This invention is a recombinant protein for the purpose of animal cell cultures that have arisen during the course of the aforementioned animal cell culture fluid of osmotic pressure increase steps, Galactose (galactosylation) is regulated, manufacturing method of the recombinant protein for the purpose or purposes of the recombinant protein to control the angry Galactose; Recombinant protein expression to the purpose of animal cell cultures to be reminded during the course of animal cell culture supplements the steps in aseupara Jin, Galactose is the purpose of recombinant proteins, which regulate the upset of a recombinant protein for the purpose of manufacturing or Galactose shoes how to adjust; Recombinant protein expression to the purpose of animal cell cultures to be reminded of the animal cell culture process of osmotic pressure rises, aseupara Jean complement steps, Galactose is the purpose of recombinant proteins, which regulate the upset of a recombinant protein for the purpose of manufacturing or Galactose shoes how to adjust; And by the way, Galactose is the purpose of recombinant proteins that regulate the upset.

[2]

Background technology

[3]

Antibody Antigen to combine the functions of antigens or Antigen, a protein that can be removed to treat, as the market expects a significant potential antibodies balghyeojime and efficient expression and antibody-based for mass production are being actively researched. These critical points in the production of antibodies, one of a group of antibodies manufactured homogeneity. In particular, the monoclonal antibody production you're in constant and reproducible per profile of monoclonal antibodies (glycoform profile) is important. However, during the process of cell culture produced a wide variety of variables of the monoclonal antibody can affect the sugar profile. This, manufactured antibodies of glycosylated, and properties such as constant content of antibodies can be manipulated in a way that has been required by the development of.

[4]

[5]

During the course of the commercial production of monoclonal antibodies is one of the important challenges produced for each placing monoclonal antibodies to maintain a consistent quality of. Monoclonal antibodies should be checked during the production process quality is no different but the antibodies of Galactose are among them, one of the most important things to be seen. Is Galactose the monoclonal antibodies are the biggest activity of the machine (MOA) is one of the CDC (complement dependent cytotoxicity) would affect the process because it is known. The above is a true story Lactobacillus go go lactose (Galactose) glico true story of chain reaction unit, and used to go the Lactobacillus room transferase (galactosyltransferase), N-acetyl Glucosamine (N-acetylglucosamine) takes place by then. Adjusting these mad Lactobacillus rooms go for manganese or Galactose in cultured and how this proposed annexation to the bar, but I (United States public Patent No. 2012-0276631), antibody to go how do I adjust the Lactobacillus true story of development is still required.

[6]

A detailed description of the invention

Technical challenges

[7]

Who invented these backgrounds in recombinant protein production for animal cell culture, recombinant proteins produced in the process of going out of Lactobacillus can effectively adjust the true story in order to develop methods for recombinant protein expression, as a result of the efforts of animal cells during the process of the cultivation of a particular culture at the time of the rise of animal cell culture fluid of osmotic pressure, or if complementing aseupara Jin, is produced in recombinant proteins of Lactobacillus can effectively produce a true story. In addition, animal cell culture fluid at the same time, the rise of osmotic pressure, aseupara Jean, if you replenish the osmotic pressure, resulting in a rise in acid water of this nature go without lactose reduced antibody-a true story, by checking to see that you can adjust the invention was complete.

[8]

Challenges in the Sudan

[9]

Purpose the purpose of this invention is one of the recombinant protein, animal cell cultures that have arisen during the course of the aforementioned animal cell culture fluid of osmotic pressure increase steps, Galactose (galactosylation) is adjusted, the purpose is to provide a way for the manufacture of recombinant proteins.

[10]

The purpose of this invention of recombinant protein to another for the purpose of animal cell cultures that have arisen during the course of the aforementioned animal cell culture fluid of osmotic pressure increase, which include the steps in the the purpose of recombinant protein of Galactose to provide a way to control the angry.

[11]

The purpose of this invention of recombinant protein to another for the purpose of animal cell cultures that have arisen during the course of the aforementioned animal cell culture supplements the steps in aseupara Jin, Galactose is the purpose of recombinant proteins, which regulate the painter's manufacturing methods.

[12]

The purpose of this invention of recombinant protein to another for the purpose of animal cell cultures that have arisen during the course of the above supplements in animal cell culture aseupara Jin, which include the steps that are for the purpose of recombinant protein of Galactose to provide a way to control the angry.

[13]

The purpose of this invention of recombinant protein to another for the purpose of animal cell cultures that have arisen during the course of the aforementioned animal cell culture fluid of osmotic pressure, elevation, and aseupara to complement the steps, Galactose is the purpose of recombinant proteins, which regulate the painter's manufacturing methods.

[14]

The purpose of this invention of recombinant protein to another for the purpose of animal cell cultures that have arisen during the course of the aforementioned animal cell culture fluid of osmotic pressure, elevation, and aseupara to supplement, which include steps for the purpose of recombinant protein of Galactose to provide a way to control the angry.

[15]

Another purpose of this invention was manufactured for the purpose of the above ways, the recombinant protein.

[16]

The effect of the invention

[17]

The method of this invention is for the purpose of recombinant protein of Galactose can be adjusted to the desired range of content effectively have an advantage. In addition, with the increase in osmotic pressure and using both saw annexation of Jin aseupara invented a way of Galactose as well as acidic water of this nature can also adjust the antibody effect, therefore, for the purpose of manufacturing the antibodies can be used effectively.

[18]

Drawing, brief description

[19]

Unlike non-thank you one is bio: Miller's expression vector to the city.

[20]

A metaphor expression 2 culture during the period of the sodium chloride aqueous solution at an infusion of city.

[21]

Also 3 oil aseupara during the period of Qin culture injection point of the expression of the city.

[22]

The best form for the implementation of the invention

[23]

For the purposes of the above one of this invention to achieve the purpose of recombinant protein expression Hamm to animal cell cultures to be reminded of the animal cell culture process of osmotic pressure increase steps, Galactose is the purpose of recombinant proteins, which regulate the upset of the manufacturing method.

[24]

[25]

Specifically, the above method is a recombinant protein for the purpose of animal cell cultures that have arisen during the course of the aforementioned animal cell culture fluid from a specific culture of osmotic pressure, step, Galactose is the purpose of recombinant proteins, which regulate the upset of the manufacturing method.

[26]

[27]

In this invention, a metaphor expression culture Petri (fed-batch culture), but is not limited to this.

[28]

In this invented term, "culture, the expression (fed-batch culture) in oil prices" is the default badge or as a badge building after it starts production of cell growth or cultivate any time of antibody production in continuous or discrete feeding in water supplying the badges to proceed with cell culture.

[29]

This invention, culture, cell culture "from the badge" or "badge" is a multicellular organism or organization outside the artificial maintenance of cells in vitro, growth, nutrient solution for hyperplasia or swelling. Cell culture badge can be optimized for a specific cell cultures, for example the default cell growth in support for the deli culture badge, or monoclonal antibodies produced in cell culture have been dispensed to promote the production of badges, nutrient enriched by high concentration enrichment have a badge. Nutrients, badge, badges, cell culture, Iran terms the elements are components that make up the meaning of the elements in this specification will be used.

[30]

Specifically, the default "default culture badge" or "badge" the term can support the growth of a minimum number of cells means a badge. The basic badge is standard inorganic salts, for example zinc, iron, magnesium, calcium and potassium, as well as trace elements, vitamins, energy, won (Buffer) system and supply of amino acids. Two variations of the nose Eagle badge of Versailles (DMEM), the default badge Eagle (BME), RPMI 1640, F-10, F-12, the default badge a badge includes, but is not limited to.

[31]

Also, specifically, "cell culture production produces badge" the badge "or" term bioreactor in monoclonal antibodies are used for the purpose of maximize the manifestation of the badge. The default is the same as the badge the badge or the production can vary, and if you vary the default badge itself enriched by certain elements of the default badge or add them in a way that can produce.

[32]

This invention was used in the "feeding (feeding) badge" and the term "additional culture badge" consisting of a specific nutrient or more nutrients as a badge can be the default badge all agricultural. Depending on the feeding cells cultured at a wide range of components and concentrations can produce.

[33]

[34]

I'm in the process of the above cultures, cell growth is the growth of the cells after inoculation when all of a sudden is in progress. In the case of People's Republic of China hamster ovary (CHO) cell temperature of 35-37 ° c, pH range from 7.0 to 7.3 have the most active cells are known to increase in number. This invention, the cell growth in culture parameters of temperature, 7.1 or 7.0 36 pH.

[35]

The above "inoculation" Iran the term cell culture in order to start injecting the badge and injecting the cells injected badge. This badge is infused in advance before injecting the cells or cells and at the same time, the cells can be injected in the reactor. A large number of animal cell culture, normally infused in advance and the badge temperature and oxygen saturation after which cells maintain a constant setting range can inject.

[36]

The above term "antibody production" monoclonal antibodies in order to maximize the production of culture process applies a change of time. This is a change of the process to maximize the production of the lower temperature and dissolved oxygen, both to replace the badge.

[37]

[38]

This invention, the osmotic pressure is rising, according to recombinant protein expression at the animal cell is produced in recombinant protein of Galactose is subject to much change of was confirmed. In other words, rising gradually over a period of osmotic pressure, cultured if not elevate, osmotic pressure, military and galactose shoes do not cultivate, on the other hand, the degree varies greatly during the period at a specific culture osmosis if the G0F content is greatly elevated sea level rise leads to the result, Galactose mad control, specifically the Galactose inhibits or angry may decline.

[39]

[40]

In addition, this invention from osmosis pressure increase step is a recombinant protein for the purpose of animal cell culture, seen to have arisen during the course of the preferable one, what happens is not limited.

[41]

In this specification, the term "culture" used in Iran, the actual production of monoclonal antibodies to cell culture phase takes place. Cell culture process, which began with a vial as a final step, this culture, this terminates the production of monoclonal antibodies of the refining process. This culture and increase, step by step, the purpose of the previous culture bulk seed (seed) can be distinguished with the term Iran.

[42]

[43]

The steps to rise above osmotic pressure for animal cell culture, this culture of the process can be performed at a certain of, specifically, this culture has seen the start of 0, 1 to 10, the cultivation of which one culture can be performed on the job, and more specifically on the basis of this culture started from 0, 1, 4, 7, and 10, which can be performed in a single culture, and, more specifically, on the basis of this culture started at 0, 4, or 7 days can be performed on the It is not limited.

[44]

In addition, the purpose of the aforementioned osmotic pressure increase step is a recombinant protein expression during the course of the cultivation of animal cells only (單 fortunate) can be performed.

[45]

[46]

In addition, the aforementioned osmotic pressure increase step is especially Petri are not limited to, the amount or the final osmotic pressure within the 460 500 mOsm/kg can be performed, so that more specifically the rise of the culture fluid seepage pressure above the final osmotic pressure within the 460 to 480 mOsm/kg so that can be performed, but is not limited to this. The final osmotic pressure within the aforementioned culture fluid culture appearing in the osmotic pressure point would say, is not limited to, specifically.

[47]

In addition, the aforementioned osmotic pressure increase step is cultivating output rose 400 to osmosis pressure within a goal 440mOsm/kg, and more specifically the 430 to 440 mOm/kg, but is not limited to this. Rising above the goal within the osmotic pressure, the liquid cultivating osmotic pressure increase at the following the steps rise to the level of the osmotic pressure, said one, is not restricted to this.

[48]

In addition, rising above the aforementioned osmotic pressure for animal cell culture fluid of sodium chloride or potassium chloride supplement and glucose in culture is made up of the military as a way to add in more ways than the selected from 1 can be done, and more specifically in cultivating sodium chloride can be done as a way to supplement, but not limited to.

[49]

Culture in sodium chloride above for more information about how to recruit, cultivate their progress on a particular day, the expression 4M culture liquid aqueous solution sodium chloride by adding up to a certain level, osmotic pressure, may be performed as a way to. This is not limited to. Sodium chloride aqueous solution through the culture, add further within the cell osmosis pressure increase offered to all active beta-go rock Toshima-Ku, Tokyo increased by monoclonal antibodies of Galactose can be suppressed angry.

[50]

This invention in "culture" or "culture output" Iran the term culture ongoing Shaker flask or bioreactor in cell, containing the liquid. Culture, culture is above the water and can be used with each other. In addition, animal cell culture, cell culture, depending on the presence or absence of fluid and can be distinguished from the badge.

[51]

[52]

This invention of recombinant protein, antibody, from, and preferable, are monoclonal antibodies.

[53]

This specification is used in a "monoclonal antibody (monoclonal antibody)" refers to the term single-antibody-forming cells that can produce antibody, antibody recognizes to determine the one won.

[54]

This invention of antibodies are not limited to, in the event that future in the field normally used for therapeutic antibodies can contain both, specifically, unlike non-thank you, Ah.

[55]

In addition, the aforementioned antibodies are antibodies and antibody fragments the battlefield in the form of a concept that contains all the above kinds of Fab antibody fragment Fv, Fab a F (ab '), '2, Fd includes all. The aforementioned Fv Fv (dsFv) disulfide double d and Fv (scFv) form a single chain includes both. Fd is a Fab short of printing, which is included in the part.

[56]

The above is for the purpose of recombinant protein expression of recombinant proteins for animal cell to a natural manifestation of the above type or transgenic cells including without limitation. For the purposes of this invention Galactose content control recombinant protein expression, which is the subject of the animal cells can be able to do, for example, People's Republic of China hamster ovary cell State (CHO) or mouse myeloma cell week (NSO), but is not limited to this.

[57]

[58]

In addition, the monoclonal antibodies of Galactose in order to adjust the culture parameter optimization of sign language and the default culture enriched the badge, optimized can be additional culture badge feeding stages.

[59]

In other words, the desired monoclonal antibodies of Galactose-Hwa, set the scope of a cell, the Lord, is a manifestation of monoclonal antibodies of Galactose-Hwa after analyzing Galactose, change the requirements stated above, depending on the degree of monoclonal antibodies of Galactose can be applied along the way to control the angry.

[60]

Specifically, the adjusted Galactose shoes with monoclonal antibodies in the incubator for the production of operational examples of changes that you can implement for dissolved oxygen (DO), acidification (pH), temperature, Agitation, including the cultivation of parameters optimization of the cultivation. Specifically, cells grow in the temperature of 22.7 ° c, dissolved oxygen is 30%, and even culture and the 7.0 acidification, antibody production in the temperature is 30 ° c, dissolved oxygen is also a 30% acidification by applying existing methods of cultivating 7.0, antibody production in temperatures of 28 ° c, 20% of the dissolved oxygen, acidification is also a 4.3 by changing culture when monoclonal antibodies of adjustable compartment of galactose.

[61]

In addition, Galactose, in order to adjust the default culture of the badge can be used in the enriched by. Specifically, there is no culture of animal origin proteins using the badge, basic badge developed for cultured expressions monoclonal antibodies of Galactose can be optimized to change shoes. Specifically, the default culture enriched the badge, specifically 0.5 times or 1.4 times the concentration used in this culture, a badge: monoclonal antibodies of adjustable of galactose.

[62]

In addition, the adjustment of Galactose can be optimized further to culture badge. This invention adds additional culture badge concentration adjustment, culture is the concentration of the metal components add badges by optimizing Galactose decline.

[63]

[64]

The above is for the purpose of recombinant proteins manufactured through the way of Galactose, N-Q-TOF or UPLC devices with glitches Khan profile (N-glycan profile) can be measured with the analysis. N-glitches Khan profile analysis of the information provided by the G0F content, GI (Galactosylation Index), NGHC (non-glycosylated Heavy Chain), non-true story (afucosylation) percent, but Foucault North content (High mannose contents) into one, this invention provides G0F content changes mainly mentioned.

[65]

[66]

In addition, the method of this invention Galactose painter reduced to manufacture antibodies can be for the collective. In other words, cells are stock manifestations monoclonal antibodies of Galactose, after analyzing the analyzed values Tue, compared to lower levels of Galactose mad thing is that if you want to manufacture a group of antibodies that can be applied to.

[67]

[68]

In this invention, the term, "antibodies" can vary per printing content, including antibodies, antibody, meaning the military purposes of the invention, the foregoing antibody group is a true story of Lactobacillus antibodies purpose go proportions, which contain Galactose means an army of angry conditioned antibodies.

[69]

[70]

This is yet another of the invention for the purpose of recombinant protein expression Hamm animal cells during the process of the cultivation of animal cell culture fluid of osmotic pressure above the rising step, the purpose of recombinant protein of Galactose is a way to control the angry.

[71]

With regard to the above and how each term is mentioned.

[72]

Specifically, the purpose of the above method of recombinant proteins in animal cell cultures that have arisen during the course of the aforementioned animal cell culture fluid seepage pressure rise in a specific culture, which can contain specific steps for the content as described above.

[73]

[74]

This is yet another of the invention for the purpose of recombinant protein expression Hamm animal cells during the process of the cultivation of the aforementioned animal cell culture supplements the steps in aseupara Jin, Galactose is the purpose of recombinant proteins, which regulate the upset of the manufacturing method.

[75]

With regard to the terminology described above, as described earlier.

[76]

The purpose of this invention of recombinant protein to animal cell cultures that have arisen during the course of the above supplements in animal cell culture aseupara Jean, a recombinant protein for the purpose of Galactose can be adjusted to get angry.

[77]

[78]

Specifically, the aforementioned aseupara Jean aseupara Jean aseupara Jean concentrate or to include culture in the form of the badge can be added.

[79]

In addition, the aforementioned aseupara's replacement to the purpose of recombinant protein expression during the course of the cultivation of certain animal cell cultures can be performed all at the circuit. Here, the aforementioned aseupara's replacement on the basis of the culture this culture starts 0 days 4-10 days, and, specifically, on the basis of cultivation of this culture started from 0, 4th, 7th and 10th, to perform both in the amount of aseupara gene in cultured concentration gradually rising.

[80]

Aseupara Jin if only circuit replacement, culture+ NH4in aqueous humor concentrations ended up being sharply increased cell growth delay and the reduction of the amount of the final protein expression to bring the yen to a disadvantage, but this aseupara according to the invention of the pendulum and feeding: won share if the annexation, these shortcomings without Galactose mad as a positive for the purpose of protein production has the advantage that can be achieved.

[81]

[82]

Animal cell culture supplements the above aseupara's output to the final concentration of 27.6 33.6 mM aseupara within a gene can do so.

[83]

Specifically, animal cell culture fluid of 33.6 mM final concentration so that the aseupara gene in aseupara Jin can complement, for example for animal cell culture, of output in addition to increasing the concentration 18mM aseupara Jean aseupara Jean, to six, you can complement the more specific example for animal cell culture, of output 6mM, 12mM and aseupara in gene concentration 18mM by adding aseupara sequentially by Jean supplement three times, but is not limited to this.

[84]

These methods applied to animal cells, the ammonium ion (NH4+) created by guiding an increase in intracellular ammonium concentration and pH of the delay in the increase in the bone marrow TRANS-beta-active Lactobacillus room transfer go la offers drop monoclonal antibody inhibition of Galactose can be angry.

[85]

[86]

In addition, as described earlier, monoclonal antibodies of Galactose in order to adjust the culture parameter optimization of sign language and the default culture enriched the badge, optimized can be additional culture badge feeding stages.

[87]

In other words, the desired monoclonal antibodies of Galactose-Hwa, set the scope of a cell, the Lord, is a manifestation of monoclonal antibodies of Galactose-Hwa after analyzing Galactose, change the requirements stated above, depending on the degree of monoclonal antibodies of Galactose can be applied along the way to control the angry.

[88]

[89]

This is yet another of the invention for the purpose of recombinant protein expression Hamm animal cells during the process of the cultivation of the aforementioned animal cell culture supplements the steps in aseupara Jin, a recombinant protein for the purpose of Galactose is a way to control the angry.

[90]

With regard to the above and how each term is mentioned.

[91]

[92]

This is yet another of the invention for the purpose of recombinant protein expression Hamm animal cells during the process of the cultivation of the aforementioned animal cell culture fluid of osmotic pressure, elevation, and aseupara to complement the steps, Galactose is the purpose of recombinant proteins, which regulate the upset of the manufacturing method.

[93]

With regard to the terminology described above, as described earlier.

[94]

This is the purpose of the invention of recombinant proteins, monoclonal antibodies, animal cell cultures that have arisen during the course of animal cell culture fluid of osmotic pressure, ensuring you are adding aseupara Jin, osmotic pressure rise due to the decrease of galactose and at the same time, the rise can reduce according to the osmotic pressure, acid content of this nature antibody aseupara's addition was determined that increased by.

[95]

[96]

The above methods above, osmosis pressure rise and aseupara's replacement can be carried out at the same time or sequentially. For example, the culture fluid pressure, osmosis in sodium chloride, such as how to complement the rise and aseupara Jean aseupara Jean complement or supplement, cultured, liquid seepage pressure within the sodium chloride rose, or in such a way that the annexation of the aseupara Qin supplements and culture at the same time, a rise in the amount to be applied within the osmotic pressure.

[97]

This is about the rise of the osmotic pressure, as described earlier, can be performed by applying the method.

[98]

The rise above the osmosis pressure culture output, the final osmotic pressure within the 460 to 500 mOsm/kg can be performed, so that the above aseupara's animal cell culture fluid supplement is aseupara within the gene so that the final concentration is m M 27.6 to 33.6 aseupara Jean.

[99]

[100]

In addition, the aforementioned recombinant protein antibodies, specifically monoclonal antibodies can be seen, such as the invention of the methods above, the purpose of recombinant antibodies at the same time, the regulation of Galactose manufactured antibodies as a way to remind a group of acid water of antibodies to this property. In other words, the osmotic pressure is on the rise along the Galactose shoes reduces at the same time, can reduce, depending on the rising osmotic pressure, acid aseupara content of this nature antibodies can increase as the Qin added.

[101]

Therefore, you can use the same method as above and for the purpose of antibody of Galactose can adjust how and simultaneously to the purpose of this collective acid antibodies antibody content also has the advantage that you can adjust.

[102]

The above acid this antibody is a kind of antibodies to this property. This section is part of the main active antibody, amino acids ceran Tal-amine modified by oxidation or antibody, and acid and this acid antibodies and the nature of this nature include the kinds of antibodies. For example, the amino acid asparagine (asparagine) ride of the amine is asparagus Tate (aspartate), the properties of the antibody, amino acids (methionine) oxidation, o Ninh mecchi is mecchi o Nin cell fate (methionine sulfate), the properties of antibodies. In addition, the N extremity of writing in the breakers of the Ruta mate (glutamate), if it exists, the aforementioned article ruta mate Pentagram Ring structure formed by writing Chase par ruta mate (pyruglutamate) has been transformed into this property contains an antibody.

[103]

The analysis of these properties antibody chromatography can be performed using this invention uses a cation exchange resin chromatography analysis was carried out. Monoclonal antibodies, depending on its own characteristics of acid (acidic), the primary active (main), basic (basic) isomer content is quite diverse. Monoclonal antibodies, cell culture conditions that have arisen (culture parameters, producing badges, etc.) in accordance with monoclonal antibodies of these properties can vary the antibody content. Therefore, the above method involves Galactose content adjustment you want at the same time, the acid water of antibodies to the desired range of this nature you can adjust the advantage.

[104]

The form for the implementation of the invention

[105]

This is an example to explain in detail by the invention. Just an example to illustrate the invention, intended to this as well, for example, the scope of this invention is restricted by.

[106]

[107]

Implementation example 1: osmosis pressure caused by the rising artificially Galactose of the control

[108]

[109]

Oil expression culture Petri of the osmotic pressure is in progress, the elevation of a variety of ways. Excessive glucose culture fluid, how to add potassium chloride or sodium chloride aqueous solution, and how to add the actor to cultivate. This invention is a prime way for the seepage pressure rise 4M culture liquid aqueous solution sodium chloride added to how to apply.

[110]

[111]

A description of the manufacturing method, and in furtherance of the badge

[112]

[113]

This invention in culture fluid for osmotic pressure rise aseupara additional applied monoclonal antibodies that are otherwise non-Holy Biosimilars antibody, unlike non-thank you Ah is abbott developed rheumatoid arthritis, Crohn's disease cure. US 6, 090, 382 children of chains and non-running thanks to antibody light chains of amino acid sequences in contrast to non-thank you refer to Biosimilars DNA, polymerase chain reaction amplification, after you have created through its vector, the pGL3 developed by CUCBin using the promoter of pCB-Am2.77_v5.4 (1) CHO-DXB11 cells, and produced a week in contrast to non-transgenic thank Biosimilars expression cells produced a week. Its pCUCBin is developed by the Korea patent (the new fusion promoter and recombinant vector, containing the registration number 10-1038126) is one of the vectors that are mentioned.

[114]

[115]

This is the default culture used in the invention of culture, badges, badges, this badge (culture badge), additional production of culture is a kind of badge (feeding badge).

[116]

The default culture is used for the purpose of the subculture of badges badges. The badge after the production done for a full-blown subculture are antibody-producing culture (culture) as a badge that is used for the default culture, enhance or add new elements, certain elements in the building. This invention provides 1.4 times the default culture used to be enhanced to produce badges for the badge. Additional culture promotes cell growth and cell culture progress badge antibodies are added to the culture medium for the purpose of increasing manifestations of the badge. This invention provides a 3.3 times the default culture, add the powdered and used as the badge the badge culture. Add the default culture to produce badges, badge, badges, all cultured IMDM (Dulbecco's Modified Medium Iscove's) as a badge badge configuration elements that are transformed in table 1.

[117]

Table 1 [table 1] default culture badge components
District minutes Sung min.
IMDM modified medium Merck 100131
Buffer HEPES
Buffer Sodium bicarbonate
Carbon source Glucose
Nitriogen source Glutamine
Etc Sodium chloride
Growth hormone Insulin
Etc MTX
Vitamin L-ascorbic acid
Vitamin Biotin
Vitamin Choline chloride
Vitamin Folic acid
Peptone Sheff CHO plus pf acf (Kerry)
Metal Boric acid
Metal Cobalt Chloride Hexahydrate
Metal Copper Sulfate Pentahydrate
Metal Ferric Citrate
Metal Magnesium chloride anhydrous
Metal Manganese sulfate monohydrate
Metal Potassium nitrate
Metal Sodium selenite pentahydrate
Metal Zinc sulfate heptahydrate

[118]

[119]

For more information about how this invention used in the manufacture of antibodies through the animal cell culture bioreactor animal cell culture grown from animal cells in culture badge and special measures (e.g., lowering the temperature) via an antibody to the manifestation process. For more information about how batch culture, cultivate cultivate cultivate continuous expression expression, culture, variety of interchangeable one badge, unlike non-Ah thank you used in the invention of Biosimilars cell culture method is the expression in oil culture. The price of oil, the expression is in progress, the production as a badge to cultivate cultivate cultivate cultivate badge added to a specific, one-time or ongoing culture while adding more than twice.

[120]

[121]

For more information about how common the expression in oil used for cultivating embodiment as culture 1, 4, 7, 10 days in the car production volume of 5% of the current culture of the amount corresponding to the amount of additional culture badge 4 times is to add across.

[122]

[123]

Conducted for 1.1: osmotic pressure rise resulting Galactose at the time of the change

[124]

[125]

250 mL Shaker flask using a real 30 mL scale crude expression culture. Culture of feeding strategies and marketable expressions, addition of aqueous solution sodium chloride point 2 and. Started by 320 mOsm/kg osmotic pressure culture on the water, 4M sodium chloride (NaCl) solution to feeding your mOms/430 kg of artificially osmotic pressure to rise up to the final osmotic pressure is 450 kg to be mOms/. Add this badge to cultivate a culture day 1, the primary feed every 3 days after a total of 4 times by conducting additional feeding feeding, carried out either once or 4 capsule (rose gradually during the osmotic pressure, cultivate) 4 M sodium chloride (NaCl) solution by adding rose water osmotic pressure and cultivate cultivation progress, monoclonal antibodies and manifestations of Galactose, analyze the results, angry to have indicated in table 2.

[126]

Table 2 [table 2] Osmosis pressure rise due to the point of change of the antibody quality
Culture conditions The amount (mg/L) antibody expression G0F(%)* Acidic(%) The final osmotic pressure (mOsm/kg)
Rose gradually during the osmotic pressure Sikkim culture 1392.0 60.4 20.1 453.0
The primary feeding (culture 1) osmotic pressure elevation of Sikkim 1215.5 66.1 17.6 445.0
The second feeding (culture 4 days) osmotic pressure rise Sikkim 1268.3 68.8 16.7 451.0
3rd feeding (culture 7 days) osmotic pressure rise Sikkim 1438.7 67.1 16.4 451.0
The fourth feeding (culture) osmotic pressure rise Sikkim 1580.1 66.2 17.4 454.0
Osmotic pressure does not elevate 1771.8 63.5 18.5 364.0

[127]

*G0F(%) = G0F/(G0F+G1F+G2F)

[128]

[129]

As a result, as indicated in table 2 above, 4M sodium chloride (NaCl) solution to feeding at the time based on monoclonal antibodies of Galactose had a difference in sign language. In the case of the cultivation culture flask Shaker during the process, dissolved oxygen (DO) and acidification (pH) is not easy to directly regulate the difference in G0F between experimental conditions may be negligible. But bioreactor culture, dissolved oxygen and can also directly control of acidification, G0F the difference can be maximized.

[130]

[131]

Conducted for 1.2: osmosis pressure rises due to a difference in the range of monoclonal antibody Galactose shoes change

[132]

[133]

The culture of water and osmotic pressure for implementation 1.1 increase depending on the point of monoclonal antibodies of Galactose showed that you can vary the upset. Add feed badges secondary feeding (culture 4 days) and tertiary feeding (culture 7 days) aqueous solution of sodium chloride at the time when he grew up feeding G0F. In addition, there is a difference of the level of water seepage pressure rise cultivate monoclonal antibodies of Galactose to learn the impact on second angry Shaker flask crude expression culture. Culture of the early pressure of 320 mOsm/kg is osmosis, osmotic pressure rise depending on amount of goals 4 M sodium chloride (NaCl) solution to the primary Shaker flask culture similar to the cultivation culture in 4 days or 7 days after adding the culture, monoclonal antibodies and galactose manifestation of mad.

[134]

Table 3 [table 3] Osmosis pressure rise due to the difference between the scope of change of antibody quality
Culture conditions Seepage pressure rise (mOsm/kg) The amount (mg/L) antibody expression G0F(%)* Acidic(%) The final osmotic pressure (mOsm/kg)
Seepage pressure rise X X 1136.2 67.4 15.6 365.0
The second feeding (culture 4 days) osmotic pressure rise Sikkim Up to 400 948.9 70.4 15.0 424.0
Up to 440 824.1 71.0 14.6 460.0
Up to 480 573.7 67.9 14.5 505.0
Up to 520 543.8 66.5 13.9 539.0
3rd feeding (culture 7 days) osmotic pressure rise Sikkim Up to 400 1053.4 70.5 14.9 416.0
Up to 440 927.4 69.5 14.5 454.0
Up to 480 851.1 68.5 14.3 497.0
Up to 520 775.1 66.9 14.2 533.0

[135]

*G0F(%) = G0F/(G0F+G1F+G2F)

[136]

[137]

Culture of the osmotic pressure rise, depending on the level of monoclonal antibodies of Galactose showed a wide variety of upset. Cultivate cultivate water osmotic pressure in progress, 480 mOsm/kg rise above will G0F content decreases. In other words, the monoclonal antibodies increases for up to G0F of the content of osmotic pressure was a reasonable range (400-480 mOsm/kg in progress to cultivate up to Sikkim).

[138]

[139]

Implementation example 1.3: bio-reactor (bio-reactor) experiments

[140]

[141]

The first and second Shaker flask culture based on the results of the checks conducted an experiment in the bio-reactor, subject to the terms and conditions of the rise of the osmotic pressure of the monoclonal antibody Galactose were able to confirm the changing of the back.

[142]

1.4 L bioreactor scale, secondary feeding real 1 L (culture 4 days), each with 4 M sodium chloride aqueous solution by adding water to artificially cultivate osmotic pressure, 440, 500, mOsm/kg up to 523 after cultivation.

[143]

Table 4 [table 4] in seepage pressure rise bioreactor range due to the differences in antibody quality change
The second feeding: osmotic pressure (mOsm/kg) climbing conditions Manifestations of the volume (mg/L) G0F(%)* Acidic(%) NH4+ concentration (mM) The final osmotic pressure (mOsm/kg)
Do not let rise 1980.6 63.7 26.5 3.6 321
Sikkim to rise up to 440 1740.2 71.8 18.4 7.7 479
500 up to Sikkim 1361.6 58.3 18.4 7.3 549
523 rise up to Sikkim 1361.7 54.8 19.3 5.6 567

[144]

*G0F(%) = G0F/(G0F+G1F+G2F)

[145]

[146]

The secondary culture of osmotic pressure feeding: 500 kg or more if you increase mOsm/(549, 567 final osmotic pressure mOsm/kg), but rather the reduction of G0F content. The second feeding: osmotic pressure, 440 mOsm/kg in the case of the experiment until bioreactor G0F 71.8% of the content.

[147]

[148]

Implementation example 2: aseupara Jin, excessive amount of cultivation through the add-in creates ammonium

[149]

[150]

According to the results of this culture, of the applicant (table 4), excessive aseupara in addition to culture culture liquid ammonium ion in (NH4+) concentration in the atmosphere has increased by leaps and bounds. Excessive aseupara of Jin culture indirectly through annexation output4NH+ concentration within the increasing way of the monoclonal antibody Galactose mad.

[151]

Table 5 [table 5] ammonium aseupara in water, resulting in the annexation of Jin culture increase
Culture conditions Added aseupara Jean depth (mM) The final ammonium ion (mM)
Aseupara-Jin added. 0 3.4
Aseupara Jean addition 18.9 8.1

[152]

[153]

Conducted for 2.1: Add liquid oil enriched expression aseupara Jin culture

[154]

[155]

250 mL Shaker flask using a real 30 mL scale crude expression culture. Oil injection point of feeding strategy and cultivating the expression 3 and aseupara Jean. Aseupara Jin concentrated solution is 200 mM manufacturing. Additional culture badge injection point culture 1, 4, 7, 10, and enriched by the addition of the second solution, aseupara Jean feeding time (culture 4 days) from feeding conducted each (a total of 3 times). After you proceed to cultivate monoclonal antibodies and galactose manifestation of mad.

[156]

Table 6 [table 6] due to the annexation of Jin aseupara monoclonal antibodies changes of Galactose
Culture conditions The amount (mg/L) antibody expression G0F(%)* Acidic(%) The final NH4+(mM)
Producing badges 1 feeding: added aseupara Jean depth (mM) A total of aseupara Jin added depth (mM)
1 X 0 0 1738.5 56.3 25.9 4.1
1 X 1 3 1705.8 59.9 21.7 5.2
1 X 2 6 1535.2 61.4 19.2 6.7
1 X 4 12 1500.0 64.3 19.3 8.8
1.4 X 0 0 1729.9 60.2 21.1 5.6
1.4 X 1 3 1721.0 61.0 21.3 7.4
1.4 X 2 6 1588.5 61.5 20.1 7.3
1.4 X 4 12 1563.7 61.9 19.3 9.0

[157]

*G0F(%) = G0F/(G0F+G1F+G2F)

[158]

[159]

Aseupara in culture fluid feeding more G0F content add gene was increased. Aseupara added increasing the options simplifies the culture fluid within the final concentrations of NH's4+ concentration is increased. 1.4 X production produced 1X aseupara Jean badge in commitment compared with badges for G0F content was increased further. Culture pause early in the aseupara gene, when added to cultures from the NH4+ concentration increased cell growth is delayed, and in the end, the final amount of low antibody expression, so add aseupara to feeding: half adopted the approach.

[160]

[161]

Conducted for 2.2. : Aseupara of culture, this excessive added badge feeding

[162]

[163]

Shaker flask crude expression culture, unlike the aseupara gene expression in oil culture bioreactors enriched solution included additional culture without creating a separate badge. Aseupara-feeding every 6 mM 120 mM additional commitment plan, aseupara Jean added additional culture after feeding produced a badge. 1.4 L 1 L scale bioreactors and proceed to the actual culture, then the manifestations of monoclonal antibodies and galactose mad.

[164]

Table 7 [table 7] aseupara add culture added this excessive badges using a bioreactor experiment
Culture conditions The amount (mg/L) antibody expression G0F(%) Acidic(%) NH4+(mM) The final osmotic pressure (mOsm/kg)
Aseupara Jin does not add 1980.6 63.7 26.5 3.6 321.
Since the second feeding feeding every 6 mM aseupara-Jin added (total 18 mM) 1281.8 73.9 20.6 13.5 379

[165]

Aseupara add this place with excessive added water content badge in G0F Petri 10.2% increase. Aseupara-excessive culture, add the amount of NH4+ concentration increases when you consider that the 9.9 mM, monoclonal antibodies reduced the cultivation of Galactose shoes+ NH4in aqueous humor concentration due to a change, you can see that. Osmotic pressure is the amount of cultivation in excessive aseupara to add 58 mOsm/kg.

[166]

[167]

Implementation example 3: Add an artificial combination of seepage pressure rise aseupara Jin

[168]

[169]

Oil prices rose during the osmotic pressure of artificially cultivating the expression or aseupara-Jin added by applying the monoclonal antibodies were able to elevate their G0F content. Monoclonal antibodies of galactose and charge this antibody (charge variants) to learn the impact of artificial seepage pressure rise and aseupara Jin is a combination of annexation.

[170]

[171]

Example 3.1: osmotic pressure rise and aseupara conducted by Jean added at the same time, applied bio-reactor experiment

[172]

[173]

1.4 L bioreactor scale osmotic pressure in 1 L artificially rise or osmotic pressure rise and aseupara gene expression in oil prices by applying at the same time, add culture. Osmosis pressure applied at the same time, the rise and fall of Jin aseupara annexation, in the case of a bio-incubator aseupara Jean commit (add 120mM add aseupara to cultivate the badge) increases the effect of the osmotic pressure to take into account the culture of water and osmotic pressure increased up to 423 mOsm/kg. After you proceed to cultivate monoclonal antibodies and galactose manifestation of mad.

[174]

Table 8 [table 8] in osmotic pressure rise aseupara bioreactor Jean Galactose applied the same time annexation of change
Culture conditions The amount (mg/L) antibody expression G0F(%) Acidic (%) NH4+(mM) The final osmotic pressure (mOsm/kg)
Osmotic pressure of 450 up to Sikkim 1511.6 73.6 20.6 8.80 502.0
Osmotic pressure rise until the annexation of Sikkim + 423 aseupara Jin 1548.1 75.4 23.1 10.5 490.0

[175]

[176]

[177]

Osmotic pressure and osmotic pressure and only apply to the aseupara gene is one of two conditions apply to a combination of process-to-G0F did not differ G0F content has increased even further. If the add-in to add water aseupara not a Petri culture compared to acidic water, this property reduces the ratio of antibody (acidic variant), but (as shown in table 6, 7) osmotic pressure and the result of applying a combination of Jin aseupara acid this antibodies rather than increased.

[178]

[179]

G0F content to increase the osmotic pressure of artificial lift, aseupara's addition, osmotic pressure of artificial lift and at the same time, you can apply the method of annexation, aseupara's G0F content adjustment as well as charge-profile (charge profile) of the acid specific gravity of antibodies increase this property if the osmotic pressure of the rise and fall of aseupara applied to the same time annexation strategy of Jin would be useful.

[180]

[181]

From the description above, the invention belongs to the invention that the technology sector saw the party's essential features, technical or any other specific form without having to change can be carried out is understandable. In this regard, the conduct described above illustrate that it is restrictive on all sides examples are not to be understood. The scope of this invention, the foregoing detailed description, rather than later, meaning and scope of the range of the patent claims, and that any change or the equivalent concept is drawn from the modified subsume this invention in the form of a range, shall be construed as being.

The scope of a claim

[Claims]

Recombinant protein expression to the purpose of animal cell cultures to be reminded of the animal cell culture process of osmotic pressure increase in a specific culture, including the steps, Galactose (galactosylation) is regulated, manufacturing method of the recombinant protein for the purpose.

[Billing section 2]

In paragraph 1, above, a metaphor expression culture Petri (fed-batch culture), which is, in a way.

[Billing section 3]

In paragraph 1, above, osmosis pressures increase step is seen as the start of 0, this cultivation culture 1st to 10th, one culture will be performed on the job, in a way.

[Billing section 4]

In paragraph 1, above, osmosis pressures increase step is started this culture on the basis of 1, 0, 4, 7, and 10, one culture will be performed on the job, how to.

[Billing section 5]

Paragraph (4) above the osmotic pressure increase, the phase is started this culture on the basis of the 4, or 0 to 7 are to be performed on the job, in a way.

[Claim 6]

In paragraph 1, above, the increase in osmotic pressure, the final osmotic pressure within a cell culture fluid 460 to 500 mOsm/kg, which is to be performed, so that way.

[Claim 7]

In paragraph 1, above, the increase in osmotic pressure above animal cell culture in sodium chloride or potassium chloride supplement and glucose in culture is made up of the military as a way to add in more ways than the selected from 1, which is being done.

[Billing section 8]

In paragraph 1, the protein chain, reassemble the above methods.

[Claims 9]

Section 8, the above method is Galactose painter reduced to mass manufacture the antibodies, methods.

[Claims 10]

In paragraph 1, above, for the purpose of the step increase osmotic pressure of recombinant protein to animal cell cultures that have arisen during the course of a single circuit will, in a way that is performed.

[Billing section 11]

Recombinant protein expression to the purpose of animal cell cultures to be reminded of the animal cell culture process of osmotic pressure rise to a specific culture, including the steps that are for the purpose of recombinant protein a, Galactose-Hwa how to adjust.

[Billing section 12]

Recombinant protein expression to the purpose of animal cell cultures to be reminded during the course of animal cell culture supplements the steps in aseupara Jin, Galactose (galactosylation) is regulated, manufacturing method of the recombinant protein for the purpose.

[Billing section 13]

In the 12th paragraph of the above aseupara Jean aseupara Jean aseupara Jean concentrate or to include culture, in the form of the badge.

[Claim 14]

Article 12, the purpose of the aforementioned aseupara's replacement to the recombinant protein expression during the course of the cultivation of certain animal cell culture will be performed at different circuits, in a way.

[Section 15 claim]

Article 14, paragraph above aseupara's replacement on the basis of the culture this culture starts 0 days 4-10 days will be performed.

[Claims 16]

Article 14, paragraph aseupara above started this culture's replacement on the basis of culture, 0, 4, 7, and 10 are to be performed on the job.

[Claims 17]

For more information about how my 12 animal cell culture, the above amount to the final concentration of 27.6 33.6 mM aseupara within the gene so that the aseupara gene is to supplement that.

[Claim 18]

The seventeenth paragraph, animal cell culture fluid aseupara in the above way to gin up the final concentration of 33.6 mM aseupara so how to supplement.

[Billing section 19]

No. 12, or the 17th paragraph, animal cell culture fluid aseupara in the above way, Jin 6mM, 12mM and 18mM each concentration to be added sequentially to aseupara to supplement three times a day, in a way.

[Claims 20]

Recombinant protein expression to the purpose of animal cell cultures to be reminded during the course of animal cell culture supplements the steps in aseupara Jin, is the purpose of recombinant protein of Galactose to control angry.

[Claim 21]

Recombinant protein expression to the purpose of animal cell cultures to be reminded of the animal cell culture process of osmotic pressure rises, aseupara Jean complement steps, Galactose (galactosylation) is regulated, manufacturing method of the recombinant protein for the purpose.

[Claim 22]

The 21st paragraph, above the increase in osmotic pressure and aseupara's replacement at the same time or sequentially, it would, in a way that is performed.

[Billing section 23]

The 21st paragraph, the increase in osmotic pressure above the animal cell culture fluid above sodium chloride or potassium chloride supplement and glucose in culture is made up of the military as a way to add in more ways than the selected from 1, which is being done.

[Claims 24]

The 21st paragraph, above the increase in osmotic pressure, the final osmotic pressure within a cell culture fluid 460 to 500 mOsm/kg, which is to be performed, so that way.

[Claims 25]

The 21st paragraph, above aseupara's animal cell culture liquid supplement is the final concentration of 27.6 33.6 mM not aseupara within the gene so that the aseupara gene is to supplement that.

[Claim 26]

On the 21st, the chain, the aforementioned recombination proteins.

[Claims 27]

Article 26, paragraph above way to recombinant antibodies for the purpose of controlling of galactose and manufactured in a way is a collective of acid properties of antibodies, antibodies to control content, in a way.