, in which gas was not sprayed when there was no need to supplement oxygen, and in other cases, gas was sprayed at a constant spray rate, the degree of foam formation in the culture medium was found to be the highest.

On the other hand, in Examples 1 and 2, where the gas injection rate was reduced after the 5th day of culture regardless of DO output, foam formation was shown to be suppressed. In addition, in Experimental Examples 1 and 2, process convenience was secured because gas was sprayed at the same injection speed before and after the 5th day of culture, regardless of the DO output value.

On the other hand, in Experimental Example 1, a slight decrease in pH was observed to the deadband level of the upper pH value in the latter half of the culture, showing a slight decrease in yield. However, in Experimental Example 2, unlike Experimental Example 1, the pH decreased in the latter half of the culture. The phenomenon did not occur, and the yield appeared to be maintained.

Example 3. Changes in protein expression level depending on the sugar source and protein expression promoter supplied to the culture medium

In Preparation Example 2, sampling was performed every day from the third day of culture until the end of culture to measure cell number and cell survival rate, and check microbial contamination and glucose concentration in the culture medium. A solution was prepared by dissolving brand name Hyclone Cellboost 7A and brand name Hyclone Cellboost 7B in a neutral solvent and a basic solvent, respectively. A mixture of prepared Hyclone Cellboost 7A and brand name Hyclone Cellboost 7B solutions in a volume ratio of 10:1 (i.e. a mixture of brand name Hyclone Cellboost 7A/7B) and 45% (w/v) as a sugar source. While adding the D-glucose solution, the glucose concentration in the culture medium was maintained at 5.0 to 7.0 g/L.

Meanwhile, unlike the case where only the mixture of the brand name Hyclone CellBoost 7A/7B is supplied during the culture process, when the mixture of the brand name Hyclone CellBoost 7A/7B is added to the culture medium alternately with 45% (w/v) D-glucose solution. In order to check whether the expression level of the target protein was increased, the cell line expressing the target protein was cultured under the input conditions shown in Table 4 below, and the culture was terminated on the 16th day of culture.

batch number Input conditions control group ICR322019, ICR322021 ·Maintain the glucose concentration in the culture medium at 6g/L
·Supply a mixture of brand name Hyclone Cellboost 7A/7B from the 2nd day of culture to the 15th day of culture. experimental group ICR322020, ICR322022 ·Maintain the glucose concentration in the culture medium at 6g/L
·Fed a mixture of brand name Hyclone Cellboost 7A/7B on days 2, 4, 6, 8, 10, 12 and 14 of culture, and on days 3, 5, 7, 9 and 11. 45% (w/v) D-glucose solution was given on days 1, 13, and 15.

After preparing an antibody that binds to the target protein having the amino acid sequence shown in SEQ ID NO: 1, in order to confirm the difference in the production of the target protein produced in the control and experimental groups through a Western blot test using the antibody, on the 9th day of culture, A portion of the culture medium was recovered on days 11, 13, 14, and 15, and Western blot was performed. Regardless of the time of culture medium recovery for Western blot, the experimental group (batch ICR32220 and ICR322022, 20 and 22 in Figure 5) The target protein production was found to be higher than that of the control group (batch ICR322019 and ICR322021, 19 and 21 in Figure 5) (see Figure 5). Therefore, rather than using the mixture of brand name Hyclone CellBoost 7A/7B alone in the culture medium, using the mixture of brand name Hyclone CellBoost 7A/7B alternating with 45% (w/v) D-glucose solution increases the protein concentration. It has been confirmed that production can be increased.

In addition, the culture medium of the control and experimental groups (i.e., ICR322019 to ICR 322022) was recovered twice at each time point on day 0, day 9, and day 15 of culture, and cell viability and viable cell density (Viable Cell) in the culture medium were collected. Density, VCD) was measured, and the measured values at each time point were averaged.

When compared to the cell viability and VCD of the control group administered only the Hyclone Cell Boost 7A/7B mixture from the second day of culture, the cell viability and VCD of the experimental group were found to be higher (see Figure 6). Therefore, rather than using the mixture of brand name Hyclone CellBoost 7A/7B alone in the culture medium, protein production can be increased by using a mixture of brand name Hyclone CellBoost 7A/7B in alternation with 45% (w/v) D-glucose solution. It has been confirmed that it can increase.

Example 4. Change in production volume according to the cross-injection cycle of the mixture of brand name Hyclone Cellboost 7A/7B and 45% (w/v) D-glucose solution supplied to the culture medium.

In order to confirm the difference in production by cross-injection cycle of the mixture of brand name Hyclone Cellboost 7A/7B and 45% (w/v) D-glucose solution supplied to the culture medium, Preparation Example 2 was prepared except for the input conditions listed in Table 5. And the cell line expressing the target protein was cultured in the manner described in Example 3.

Cross-dosing cycle of mixture of brand name Hyclone Cellboost 7A/7B and 45% (w/v) D-glucose solution batch number Incubation period (Day) 0, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ICR322035 C D C D C D C D C D C D C collect ICR322036 C D D C D D C D D C D D C collect ICR322037 C D D D C D D D C D D D C collect

C: Blend of brand name Hyclone Cellboost 7A/7B

D: 45% (w/v) D-glucose solution

Meanwhile, on the 0th, 10th, and 15th days of culture, a portion of the culture medium of the batches ICR322035 to ICR 322037 was recovered twice at each time point, and cell viability in the culture medium, which is one of the indicators for confirming the increase in protein production, and The viable cell density was measured, and the values measured at each time point were averaged. Cell viability and VCD were found to be significantly higher in all experimental groups (i.e., batches ICR322035 to ICR322037) compared to batch ICR322019 (control group) of Example 1, which was administered only the mixture of brand name Hyclone Cellboost 7A/7B from the second day of culture. (See Figure 7).

In addition, after recovering the culture medium from the batches ICR322035 to ICR 322037 on the 10th, 11th, 12th, 13th, 14th, and 15th days of culture, SDS-PAGE was performed to confirm the production amount of the target protein expressed at each time point. and Western blot were performed. The expression level of the target protein measured by SDS-PAGE was found to be high in all experimental groups (i.e., batches ICR322035 to ICR322037, 35 to 37 in Figure 8) on days 10, 11, 12, 13, 14, and 15 of culture. However, the highest expression level of the target protein was found in the ICR322035 batch administered with a mixture of brand name Hyclone Cellboost 7A/7B and 45% (w/v) D-glucose solution every other day (see Figure 8). In addition, the expression level of the target protein measured by Western blot was high in all experimental groups (i.e., batches ICR322035 to ICR322037, 35 to 37 in Figure 9) on days 10, 11, 12, 13, 14, and 15 of culture. However, the highest expression level of the target protein was found in the ICR322035 batch administered with a mixture of brand name Hyclone Cellboost 7A/7B and 45% (w/v) D-glucose solution every other day (see Figure 9).

Optimization of recovery and purification process for Component A

Example 1. Changes in recovery amount and impurities of target protein according to differences in centrifugation feed flow

In order to quickly produce a large amount of the target protein, a large-scale bioreactor can be used or the volume of the culture medium can be increased. However, it takes longer to input the culture medium to the centrifuge during the recovery process than when culturing in a small-scale bioreactor or a small volume. There was a disadvantage in that the production period increased because the waiting time in the centrifuge increased.

Accordingly, in a bioreactor (2000L) used for large-scale production of proteins, 1400L of medium was filled, culture conditions such as dissolved oxygen and pH were calibrated, and then culture of cells expressing the target protein was started with 1600L of medium. Culture additives for production were added during the culture process so that the culture volume on the 10th day of culture was 2000 L. By using an industrial continuous centrifuge (capable of separating 2000L) and shortening the time required for centrifugation by increasing the feed flow rate at which the culture medium is supplied to the centrifuge, impurities are removed without affecting the yield of the target protein recovered after centrifugation. To determine whether it was possible to reduce the concentration of the target protein in the supernatant, yield, impurities in the supernatant, and turbidity of the supernatant were measured.

Total protein concentration in the supernatant was measured using the BCA assay using the Pierce™ BCA protein assay kit (catalog number 23225) provided by ThermoFisher Scientific. Additionally, the concentration of the target protein in the supernatant was measured using HIC-HPLC.

The content of impurities in the supernatant was confirmed by measuring host cell proteins and host cell-derived DNA in the supernatant. At this time, the host cell protein concentration in the supernatant was determined by ELISA analysis using the Chinese hamster ovary cell (CHO) Host Cell Protein (HCP) ELISA kit (catalog number: F550-1) from Cygnus Technologies. It was measured through. Furthermore, the concentration of host cell derived DNA (HCD) in the supernatant was determined by RT-PCR using Thermo Fisher Scientific's Applied Biosystems (trade name: Applied Biosystems) resDNASEQ quantitative CHO DNA kit (catalog number: 4402085). It was analyzed through. Additionally, the turbidity of the supernatant was measured using a turbidity meter (Naphelometer).

Table 6 shows the measurement results of total protein concentration in the supernatant, target protein concentration, yield of the target protein, impurities in the supernatant, and turbidity of the supernatant according to changes in the feed flow rate at which the culture medium is supplied to the centrifuge.

Concentration, impurities and turbidity of target protein in supernatant according to difference in feed flow rate Supply flow rate (L/h) Total protein concentration in supernatant (μg/ml) target protein
Concentration (μg/ml) Host cell protein (HCP) (ng/ml) Host cell derived DNA (HCD) (ng/ml) Turbidity (NTU) 200 5639.778 1585 369140 26473.4 56.6 300 5794.096 1568.1 359130 27045.6 73.8 400 6081.003 1598.6 389700 32786 154

When the feed flow rate during centrifugation was 200 L/h, the centrifugation time for 2000 L of culture medium was 10 hours, whereas when the feed flow rate was increased to 300 L/h, it took about 6.7 hours to centrifuge 2000 L culture medium. Additionally, when the feed flow rate was 400L/h, the time required to centrifuge 2000L culture medium was shortened to about 5 hours.

Meanwhile, even if the feed flow rate during centrifugation increased from 200 L/h to 300 L/h or 400 L/h, the content of the target protein in the recovered supernatant did not appear to change significantly. In addition, even when the feed flow rate increased from 200 L/h to 300 L/h during centrifugation, host cell proteins and host cell-derived DNA, which are impurities in the supernatant, did not appear to significantly increase or decrease. Although, when the feed flow rate during centrifugation increased from 200 L/h to 300 L/h, the turbidity in the supernatant increased, but this increase in turbidity was improved by applying a depth filter to the supernatant obtained by centrifugation. It was confirmed that

Furthermore, when the feed flow rate during centrifugation increased from 200 L/h to 400 L/h, host cell proteins and host cell-derived DNA tended to increase, but by using a depth filter after centrifugation, host cell proteins and host cell-derived DNA increased. It was confirmed that a significant number of was removed and turbidity was significantly lowered.

On the other hand, if the supply flow rate during centrifugation is lower than 200 L/h, the centrifugation time increases to 10 hours or more, which inevitably causes the problem of delayed production period. In addition, if the supply flow rate during centrifugation is higher than 400 L/h, the centrifugation time can be reduced to less than half compared to when the supply flow rate is 200 L/h, but as impurities and turbidity in the supernatant increase, it must be used to remove impurities. A problem arose as the number of filters used increased rapidly.

Therefore, if the supply flow rate is 200 L/h to 400 L/h when centrifuging the culture medium of a cell line expressing the target protein, the effect of shortening the production period and reducing production costs is achieved without affecting the concentration of the target protein in the supernatant. It was confirmed that

Example 2. Turbidity change according to difference in centrifugation feed flow

Start culturing cells expressing the target protein with 1600 L medium in a 2000 L bioreactor, and add culture additives for the production of the target protein during the culture process so that the culture volume is 2000 L on the 10th day of culture, and then continue industrial use. The supernatant was recovered using a centrifuge. The turbidity of the supernatant obtained by centrifugation with a supply flow rate of 200 L/h, 250 L/h, or 300 L/h and a discharge interval of 252 seconds, 201 seconds, or 168 seconds, respectively, was measured using a turbidity meter, and the measurement results are shown in the table. It is described in 7.

Turbidity change in supernatant due to difference in supply flow rate Supply flow rate (L/h) Discharge interval during centrifugation Turbidity (NTU) 200 252 seconds 134 250 201 seconds 135 300 168 seconds 166

Even if the feed flow rate increased from 200 L/h to 250 L/h during centrifugation, the turbidity in the supernatant hardly changed, and even if the feed flow rate increased from 200 L/h to 300 L/h, the change in turbidity was about 30 L/h. It was confirmed that there was no significant difference from the change (about 20) that occurred when the supply flow rate was increased from 200 L/h to 300 L/h in Example 1.

Although turbidity was not measured when the feed flow rate during centrifugation was 400 L/h, considering the turbidity at 200 L/h and 300 L/h, turbidity was similar to that measured in Example 1. It was expected. Therefore, it was confirmed that even if the culture volume increases during commercial cultivation of cells expressing proteins, it is possible to obtain a supernatant with low turbidity due to the small amount of impurities while shortening the production period by using a supply flow rate of 200 L/h to 400 L/h. It has been done.

Example 3. Purification of target protein and removal of impurities according to the use of filters used in filtration of centrifugal supernatant

When the centrifuged supernatant obtained in Example 1 was filtered using a filter, the change in the content of the target protein in the filtrate and the effect of reducing impurities were confirmed.

The centrifuged supernatant obtained in Example 1 was filtered using an A1HC depth filter, a combination of a filter with a pore diameter of 0.5 ㎛ and a 0.2 ㎛ filter, a filter with a pore diameter of 0.45 ㎛, or a filter with a pore diameter of 0.45 ㎛. When this was done, the change in the content of the target protein in the filtrate and the effect of removing impurities were measured through Western blot.

When a filter is applied to the centrifugation supernatant, the target protein content in the filtrate is similar to the target protein content in the centrifugation supernatant without applying a filter, and the target protein monomer and target protein dimer band sizes are similar, thereby increasing the target protein content. was found to be maintained (see Figures 10 and 11).

On the other hand, when a filter was applied to the centrifugation supernatant, impurities were reduced, and the target protein monomer and target protein dimer bands were more distinct than the target protein monomer and target protein dimer bands observed in the centrifugation supernatant without a filter. The clarity of other bands (i.e., impurities that are proteins other than the target protein) was reduced and they were observed relatively blurry (see FIGS. 10 and 11).

Example 4. Nuclease concentration, treatment time, and Mg of the culture medium treated with the culture medium ²⁺ Confirmation of changes in host cell DNA removal rate depending on ion concentration

In the nuclease treatment step of Preparation Example 2, the following experiment was performed to derive optimized process conditions for effectively removing host cell DNA, which is an impurity derived from cell lines.

More specifically, (1) nuclease benzonase concentration (unit/ml), (2) nuclease treatment time (hr), and (3) culture medium were identified as factors that significantly affect the removal of impure DNA. Concentration of Mg ²⁺ ions (mM) A total of three factors are set as variables, and conditions are set to maximize the DNA removal rate through Design of Experiments (DoE) multivariate experiments using JMP14 software. was explored. The DoE platform allows investigators to change multiple parameters simultaneously, rather than changing each parameter individually and then considering each optimized parameter for the overall optimized result. In cases where secondary effects between parameters can affect the results, using a DoE platform that changes all candidate parameters simultaneously can yield more efficient and accurate results. Experiments using the DoE platform also require fewer runs and are more economical than traditional experimentation approaches. See Kauffman et al., Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs, NanoLetters 15:7300-7306 (2015) and the supplementary material for a theoretical discussion of the DoE platform. .

(1) nuclease (benzonase) concentration (unit/ml) of 1 to 20 unit/ml, (2) nuclease treatment time (hr) of 1 to 6 hr, and (3) Mg ²⁺ in the culture medium. When the ion concentration (mM) was set in the range of 0 to 2 mM, the augment design test design results derived from the DoE program (JMP, Version: 14.0) are shown in FIG. 12.

As shown in Figure 12, the conditions for maximizing the DNA removal rate were found to be a benzonase concentration of 13.5 unit/mL, a benzonase reaction time of 5.5 hours, and a Mg ²⁺ concentration of 2 mM. In addition, when performing 100,000 monte-carlo simulations in the JMP program under the above conditions, when a DNA removal rate of 80% or more is set as the target range, the probability of exceeding the range is 0.007%, confirming very stable results. From this, it was confirmed that the optimal conditions were a benzonase concentration of 13.5 unit/mL, a benzonase reaction time of 5.5 hours, and a Mg ²⁺ concentration of 2 mM in terms of DNA removal rate.

However, the concentration of benzonase has a direct effect on increasing the cost of producing vaccine antigens, and if the benzonase is used at a concentration of 13.5 unit/mL, there is a problem in that the cost is so high that commercial production is practically impossible.

Example 5. Confirmation of conditions that reduce costs that will occur when applied to commercial production

In addition to the conditions of maximizing the DNA removal rate derived in Example 4, by additionally considering the costs incurred when applying commercial production, such as production model and process cost, a DNA removal efficiency that is economical and excellent enough to be applied to commercial production is achieved. We attempted to derive conditions.

More specifically, when designing the DoE experiment, the benzonase reaction time was divided into groups of 2 hours or 4 hours, and the final concentration of benzonase for each group was adjusted to 1 unit/mL, 2 unit/mL, or 4 unit/mL, respectively. The reduction rate of DNA in the culture medium after treatment compared to the culture medium before nuclease treatment was measured. At this time, other process conditions except benzonase treatment time and concentration, especially Mg ²⁺ concentration, were fixed at 2 mM.

The results are shown in Table 8 below.

Sample DNA concentration (ng/mL) DNA reduction rate (-%) Benzonase processing time Benzonase concentration Culture filtrate 18382 2 hours 1 unit/mL 10641.5 42.1 2 units/mL 9938.7 45.9 4 units/mL 5264.6 71.4 4 hours 1 unit/mL 6462.3 64.8 2 units/mL 3542.1 80.7 4 units/mL 1360.9 92.6

Commercial production can be applied only when the DNA reduction rate (-%) is achieved at least 80%. Among the benzonase treated groups for 4 hours in Table 8, the DNA reduction rate (-%) was 80 at a concentration of 2 or 4 units/mL of benzonase. It can be seen that more than % has been achieved.

Next, when applying commercial production under the benzonase concentration condition of 2 or 4 unit/mL, which achieves the DNA reduction rate (-%) of 80% or more, and the 13.5 unit/mL concentration condition confirmed in Example 1, in the benzonase treatment process The costs incurred were compared.

Benzonase product information used in Preparation Example 2 is shown in Table 9 below.

unit unit/μL mL Product price (10,000 won) Amount (10,000 won/mL) Culture medium (unit/L) Throughput (μL/L) 5,000,000 250 20 1,800 90 2,000 8

Next, the amount and amount of benzonase that must be treated when adjusting the benzonase concentration in the 1800L culture medium are shown in Table 10 below.

Final benzonase control concentration (unit/ml) Benzonase throughput (ml) Total (10,000 won) 2 14.4 1,296 4 28.8 2,592 13.5 97.2 8,748

In order to adjust the final benzonase concentration to 2 units/ml in 1800L culture medium, 14.4 ml of 250 unit/μl benzonase product must be processed. Considering that benzonase costs 900,000 won per ml, the cost of adjusting the benzonase concentration is approximately 12.96 million won is derived. The cost incurred when adjusting the final benzonase concentration to 4 or 13.5 unit/ml through the same process can also be derived.

If the benzonase treatment process is performed by adjusting the benzonase concentration to 2 unit/mL, antigen can be produced at a cost savings of approximately 12.96 million won compared to adjusting the benzonase concentration to 4 unit/mL, and the cost is approximately 13.5 unit/mL. It can be seen that antigens can be produced at a reduced cost of 74.52 million won.

It can be seen that as the amount of antigen to be produced increases and as the process cycle is repeated, the cost gap will become very large.

In summary, when the benzonase treatment process is performed under the conditions of a benzonase concentration of 2 unit/mL, a benzonase reaction time of 4 hours, and a Mg ²⁺ ion concentration of 2 mM, the DNA removal efficiency is economical enough to be applied to commercial production and has excellent DNA removal efficiency. achieved.

Optimization of Component B expression cell culture conditions

Example 1. Optimization of glycerol content of growth phase medium

Glycerol was added to the medium in the growth phase of Preparation Example 4 to confirm the effect on the growth and protein expression of the strain.

Glycerol was added at concentrations of 0% (not added), 2%, and 4% (v/v), and cell growth according to concentration is shown in Figure 4 and Table 11, and the cell weight was confirmed after recovery. As a result of the experiment, it was confirmed that maximum cell growth was improved when glycerol was added.

In addition, the effect of glycerol concentration on protein expression was confirmed according to the design of experiment (DoE), and is shown in Table 11.

As a result of the experiment, the protein expression level reached the maximum when 2% of glycerol was added, and an increase in protein expression level of about 25% was confirmed compared to the group without glycerol added.

classification Glycerol concentration (%) Maximum cell growth (OD600) Test group 1 0 23 test group 2 2 56 test group 3 4 50

Example 2. Optimization of induction phase conditions

In the induction phase, the optimal conditions were confirmed by adjusting the amount of IPTG added (0, 0.5mM, 1mM, 2mM: Figure 15).

Sample information Relative
Expression (%) Main fermentation Result Time (h) Harvest O.D. IPTG concentration 0mM 100.0 18.5 53 0.5mM 104.6 18.5 51 1mM 107.7 18.5 50 2mM 104.8 18.5 50

As a result of the experiment, protein expression increased in all IPTG-added groups, and it was confirmed that the optimal expression conditions were especially when 0.5mM to 1mM was added (FIG. 15).

In addition, according to the test design method, the degree of cell growth when entering the induction phase (early log phase, mid log phase, late log phase, respectively indicated as -1, 0, and 1 in Figure 4) and the purpose according to the duration of the induction phase The relative expression levels of proteins are shown in Figure 16.

As a result of the test, it was confirmed that entering the induction period in the late log phase increased the target protein by about 25% compared to entering the induction period in the early log phase. In the case of the induction period duration, it was confirmed that the 10-hour induction period showed the maximum expression of the target protein.

Optimization of recovery and purification process for Component B

Example 1. Optimization of cell disruption and protein separation process

To optimize protein yield, cell suspension volume, disruption pressure, and benzonase treatment parameters were changed.

The experiment was designed using the JMP® Design of Experiment (DOE) platform. The DOE platform allows investigators to change multiple parameters simultaneously, rather than changing each parameter individually and then considering each optimized parameter for the overall optimized result. In cases where secondary effects between parameters can affect the results, more efficient and accurate results can be obtained by using a DOE platform that changes all candidate parameters simultaneously. Experiments using the DOE platform also require fewer runs and are more economical than traditional experimental approaches. See Kauffman et al., Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs, NanoLetters 15:7300-7306 (2015) and the supplementary material for a theoretical discussion of the DOE platform. .

The volume of cell suspension per 1g of cells was set to 5 to 15 ml, and the crushing pressure was set to 13,000 to 21,000 psi. Protein recovery according to each parameter was confirmed and is shown in Figure 17.

As a result of the experiment combining the two parameters, it was confirmed that the cell suspension had the highest protein yield in a volume of about 8 ml to 14 ml, specifically about 10 ml, and the crushing pressure was about 14000 to 21000 psi, specifically about 17000 psi.

In addition, using the JMP® Design of Experiment (DOE) platform, we changed the benzonase treatment conditions in the range of 0-10 Unit/ml, incubation time 0-300 min, and pH 7-9 to determine the removal rate of host cell DNA as an impurity. Optimal conditions were confirmed by combining host cell DNA elimination rate (HCD) and process yield (FIG. 18).

As a result of the experiment, it was confirmed that the conditions of approximately 5 Unit/mL, 3hr, and pH 8.0 were the optimal conditions for the highest impurity removal rate.

In addition, the effect of solubilization time and buffer volume parameters of the solubilization process on protein recovery and impurities was determined (Figure 19), and the volume of solubilization buffer was confirmed to be optimal at 10 mL.

Example 2. Optimization of primary chromatography conditions

Example 2.1. Confirmation of impurity content depending on whether first chromatography is performed

Changes in impurity levels were confirmed depending on whether first chromatography was performed in negative mode. If negative mode chromatography was not performed, it was labeled as Group 1, and if negative mode chromatography was performed, it was labeled as Group 2. The overall purification conditions are as follows.

Group Chromatography application Product quality Negative mode Positive mode Endotoxin content (EU/mg of protein) Host cell DNA content (ng/mg of protein) Productivity (mg/L of fluid) Group 1 X O 11,924 2.9 62 Group 2 O O 517 1.6 89

As a result of the experiment, the protein yield and amount of impurities were as shown in Table 13 above, and it was confirmed that the content of endotoxin and nucleic acid, which are impurities, was lowered by the addition of the first chromatography process performed in negative mode. did.

Example 2.2. Optimization of first chromatography performance conditions

Next, the design of experiment (DoE) was performed to confirm the impurity removal rate by adjusting the loading ratio, linear velocity, and circulation time (CV) of the sample loaded into the unit resin when performing the first chromatography (Figure 20 ). As a result of the experiment, it was confirmed that the yield increased at 10CV, which is the condition of reloading about 10 times the volume of the resin.

Example 3. Optimization of secondary chromatography conditions

Example 3.1. Confirmation of purification yield depending on whether or not secondary chromatography buffer contains detergent

The purification yield was confirmed depending on whether detergent was included in the equilibration, washing, and elution buffer used in the second chromatography. Other components in the buffer solution are as described in the preparation example.

Group Test conditions Product quality Triton concentration in buffer (%) Endotoxin content (EU/mg of protein) Host cell DNA content (ng/mg of protein) Productivity (mg/L of fluid) Group 1 0 343 1.7 48 Group 2 0.1 662 11.7 212

The experimental results are as shown in Table 14 above.

Through this, it was confirmed that when detergent was used at 0.1% content in the secondary chromatography, protein productivity increased compared to the case where detergent was not included.

Example 3.2. Control of salt content in secondary chromatography wash buffer

Second chromatography In the composition of the second washing buffer, the NaCl content was changed to minimize the content of impurities and the composition was confirmed to minimize the decrease in yield. Other components in the buffer solution are as described in the preparation example.

The experimental results, protein yield and impurity content are shown in Table 15 below.

Group Test condition Product quality NaCl (mM) in washing buffer Endotoxin content (EU/mg of protein) Host cell DNA content (ng/mg of protein) Productivity (mg/L of fluid) Group 1 0 25,100 6,466 127 Group 2 200 11,924 2.9 62 Group 3 250 11,099 17.2 10

Through this, it was confirmed that the optimal content of NaCl in the secondary washing buffer for secondary chromatography was about 200mM.

Example 4. Optimization of UF/DF conditions

By changing the composition of the buffer solution in the ultrafiltration/diafiltration process, we attempted to identify the buffer that showed the optimal yield.

Example 4.1. Determination of optimal composition of buffer solution

As a result of using the four compositions in Table 16 below, it was confirmed that the composition of Group 4 (50mM Tris, 500mM NaCl, 0.75% CHAPS (pH 7.5)) had the best yield.

Group Diafiltration buffer composition Process yield (%) Whether aggregation occurs during diafiltration One 50mM Tris + 5% sucrose + 100mM Arginine + 150mM NaCl 100 O 2 50mM Tris + 100mM Arginine + 150mM NaCl 120 O 3 50mM Tris + 5% sucrose + 500mM Arginine + 150mM NaCl 136 O 4 50mM Tris + 500mM NaCl + 0.75% CHAPS 137 X

Specifically, in the case of Groups 1 to 3, sediment was formed over storage time, but Group 4 was confirmed to be stably maintained without sediment.

Example 4.2. Check protein yield according to the type of detergent included in the buffer solution

In the composition of Group 4 selected through the above experiment, the type of detergent was changed to PS80 and Triton to confirm the yield.

Group Composition Protein content reduction rate (%) One 50mM Tris, 500mM NaCl 21.6 2 50mM Tris, 500mM NaCl, 0.75% CHAPS 1.88 3 50mM Tris, 500mM NaCl, 0.05% PS80 8.81 4 50mM Tris, 500mM NaCl, 0.1% Triton 22.1

As a result of the experiment, it was confirmed that the protein content reduction rate was lowest when Group 2, that is, CHAPS, was used.

Through this, it was confirmed that 50mM Tris, 500mM NaCl, and 0.75% CHAPS were the optimal composition of the buffer solution.

Optimization of Component A and Component B assembly processes

In order to optimize the assembly reaction of Preparation Example 6, the optimal mixing ratio and buffer composition were changed.

Example 1. Setting the optimal mixing ratio

A reaction was performed by mixing Component A stock solution and Component B stock solution at room temperature for 2 hours and a stirring speed of 80 rpm. When the reaction was completed, the reaction solution was filtered through a 0.2 μm filter.

Meanwhile, in order to confirm the optimal mixing molar ratio, the molar concentration ratio of Component A and Component B was changed and the particle size and polydispersity index were measured, and the results are shown in the table below and Figure 21.

Group reaction ratio
(A:B) Particle size
(d,nm) Polydipersity Index (PDI) One 1:1.1 54.6 0.210 2 1:2 68.9 0.310 3 1:3 84.3 0.432

Group reaction ratio
(A:B) Particle size
(d,nm) Polydipersity Index (PDI) One 1.5:1 72.7 0.496 2 1.1:1 56.8 0.275 3 1:1.5 69.4 0.306

As shown in Tables 18 and 19 above, it can be seen that the particle size and polydispersity index also change as the molar concentration ratio of Component A and Component B is changed. From this, it was confirmed that the optimal mixing molar ratio is 1:1.1 molar concentration ratio, which secures the most desirable particle size and PDI value for vaccine antigen use.

Example 2. Optimal mixing time setting

Self-assembly of Component A and Component B was induced using the optimal molar concentration ratio confirmed above, but only the reaction time was set differently to evaluate the final particle size and yield. The results are shown in the table below and Figure 22.

Group Reaction time (hr) Particle size
(nm) Process yield (%) One 0.5 60.2 28 2 One 67.8 26 3 2 53.4 49 4 4 58.3 47

As shown in Table 20 above, it can be seen that the particle size and yield change as the self-assembly induction reaction time of Component A and Component B is changed. From this, it was confirmed that the most desirable particle size for vaccine antigen use was secured and that the reaction time with the best yield was 2 hours.

Example 3. Optimization of buffer composition

By mixing the Component A and B stock solutions, the buffer in which self-assembly is induced corresponds to the 50 mM Tris buffer (500 mM NaCl, containing 0.75 w/v% CHAPS) used when performing the buffer exchange in the above-described preparation example. Polysorbate-80 (PS80) was added as an additional stabilizer to the buffer solution, and an attempt was made to derive the optimal concentration of the stabilizer in the buffer solution to induce self-assembly. The yield, particle size, and polydispersity index were confirmed without adding polysorbate-80 (PS80) or changing the content from 0.05 v/v% to 0.01 v/v%, and are shown in the table below.

Test group PS80 concentration (v/v%) Process yield
(%) Particle size (DLS) Z-average PDI Group 1 0.050 67.3 82.57 0.556 Group 2 0.025 62.0 41.92 0.263 Group 3 0.010 61.1 40.24 0.270 Group 4 0.000 56.6 62.09 0.635

As shown in the table above, Group 4, which does not contain PS80, has a relatively low yield and high PDI value, and Group 1, which has a PS80 content of 0.05 v/v%, has an average particle size value that is too large and is not suitable for use as a vaccine antigen. It was confirmed that it was not. In addition, when PS80 was not included in the self-assembly induction buffer or included at a concentration of 0.05 v/v%, intensity and mass peaks of various sizes were observed.

On the other hand, in the case of Groups 2 and 3, which included PS80 at a concentration of 0.025 v/v% or 0.010 v/v% in the self-assembly induction buffer, a yield higher than 60% was achieved when considering the scale-up process, and at the same time, Z- It was confirmed that the average and PDI values were secured in a desirable range for use as a vaccine antigen. The polydispersity index PDI refers to the weight average molecular weight (Mw) divided by the number average molecular weight (Mn) (Mw/Mn). A high polydispersity index means that the size, shape, or mass distribution of the manufactured particles is not constant. It is non-uniform, meaning that the quality is not uniform and processability is reduced.

From this, it was confirmed that the preferred concentration range of the stabilizer in the self-assembly inducing buffer is 0.010-0.025 v/v%. Ultimately, considering the possibility of loss during the process, 0.025% v/v% was set as the most desirable stabilizer concentration in the self-assembly induction buffer.

Optimization of purification process for multimeric protein assembly nanoparticles

As a result of performing the UF/DF process in Preparation Example 6, it was confirmed that Component A, in which the assemble reaction did not proceed, was removed (FIG. 23). To determine the optimal conditions for ultrafiltration and diafiltration, (i) the initial protein concentration during diafiltration, (ii) the molecular weight cutoff (MWCO) of the membrane, and (iii) the protein load per filtration membrane area (protein/membrane load ratio) were changed. Yield and quality were confirmed.

Example 1. UF/DF (Ultrafiltration/diafiltration) process

Example 1.1. Diafiltration protein concentration optimization

In order to optimize the protein concentration during diafiltration, yield and quality were confirmed when performed at a concentration of 1 to 6 mg/mL (FIG. 24).

Group Test condition Result Target UFDF conc. (mg/mL) Yield (%) z average (d.nm) PDI One One 62.4 61.51 0.23 2 2 64.1 59.65 0.21 3 4 67.7 75.66 0.35 4 6 46.5 95.77 0.50

As a result of the experiment, the yield and quality were maintained at an appropriate level at a concentration of about 1 to 2 mg/mL, but at a concentration of 4 mg/mL or more, the z average (d, nm) increased to more than 70 nm and the polydispersity index (PDI) also increased, lowering the quality. It was confirmed that a decrease occurred. Through this, the optimal protein concentration during diafiltration was set to less than 4 mg/mL.

Example 1.2. Membrane molecular weight cutoff (MWCO) optimization for UF/DF process

The yield was confirmed by changing the membrane molecular weight cutoff (MWCO) in the UF/DF process (FIG. 25).

MWCO Yield (%) DLS Z-average PDI 300 kDa 47.59 38.68 0.187 500 kDa 3.85 101.5 0.662

As a result of the experiment, it was confirmed that when the membrane molecular cutoff was 500 kDa, the yield decreased significantly and the quality also decreased. Through this, it was confirmed that the optimal range of membrane molecular weight fraction in ultrafiltration and diafiltration processes is about 300 kDa.

Example 1.3. Check yield and quality changes according to protein load ratio per filtration membrane area in the UF/DF process

In the UF/DF process, process yield and quality were confirmed according to the protein/membrane load ratio per filtration membrane area (FIG. 26).

Batch Test condition Yield (%) QA Protein loading (mg/0.1m ² ) Particle size
(z average, d.nm) PDI NPA210720-01 300 78.2 63.65 0.27 NPA210720-02 750 86.4 67.56 0.24 NPA210720-03 1250 96.3 79.56 0.26

As a result of the experiment, it was confirmed that the yield increased as the protein/membrane area load ratio increased, but there was a problem in that the size of the multimeric protein assembly increased above 1250 mg/0.1m ² . Through this, the optimal range was set at 300 to 750 mg/0.1m ² .

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

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150 155 160 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 165 170 175 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 180 185 190 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 195 200 205 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 210 215 220 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Gly Gly Ser Gly 225 230 235 240 Gly Ser Gly Ser Gly Gly Ser Gly Gly Ser Gly Ser Glu Lys Ala Ala 245 250 255 Lys Ala Glu Glu Ala Ala Arg Lys Met Glu Glu Leu Phe Lys Lys His 260 265 270 Lys Ile Val Ala Val Leu Arg Ala Asn Ser Val Glu Glu Ala Ile Glu 275 280 285 Lys Ala Val Ala Val Phe Ala Gly Gly Val His Leu Ile Glu Ile Thr 290 295 300 Phe Thr Val Pro Asp Ala Asp Thr Val Ile Lys Ala Leu Ser Val Leu 305 310 315 320 Lys Glu Lys Gly Ala Ile Ile Gly Ala Gly Thr Val Thr Ser Val Glu 325 330 335 Gln Ala Arg Lys Ala Val Glu Ser Gly Ala Glu Phe Ile Val Ser Pro 340 345 350 His Leu Asp Glu Glu Ile Ser Gln Phe Ala Lys Glu Lys Gly Val Phe 355 360 365 Tyr Met Pro Gly Val Met Thr Pro Thr Glu Leu Val Lys Ala Met Lys 370 375 380 Leu Gly His Thr Ile Leu Lys Leu Phe Pro Gly Glu Val Val Gly Pro 385 390 395 400 Gln Phe Val Lys Ala Met Lys Gly Pro Phe Pro Asn Val Lys Phe Val 405 410 415 Pro Thr Gly Gly Val Asn Leu Asp Asn Val Ala Glu Trp Phe Lys Ala 420 425 430 Gly Val Leu Ala Val Gly Val Gly Ser Ala Leu Val Lys Gly Thr Pro 435 440 445 Asp Glu Val Arg Glu Lys Ala Lys Ala Phe Val Glu Lys Ile Arg Gly 450 455 460 Ala Thr Glu 465 <210> 2 <211> 1404 <212> DNA <213 > Artificial Sequence <220> <223> Component A <400> 2 atgggaatcc tgccaagccc tggaatgcca gccctgctgt ccctggtgtc tctgctgagc 60 gtgctgctga tgggatgcgt ggcagagacc ggaacaaggt tccctaacat caccaacctg 120 tgcccattcg gcga ggtgtt taacgccaca cgctttgcct ccgtgtatgc ctggaaccgg 180 aagagaatct ctaattgcgt ggccgactat agcgtgctgt acaatagcgc ctccttctct 240 acctttaagt gctatggcgt gtctcccacc aagctgaacg acctgtgctt cacaaacgtg 300 tacgccgaca gctttgtgat ccggggcgat gaggtgagac agatcgcacc aggacagacc 360 ggcaagatcg cagactacaa ctataagctg cctgacgatt tcacaggctg cgtgatcgcc 420 tggaatagca acaatctgga ttccaaagtg ggcggcaact acaattatct gtacaggctg 4 80 ttccgcaaga gcaacctgaa gccatttgag cgggacatca gcaccgagat ctaccaggca 540 ggctccacac catgcaacgg agtggagggc ttcaattgtt attttcccct gcagagctac 600 ggcttccagc ctaccaatgg cgtgggctat cagccataca gagtggtggt gctgtccttt 66 0 gagctgctgc acgcaccagc aaccgtgtgc ggacctaaga agtccacagg cggctctgga 720 ggaagcggat ccggaggatc cggaggatct ggaagcgaga aggcagcaaa ggcagaggag 780 gcagcaagga agatggagga gctgttcaag aagcacaaga tcgtggccgt gctgagagcc 840 aactctgtgg aggaggccat cgagaaggca gtggccgtgt tcgcaggagg agtgcacctg 900 atcgagatca cctttacagt gcccgacgcc gataccgtga tcaaggccct gtccgtgctg 960 aaggagaagg gagcaatcat cggagcagga accgtgacat ctgtggagca ggcaaggaag 1020 gcagtggagt ccggagccga gtttatcgtg tctcctcacc tggatgagga gatctcccag 1080 ttcgccaagg agaagggcgt gttttacatg cctggcgtga tgaccccaac agagctggtg 1140 aaggccatga agctgggcca caccatcctg aagctgttcc caggagaggt ggtgggacca 1200 cagtttgtga aggccatgaa gggcccattc cccaatgtga agtttgtgcc tacaggcggc 1260 gtgaacctgg acaatgtggc agagtggttc aaggcaggcg tgctggcagt gggagtggga 1320 tctgccctgg tgaagggaac cccagatgag gtgagggaaa aggccaaggc ctttgtggag 1380 aagatcaggg gagcaacaga gtga 1404 <210> 3 <211> 10704 <212> DNA <213> Artificial Sequence <220> <223> Plasmid (M-2560) <400> 3 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg t cggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420 tgcgtcgaga tgtttaaact cccgccccta actccgccca gttccgccca ttctccgccc 480 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta 540 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctagctggt 600 tctttccgcc tcagaaggta cctaaccaag ttcctctttc agaggttatt tcaggcc acc 660 ttccaccatg gccacctcag caagttccca cttgaacaaa aacatcaagc aaatgtactt 720 gtgcctgccc cagggtgaga aagtccaagc catgtatatc tgggttgatg gtactggaga 780 aggactgcgc tgcaaaaccc gcaccctgga ctgtgagccc aagtgtgtag aagagttacc 840 tgagtggaat tttgatggct ctagtacctt tcagtctgag ggctccaaca gtgacatgta 900 tctcagccct gttgccatg t ttcgggaccc cttccgcaga gatcccaaca agctggtgtt 960 ctgtgaagtt ttcaagtaca accggaagcc tgcagagacc aatttaaggc actcgtgtaa 1020 acggataatg gacatggtga gcaaccagca cccctggttt ggaatggaac aggagtatac 1080 tctgatgg ga acagatgggc acccttttgg ttggccttcc aatggctttc ctgggcccca 1140 aggtccgtat tactgtggtg tgggcgcaga caaagcctat ggcagggata tcgtggaggc 1200 tcactaccgc gcctgcttgt atgctggggt caagattaca ggaacaaatg ctgaggtcat 1260 gcctgcccag tgggaatttc aaataggacc ctgtgaagga atccgcatgg gagatcatct 1320 ctgggtggcc cgtttcatct tgcatcgagt at gtgaagac tttggggtaa tagcaacctt 1380 tgaccccaag cccattcctg ggaactggaa tggtgcaggc tgccatacca actttagcac 1440 caaggccatg cgggaggaga atggtctgaa gcacatcgag gaggccatcg agaaactaag 1500 caagcggcac cggtaccaca ttcgagccta cgatcc caag gggggcctgg acaatgcccg 1560 tcgtctgact gggttccacg aaacgtccaa catcaacgac ttttctgctg gtgtcgccaa 1620 tcgcagtgcc agcatccgca ttccccggac tgtcggccag gagaagaaag gttactttga 1680 agaccgccgc ccctctgcca attgtgaccc ctttgcagtg acagaagcca tcgtccgcac 1740 atgccttctc aatgagactg gcgac gagcc cttccaatac aaaaactaac gcccgcccca 1800 cgacccgcag cgcccgaccg aaaggagcgc acgaccccat gcatcgcaca catcataaga 1860 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaaatg ctttatttgt 1920 gaaatttgtg at gctattgc tttattgta accttataa gctgcaataa acaagttaac 1980 aacaacaatt gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttaa 2040 agcaagtaaa acctctacaa atgtggtaga attctacgta gataaaagtt ttgttacttt 2100 atagaagaaa ttttgagttt ttgttttttt taataaataa ataaacataa ataaattgtt 2160 tgttgaattt attattagta tgtaagtgta aatataataa aacttaatat ct attcaaat 2220 taataaataa acctcgatat acagaccgat aaaacacatg cgtcaatttt acacatgatt 2280 atctttaacg tacgtcacaa tatgattatc tttctagggt taatctagct gcgtgttctg 2340 cagcgtgtcg agcatcttca tctgctccat cacgctgtaa a acacatttg caccgcgagt 2400 ctgcccgtcc tccacgggtt caaaaacgtg aatgaacgag gcgcgctcat atcatgatta 2460 cgccaagcgc gcccgccggg taactcacgg ggtatccatg tccatttctg cggcatccag 2520 ccaggatacc cgtcctcgct gacgtaatat cccagcgccg caccgctgtc attaatctgc 2580 acaccggcac ggcagttcca tttaaatggc tgtcgccggt attgttcg gg ttgctgatgc 2640 gcttcgggct gaccatccgg aactgtgtcc ggaaaagccg cgacgaactg gtatcccagg 2700 tggcctgaac gaacagttca ccgttaaagg cgtgcatggc cacaccttcc cgaatcatca 2760 tggtaaacgt gcgttttcgc tcaacgtcaa t gcagcagca gtcatcctcg gcaaactctt 2820 tccatgccgc ttcaacctcg cgggaaaagg cacgggcttc ttcctccccg atgcccagat 2880 agcgccagct tgggcgatga ctgagccgga aaaaagaccc gacgatatga tcctgatgca 2940 gctagattaa ccctagaaag atagtctgcg taaaattgac gcatgcattc ttgaaatatt 3000 gctctctctt tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc tcagtcgccg 3060 cttggagctc ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt ttgaactata 3120 acgaccgcgt gagtcaaaat gacgcatgat tatcttttac gtgactttta agatttaact 3180 catacgataa ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata 3240 ttttcttgtt atagatatca agcttataga tctggggaca gccccccccc aaagccccca 3300 gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct 3360 ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg 3420 gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag 3480 acacctgggg ggatacgggg aaaaagcttt aggctgaaag agagatttag aatgacagaa 3540 tcatagaacg gcctgggttg caaaggagca cagtgctcat ccagatccaa ccccctgcta 3600 tgtgcagggt catcaaccag cagcccaggc tgcccagagc cacatccagc ctggccttga 36 60 atgcctgcag ggatggggca tccacagcct ccttgggcaa cctgttcagt gcgtcaccac 3720 cctctggggg aaaaactgcc tcctcatatc caacccaaac ctcccctgtc tcagtgtaaa 3780 gccattcccc cttgtcctat caagggggag tttgctgtga cattgttggt ctggggtgac 3840 acatgtttgc caattcagtg catcacggag aggcagattt ggggataagg aagtgcagga 3900 cagcatggac gtggga catg caggtgttga gggctctggg acactctcca agtcacagcg 3960 ttcagaacag ccttaaggat aagaagatag gatagaagga caaagagcaa gttaaaaccc 4020 agcatggaga ggagcacaaa aaggccacag acactgctgg tccctgtgtc tgagcctgca 4080 tgtttgatgg t gtctggatg caagcagaag gggtggaaga gcttgcctgg agagatacag 4140 ctgggtcagt aggactggga caggcagctg gagaattgcc atgtagatgt tcatacaatc 4200 gtcaaatcat gaaggctgga aaagccctcc aagatcccca agaccaaccc caacccaccc 4260 accgtgccca ctggccatgt ccctcagtgc cacatcccca cagttcttca tcacctccag 4320 ggacggtgac ccccccacct ccgtgggcag ctgtgccact gcagcaccgc tctttggaga 4380 aggtaaatct tgctaaatcc agcccgaccc tcccctggca caacgtaagg ccattatctc 4440 tcatccaact ccaggacgga gtcagtgagg atggggctct agagtcaaca ggaaagttcc 4500 attggagcca agtacattga gt caataggg actttccaat gggttttgcc cagtacataa 4560 ggtcaatggg aggtaagcca atgggttttt cccattactg gcacgtatac tgagtcatta 4620 gggactttcc aatgggtttt gcccagtaca taaggtcaat aggggtgaat caacaggaaa 4680 gtcccattgg agccaagtac actgagtcaa tagggacttt ccattgggtt ttgcccagta 4740 caaaaggtca atagggggtg agtcaatggg tttttcccat tattggcacg tacataaggt 4800 caataggggt gagtcattgg gtttttccag ccaatttaat taaaacgcca tgtactttcc 4860 caccattgac gtcaatgggc tattgaaact aatgcaacgt gacctttaaa cggtactttc 4920 ccatagctga ttaatgggaa agtaccgttc tcgagcca at acacgtcaat gggaagtgaa 4980 agggcagcca aaacgtaaca ccgccccggt tttcccctgg aaattccata ttggcacgca 5040 ttctattggc tgagctgcgt tctacgtggg tatataagca gagctctccc tatcagtgat 5100 agagatctcc ctatcagtga tagagatcga gctcagcgtc ggtaccgtac ctcttccgca 5160 tcgctgtctg cgagggccag ctgttggggt gagtggcggg t gtggcttcc gcgggccccg 5220 gagctggagc cctgctctga gcgggccggg ctgatatgcg agtgtcgtcc gcagggttta 5280 gctgtgagca ttcccacttc gagtggcggg cggtgcgggg gtgagagtgc gaggcctagc 5340 ggcaaccccg tagcctcgcc t cgtgtccgg cttgaggcct agcgtggtgt ccgccgccgc 5400 gtgccactcc ggccgcacta tgcgtttttt gtccttgctg ccctcgattg ccttccagca 5460 gcatgggcta acaaagggag ggtgtggggc tcactcttaa ggagcccatg aagcttacgt 5520 tggataggaa tggaagggca ggaggggcga ctggggcccg cccgccttcg gagcacatgt 5580 ccgacgccac ctggatgggg cgaggcctgt ggctttccga agcaatcggg cg tgagttta 5640 gcctacctgg gccatgtggc cctagcactg ggcacggtct ggcctggcgg tgccgcgttc 5700 ccttgcctcc caacaagggt gaggccgtcc cgcccggcac cagttgcttg cgcggaaaga 5760 tggccgctcc cggggccctg ttgcaaggag ct caaaatgg aggacgcggc agcccggtgg 5820 agcgggcggg tgagtcaccc acacaaagga agagggcctt gcccctcgcc ggccgctgct 5880 tcctgtgacc ccgtggtcta tcggccgcat agtcacctcg ggcttctctt gagcaccgct 5940 cgtcgcggcg gggggagggg atctaatggc gttggagttt gttcacattt ggtgggtgga 6000 gactagtcag gccagcctgg cgctggaagt cattcttgga atttgcccct tt gagtttgg 6060 agcgaggcta attctcaagc ctcttagcgg ttcaaaggta ttttctaaac ccgtttccag 6120 ctcgcggttg aggacaaact cttcgcggtc tttccagtac tcttggatcg gaaacccgtc 6180 ggcctccgaa cggtactccg ccaccga ggg acctgagcga gtccgcatcg accggatcgg 6240 aaaacctcgt cgacgccgcc accatgggaa tcctgccaag ccctggaatg ccagccctgc 6300 tgtccctggt gtctctgctg agcgtgctgc tgatgggatg cgtggcagag accggaaacaa 6360 ggttccctaa catcaccaac ctgtgcccat tcggcgaggt gtttaacgcc acacgctttg 6420 cctccgtgta tgcctggaac cggaagagaa tctctaattg cgtggccgac tatagcgtgc 648 0 tgtacaatag cgcctccttc tctaccttta agtgctatgg cgtgtctccc accaagctga 6540 acgacctgtg cttcacaaac gtgtacgccg acagctttgt gatccggggc gatgaggtga 6600 gacagatcgc accaggacag accggcaaga tcgcagacta caactataag ct gcctgacg 6660 atttcacagg ctgcgtgatc gcctggaata gcaacaatct ggattccaaa gtgggcggca 6720 actacaatta tctgtacagg ctgttccgca agagcaacct gaagccattt gagcgggaca 6780 tcagcaccga gatctaccag gcaggctcca caccatgcaa cggagtggag ggcttcaatt 6840 gttattttcc cctgcagagc tacggcttcc agcctaccaa tggcgtgggc tatcagccat 6900 acagagtggt ggtgctgtcc tttgagctgc tgcacgcacc agcaaccgtg tgcggaccta 6960 agaagtccac aggcggctct ggaggaagcg gatccggagg atccggagga tctggaagcg 7020 agaaggcagc aaaggcagag gaggcagcaa ggaagatgga ggagctgttc aagaagcaca 7080 agat cgtggc cgtgctgaga gccaactctg tggaggaggc catcgagaag gcagtggccg 7140 tgttcgcagg aggagtgcac ctgatcgaga tcacctttac agtgcccgac gccgataccg 7200 tgatcaaggc cctgtccgtg ctgaaggaga agggagcaat catcggagca ggaaccgtga 7260 catctgtgga gcaggcaagg aaggcagtgg agtccggagc cgagtttatc gtgtctcctc 7320 acctggatga ggagatctcc ca gttcgcca aggagaaggg cgtgttttac atgcctggcg 7380 tgatgacccc aacagagctg gtgaaggcca tgaagctggg ccacaccatc ctgaagctgt 7440 tcccaggaga ggtggtggga ccacagtttg tgaaggccat gaagggccca ttccccaatg 7500 tgaagt ttgt gcctacaggc ggcgtgaacc tggacaatgt ggcagagtgg ttcaaggcag 7560 gcgtgctggc agtgggagtg ggatctgccc tggtgaaggg aaccccagat gaggtgaggg 7620 aaaaggccaa ggcctttgtg gagaagatca ggggagcaac agagtgagcg gccgccacac 7680 atcataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaaatgc 7740 tttatttgtg aaatttgtga tgctattgct ttatt tgtaa ccattataag ctgcaataaa 7800 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag 7860 gttttttaaa gcaagtaaaa cctctacaaa tgtggtacac aaagtgggga gctctagagg 7920 gacagccccc ccccaaagcc cccagggatg taattacgtc cctcccccgc tagggggcag 7980 cagcgagccg cccggggctc cgctccggtc cggcgctccc cccgcatccc cgagccggca 8040 gcgtgcgggg acagcccggg cacggggaag gtggcacggg atcgctttcc tctgaacgct 8100 tctcgctgct ctttgagcct gcagacacct ggggggatac ggggaaaaag gggagctcta 8160 gagggacagc ccccccccaa agcccccagg gatgtaatta cgtccct ccc ccgctagggg 8220 gcagcagcga gccgcccggg gctccgctcc ggtccggcgc tccccccgca tccccgagcc 8280 ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa 8340 cgcttctcgc tgctctttga g cctgcagac acctgggggg atacggggaa aaaggatcca 8400 tggccggcca tctgcagatc atatgatcgg atgccgggac cgacgagtgc agaggcgtgc 8460 aagcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 8520 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 8580 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 8640 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 8700 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 8760 tatcagctca ctcaaaggcg gtaatac ggt tatccacaga atcaggggat aacgcaggaa 8820 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 8880 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 8940 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 9000 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctccctt cgg 9060 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 9120 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 9180 gtaactatcg tcttgagtcc aacccggtaa gacac gactt atcgccactg gcagcagcca 9240 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 9300 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 9360 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 9420 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 9480 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 9540 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaat gaagtt 9600 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 9660 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 9720 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagt gct gcaatgatac 9780 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 9840 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 9900 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 9960 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc gg ttcccaac 10020 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 10080 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 10140 tgcataattc tcttactgtc atgccatccg taaga tgctt ttctgtgact ggtgagtact 10200 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 10260 tacggggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 10320 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 10380 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 10440 aaacaggaag gcaaaatgcc gcaaaaaaagg gaataagggc gacacggaaa tgttgaatac 10500 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 10560 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 10 620 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata 10680 ggcgtatcac gaggcccttt cgtc 10704 <210> 4 <211> 204 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-2 RBD <400> 4 Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 1 5 10 15 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 20 25 30 Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 35 40 45 Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 50 55 60 Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 65 70 75 80 Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr 85 90 95 Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 100 105 110 Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 115 120 125 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 130 135 140 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 145 150 155 160 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 165 170 175 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 180 185 190 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr 195 200 <210> 5 <211> 204 <212> PRT <213> Artificial Sequence <220> < 223> synthetically constructed nanostructure polypeptide <400> 5 Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu Arg 1 5 10 15 Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe Ala 20 25 30 Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala Asp 35 40 45 Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile Ile 50 55 60 Gly Ala Gly Thr Val Thr Ser Val Glu Gln Ala Arg Lys Ala Val Glu 65 70 75 80 Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile Ser 85 90 95 Gln Phe Ala Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met Thr 100 105 110 Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Thr Ile Leu Lys 115 120 125 Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met Lys 130 135 140 Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn Leu 145 150 155 160 Asp Asn Val Ala Glu Trp Phe Lys Ala Gly Val Leu Ala Val Gly Val 165 170 175 Gly Ser Ala Leu Val Lys Gly Thr Pro Asp Glu Val Arg Glu Lys Ala 180 185 190 Lys Ala Phe Val Glu Lys Ile Arg Gly Ala Thr Glu 195 200 <210> 6 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> Component B <400> 6 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asn Gly Met Met Asn Val Gln Leu Asn Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Asn Tyr Asp Lys Ser Lys Ala His Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 7 <211> 474 <212> DNA <213> Artificial Sequence <220> <223> Component B <400 > 7 atgaaccagc acagtcacaa ggaccacgaa acggtaagaa tagcggtagt gcgtgcgcgt 60 tggcatgcgg aaattgtgga cgcctgtgtg agtgcgtttg aagccgcgat gcgtgatatt 120 ggcggcgatc gttttgccgt ggatgtgttt g atgtgccgg gtgcgtacga aattccactg 180 catgcgcgta ccctggcgga aaccggccgt tatggcgcgg tgttaggcac cgcctttgtg 240 gtgaatggtg gcatttatcg tcacgaattt gtggcgagcg cggttattaa cggcatgatg 300 aatgtgcagc tgaac acggg cgtgccagtg ttaagtgccg tgctgacccc acacaactat 360 gataaaagca aagcccatac cctgctgttc ttagcgctgt ttgcggtgaa aggcatggaa 420 gcggcgcgtg cctgcgtgga gattttagcg gcccgtgaaa agattgcggc gtga 474 <210> 8 <211> 5712 <212> DNA <213> Artificial Sequence <220> <223> plasmid <400> 8 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgac gttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt a caatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600 tcatatcagg attatcaata ccatattttt gaaaaaagccg tttctgtaat gaaggagaaa 660 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900 cgttatcat tcgtg attgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140 taaatt ccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 132 0 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380 cccttgtatt actgtttatg taagcagaca gtttattgt tcatgaccaa aatcccttaa 1440 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560 gtggt ttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980 ccagggggaa acgcctggta tctttatagt c ctgtcgggt ttcgccacct ctgacttgag 2040 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400 ggtcatggct gcgccccgac acccgccaac acccgct gac gcgccctgac gggcttgtct 2460 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580 gtgaagcgat tcacaga tgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000 cgaaacacgg aaaccgaaga ccattcatgt tgttgctca g gtcgcagacg ttttgcagca 3060 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240 ggcttgagcg agggcgtgca agattccgaa taccgcaagc ga caggccga tcatcgtcgc 3300 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420 ccggaaggag ctgactgggt tgaaggctct caaggg catc ggtcgagatc ccggtgccta 3480 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcc cc agcaggcgaa 3720 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780 atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840 cgcccagcgc catctgatcg ttgg caacca gcatcgcagt gggaacgatg ccctcattca 3900 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140 ggt cagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag tagg ttgagg 4740 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc 4980 gcgaaattaa tacgactc ac tataggggaa ttgtgagcgg ataacaattc ccctctagaa 5040 ataattttgt ttaactttaa gaaggagata tacatatgaa ccagcacagt cacaaggacc 5100 acgaaacggt aagaatagcg gtagtgcgtg cgcgttggca tgcggaaatt gtggacgcct 5160 gtgtgag tgc gtttgaagcc gcgatgcgtg atattggcgg cgatcgtttt gccgtggatg 5220 tgtttgatgt gccgggtgcg tacgaaattc cactgcatgc gcgtaccctg gcggaaaccg 5280 gccgttatgg cgcggtgtta ggcaccgcct ttgtggtgaa tggtggcatt tatcgtcacg 5340 aatttgtggc gagcgcggtt attaacggca tgatgaatgt gcagctgaac acgggcgtgc 5400 cagtgttaag tgccgtgctg acccc acaca actatgataa aagcaaagcc cataccctgc 5460 tgttcttagc gctgtttgcg gtgaaaggca tggaagcggc gcgtgcctgc gtggagattt 5520 tagcggcccg tgaaaagatt gcggcgtgac tcgagcacca ccaccaccac cactgagatc 5580 cggctgctaa caa agcccga aaggaagctg agttggctgc tgccaccgct gagcaataac 5640 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa 5700 ctatatccgg at 5712 <210> 9 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 9 Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Ala Glu His His Arg 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 10 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 10 Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Ala Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Ala His Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 11 <211 > 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 11 Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Gln Cys Val Arg Ala 20 25 30 Phe Glu Glu Ala Met Ala Asp Ala Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Ser Ser Arg Glu His His Glu 115 120 125 Phe Phe Arg Glu His Phe Met Val Lys Gly Val Glu Ala Ala Ala Ala 130 135 140 Cys Ile Thr Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 12 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 12 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Glu His His Arg 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Asn Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 13 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 13 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asp Gly Gly Ile Tyr Asp His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Glu Asp His Glu 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Asn Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 14 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 14 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Ala His Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 15 <211 > 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 15 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asp Gly Gly Ile Tyr Asp His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Ala Asp Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 16 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <220> <221> MISC_FEATURE <222 > (9) <223> Xaa is Tyr or His <220> <221> MISC_FEATURE <222> (82) <223> is Arg or Asp <220> <221> MISC_FEATURE <222> (105) <223> Xaa is Ser or Asp <220> <221> MISC_FEATURE <222> (119) <223> Xaa is Arg or Glu <220> < 221> MISC_FEATURE <222> (121) <223> ) <223> Xaa is His or Asp <220> <221> MISC_FEATURE <222> (128) <223> Asn <400> 16 Met Asn Gln His Ser His Lys Asp Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Ser > 205 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 17 Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Thr Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn 145 150 155 160 Leu Asp Asn Val Cys Glu Trp Phe Lys Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Ser Ala Leu Val Lys Gly Thr Pro Asp Glu Val Arg Glu Lys 180 185 190 Ala Lys Ala Phe Val Glu Lys Ile Arg Gly Cys Thr Glu 195 200 205 <210> 18 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide < 400> 18 Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn 145 150 155 160 Leu Asp Asn Val Cys Glu Trp Phe Lys Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Asp Ala Leu Val Lys Gly Asp Pro Asp Glu Val Arg Glu Lys 180 185 190 Ala Lys Lys Phe Val Glu Lys Ile Arg Gly Cys Thr Glu 195 200 205 <210> 19 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 19 Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Glu Phe Val Glu Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asp 145 150 155 160 Leu Asp Asp Val Cys Glu Trp Phe Asp Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Asp Ala Leu Val Glu Gly Asp Pro Asp Glu Val Arg Glu Asp 180 185 190 Ala Lys Glu Phe Val Glu Glu Ile Arg Gly Cys Thr Glu 195 200 205 <210> 20 <211> 205 <212> PRT <213> Artificial Sequence <220> <223 > synthetically constructed nanostructure polypeptide <400> 20 Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn 145 150 155 160 Leu Asp Asn Val Cys Lys Trp Phe Lys Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Lys Ala Leu Val Lys Gly Lys Pro Asp Glu Val Arg Glu Lys 180 185 190 Ala Lys Lys Phe Val Lys Lys Ile Arg Gly Cys Thr Glu 195 200 205 <210> 21 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <220> <221> MISC_FEATURE <222> (126) <223> Xaa is Thr or Asp <220> <221> MISC_FEATURE <222> (139) <223> Xaa is Gln or Glu <220> <221> MISC_FEATURE <222> (142) <223> Xaa is Lys or Glu <220> < 221> MISC_FEATURE <222> (160) <223> MISC_FEATURE <222> (163) <223> Xaa is Asn or Asp <220> <221> MISC_FEATURE <222> (166 ) <223> Xaa is Glu or Lys <220> <221> MISC_FEATURE <222> (169) <223> Lys or Asp <220> <221> MISC_FEATURE <222> (183) <223> Xaa is Lys or Glu <220> <221> MISC_FEATURE <222> (185) <223> <221> MISC_FEATURE <222> (192) <223> > (198) <223> Xaa is Glu or Lys <220> <221> MISC_FEATURE <222> (199) <223> Xaa is Lys or Glu <400> 21 Met Lys Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Pro Xaa Phe Val Xaa Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Gly

Claims (20)

(a) (i) cultivating a Chinese Hamster Ovary (CHO) cell line expressing fusion protein A of SEQ ID NO: 1,
glucose; and adding a culture additive containing Hyclone CellBoost 7A and Hyclone CellBoost 7B, wherein the addition includes glucose to the cell culture medium on or after the second day of culture; and alternately adding Hyclone CellBoost 7A and Hyclone CellBoost 7B at intervals of at least one day, and maintaining the pH of the culture medium at 6.75 to 7.15;
(ii) recovering and purifying the fusion protein A from the culture medium of (i), which includes centrifuging the culture medium of (i) to recover the supernatant and filtering the recovered culture medium,
After the filtration, treatment with nuclease and performing first, second and third chromatography, wherein the first chromatography step is hydrophobic interaction chromatography and the second chromatography. A purification step, wherein the step is Mixed Mode Chromatography, and the third chromatography step is Anion Exchange Chromatography;
(b) (i) seed and main culture of E. coli expressing protein B of SEQ ID NO: 6,
A culture step, wherein the growth phase culture medium after the seed fermentation step includes glycerol;
(ii) recovering and purifying the protein B from the culture medium of (i),
A purification step comprising suspending E. coli into a suspension, disrupting the suspension, treating with nuclease, and performing first anion exchange chromatography and second anion exchange chromatography; and
(c) mixing the fusion protein A obtained in (a) and protein B obtained in (b) to form a self-assembled multimeric protein assembly; and
(d) A method for producing a multimeric protein assembly, comprising the step of filtering the reaction product of assembly formation in (c),
A manufacturing method wherein (a) and (b) are performed simultaneously, performed at time intervals, performed sequentially, or performed in reverse order. The manufacturing method according to claim 1, wherein the filtration in (d) includes ultrafiltration and diafiltration (UF/DF). The manufacturing method according to claim 1, wherein (a) (i) includes seed culture and main culture steps. The manufacturing method according to claim 1, wherein in the step of centrifuging the cell culture medium of (a) (ii) and recovering the supernatant, the feed flow during centrifugation is 200 L/h to 400 L/h. . The production method according to claim 1, wherein the pH of the culture medium of (a) (i) is maintained at 7.1. The manufacturing method according to claim 1, wherein (a) (ii) includes virus filtration and ultrafiltration. The production method according to claim 1, wherein (a) (ii) secondary mixed mode chromatography is ceramic hydroxyapatite chromatography. The production method according to claim 1, wherein (a) (i) includes maintaining the glucose concentration of the culture medium at 5.0 to 7.0 g/L. The production method according to claim 1, wherein the E. coli of (b) (i) is an E. coli BL21 strain. The manufacturing method according to claim 1, wherein the air sparging rate introduced into the culture medium in the late stage of the culture in (a) (i) is less than half of the air sparging rate in the early stage of the culture. The method of claim 1, wherein (a) (ii) adjusts the concentration of nuclease to 2 unit/ml to 14 unit/ml; Adjusting the nuclease treatment time from 2 hours to 7 days; A manufacturing method comprising controlling one or more factors selected from the group consisting of controlling the concentration of Mg ²⁺ ions in the culture medium to 2mM or less. The production method according to claim 1, wherein the growth phase culture medium after the seed fermentation step of (b) (i) contains glycerol in a content exceeding 0 and less than 4% (v/v). The preparation method according to claim 1, wherein the equilibration, washing, and elution buffer used in the secondary chromatography of (b) (ii) contains detergent. The production method according to claim 1, wherein the volume of the suspension in (b) (ii) is 8 ml to 14 ml per 1 g of E. coli. The method of claim 1, wherein the filtration in (d) includes ultrafiltration and diafiltration (UF/DF),
The (a) (ii) secondary mixed mode chromatography is a preparation method of ceramic hydroxyapatite (Ceramic Hydroxyapatite) chromatography. The method of claim 1, wherein the mixing reaction of (c) includes polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, and Pollock. Samer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, arginine A manufacturing method, which is carried out in the presence of a buffer containing a stabilizer selected from sucrose or a combination thereof. According to paragraph 1,
(a) (i) includes maintaining the glucose concentration of the culture medium at 5.0 to 7.0 g/L,
The air sparging rate introduced into the culture medium at the late stage of the culture in (a) (i) is less than half of the gas sparging rate before the culture,
(a) (ii) above includes virus filtration and ultrafiltration,
In the step of recovering the supernatant by centrifuging the cell culture medium of (a) (ii), the feed flow during centrifugation is 200 L/h to 400 L/h,
In (a) (ii), the concentration of nuclease is adjusted to 2 unit/ml to 14 unit/ml; Adjusting the nuclease treatment time from 2 hours to 7 days; and controlling one or more factors selected from the group consisting of adjusting the concentration of Mg2+ ions in the culture medium to 2mM or less,
After the seed fermentation step of (b) (i), the growth phase culture medium of E. coli contains glycerol in a content exceeding 0 and less than 4% (v/v),
The volume of the suspension in (b) (ii) is 8 ml to 14 ml per gram of E. coli,
The equilibration, washing, and elution buffer used in the secondary chromatography of (b) (ii) contains detergent,
The filtration of (d) includes ultrafiltration and diafiltration (UF/DF). The method according to any one of claims 1 to 17, wherein the multimeric protein assembly has an icosahedral structure consisting of 20 trimers of fusion protein A and 12 pentamers of protein B. 18. A method of preparing an immunogenic composition comprising preparing a multimeric protein assembly according to any one of claims 1 to 17. A method for producing a vaccine composition for preventing COVID-19, comprising preparing a multimeric protein assembly according to any one of claims 1 to 17.