WO2015083853A1 - Procédé de préparation d'un anticorps par ajustement de la quantité d'anticorps hétérogènes - Google Patents

Procédé de préparation d'un anticorps par ajustement de la quantité d'anticorps hétérogènes Download PDF

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WO2015083853A1
WO2015083853A1 PCT/KR2013/011236 KR2013011236W WO2015083853A1 WO 2015083853 A1 WO2015083853 A1 WO 2015083853A1 KR 2013011236 W KR2013011236 W KR 2013011236W WO 2015083853 A1 WO2015083853 A1 WO 2015083853A1
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antibody
settling
time
rate
maturation
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PCT/KR2013/011236
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English (en)
Korean (ko)
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윤지용
김세연
김원겸
박상경
안용호
이정우
장재영
황은호
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한화케미칼 주식회사
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Publication of WO2015083853A1 publication Critical patent/WO2015083853A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature

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  • the present invention provides a method for producing an antibody, the method for producing a high purity and high quality antibody population by adjusting the content of the isomeric antibody in the population of antibodies (population of antibodies) to the target content, the antibody population prepared by the above method, the antibody population
  • the present invention relates to a method for controlling the content of isomeric antibodies in a body and a method for determining the pH settling time for controlling the content of isomeric antibodies.
  • the antibody has a structure in which two heavy chains and two light chains are disulfide bonds, and glycosylation is performed near the Fc of the heavy chain.
  • Antibodies produced using CHO cells as a host include various isomeric antibodies (Hongcheng Liu, Georgeen Stahl-Bulseco, Journal of Chromatography B, 837 (2006) 35-43), and isomeric antibodies are deamidation.
  • isparagine is an amino acid deamined as an aspatate (Boxu Yan, Sean Steen, Journal of Pharmaceutical Sciences, Vol. 98, No. 10, October 2009), Isomers in which amino acid methionine is oxidized to become methionine sulfate (Chris Chumsae, Georgeen Stahl-Bulseco, Journal of Chromatography B, 850 (2007) 285-294).
  • glutamate when glutamate is present at the N-terminus of the heavy chain, the glutamate may form a pentagonal ring structure to be transformed into pyruglutamate (William E. Werner, Sylvia Wu, Analytical Biochemistry 342 (2005) 120-125). Since these isomeric antibodies affect the biological activity of the antibody, it is necessary to control the amount of the isomeric antibody to a certain limit in the production of the antibody.
  • Isomer antibodies vary in rate of deamination depending on pH and temperature, and are generally studied for conditions under which deamination can be minimized in terms of stability.
  • the quality may be different in terms of efficacy, and thus may be a problem in terms of equivalence evaluation. Therefore, the development of a method of maintaining a constant content of isomeric antibodies in the final purification product is greatly demanded in the biosimilar antibody pharmaceutical market.
  • One object of the present invention is to (a) selecting a pH and time to prepare an isomeric antibody of a desired content; And (b) allowing the sample containing the mixed solution of the antibody to stand for a predetermined time within the pH selected in step (a), wherein the antibody population comprises a desired amount of isoform. It is to provide a method for producing.
  • Another object of the present invention is to provide an antibody population comprising the desired amount of isomeric antibodies prepared by the above method.
  • Another object of the present invention is to select a pH and time to prepare a desired amount of isomeric antibody; And (b) allowing the sample containing the mixed solution of the antibody to stand for a predetermined time within the pH selected in step (a). To provide.
  • Another object of the present invention is to select a pH at which (a) to prepare isomeric antibodies of a desired content; And (b) allowing the sample containing the mixed solution of the antibody to remain within the pH selected in step (a), to control the content of the isoform in the antibody population or to a desired content.
  • the manufacturing method of the antibody population containing an isomeric antibody it provides the method of determining pH settling time using the hourly rate of increase of specific main acidic peak%.
  • the content of isomeric antibodies can be controlled to consistently produce a high quality antibody population of interest.
  • the antibody according to the present invention can be manufactured by controlling the content of the isomeric antibody to improve the equivalence with the reference drug, and can shorten the existing process time. This can be high.
  • FIG. 1 is a diagram showing a pattern change of the isomeric antibody over time by the pH rise.
  • 1A is a diagram showing a pattern change of isomeric antibodies after 4 hours of pH maturation, and
  • FIG. 1B is 24 hours of pH maturation.
  • Figure 2 is a view showing the range of a typical acidic, main, basic portion on CEX HPLC.
  • Figure 3 is a diagram showing the pattern change of the isomeric antibody at 12 hours pH maturation.
  • 3A shows CEX HPLC Full and
  • 3B shows CEX HPLC expansion, respectively.
  • FIG. 4 is a diagram showing the pattern change of the isomeric antibody at 24 hours pH maturation.
  • 4A shows CEX HPLC Full
  • FIG. 4B shows CEX HPLC expansion.
  • 5 is a diagram comparing the content of each portion during pH maturation (12hr vs 24hr).
  • 5A shows a main acidic peak (AM)%
  • FIG. 5B shows an acidic portion%
  • FIG. 5C shows a main portion%
  • FIG. 5D shows a basic portion%.
  • FIG. 6 is a diagram showing the change in AM (main acidic peak) and acidic portion content at pH 7.5 maturation in the culture supernatant (3, 6, 9, 24hr).
  • 6A shows main acidic peaks (AMs)
  • FIG. 6B shows acidic portion%.
  • Figure 7 is a diagram showing the pattern change of the isomeric antibody during pH maturation in the culture supernatant (temperature: 25 °C, 30 °C, 37 °C, time: 24hr).
  • 7A shows CEX HPLC Full
  • FIG. 7B shows CEX HPLC expansion, respectively.
  • Figure 8 is a schematic diagram of the process flow and pH maturation insertion step of the present inventors Trastuzumab purification process.
  • 9 is a view showing a comparison of the content of each portion during pH maturation in CEX and VI step (time: 6, 11, 24hr, temperature: 4 °C, 25 °C).
  • 9A shows main acidic peak%
  • FIG. 9B shows acidic portion%
  • FIG. 9C shows main portion%
  • FIG. 9D shows Basic Portion%.
  • 10 is a diagram showing the pattern change of the isomeric antibody during pH maturation in CEX and VI (temperature: 25 °C, time: 11hr, 24hr).
  • 11 is a diagram comparing the content of each portion during pH maturation in the HIC step (time: 6, 9, 24hr, temperature: 4 °C, 25 °C).
  • 11A shows the main acidic peak (AM)%
  • FIG. 11B shows the acidic portion%
  • FIG. 11C shows the main portion%
  • FIG. 11D shows the Basic Portion%.
  • FIG. 12 is a view showing a pattern change of the isomeric antibody during pH maturation in the HIC step (temperature: 4 °C, 25 °C, time: 24hr).
  • FIG. 13 is a diagram showing the content comparison for each portion during pH maturation in UF / DF1 step (time: 6, 24hr, temperature: 4 °C, 25 °C).
  • FIG. 13A shows the main acidic peak (AM)%
  • FIG. 13B shows the acidic portion%
  • FIG. 13C shows the main portion%
  • FIG. 13D shows the Basic Portion%.
  • FIG. 14 is a view showing a pattern change of the isomeric antibody during pH maturation in UF / DF1 step (temperature: 25 °C, 4 °C, time: 24hr).
  • 15 is a diagram showing the peak containing AM in CEX HPLC.
  • 16 is a diagram showing AM growth rate verification by Plot between AM% and maturation time.
  • Figure 17 is a graph confirming the content control of isomeric antibodies in the production of Trastuzumab 1000l.
  • 17a shows CEX, UF / DF2, and reference overlap (No overlap)
  • FIG. 17B shows CEX, UF / DF2, and reference comparison (overlap).
  • the present invention provides a method for consistently producing a high-quality or equivalence antibody population, by adjusting the pH, temperature, time, etc. in the culture or purified solution to the desired content of the isomeric antibody
  • methods for producing an antibody population comprising.
  • the present invention comprises the steps of (a) selecting the pH and time to prepare the isomeric antibody of the desired content; And (b) allowing the sample containing the mixed solution of the antibody to stand for a predetermined time within the pH selected in step (a), wherein the antibody population comprises a desired amount of isoform. It is possible to provide a method for producing.
  • the term "pH settling" may be used interchangeably with pH maturation.
  • Antibody products prepared through host cells include several isomeric antibodies in addition to the main active antibody.
  • the isomeric antibody is an antibody in which some amino acids in the antibody are modified by deamine or oxidation, and the isomeric antibodies differ in biological activity.
  • Antibody products expressed through host cells may produce inconsistent amounts of such isomeric antibodies.
  • preparation of a quality similar to that of a reference drug is important. Therefore, a process for producing an antibody from host cells and then controlling the content of the isomeric antibody is required.
  • the present invention has been developed a method for controlling the content of isomeric antibodies, which surprisingly only raises a specific peak of the isomeric antibodies when the culture or purified liquid used for the production of the antibody is left at a certain pH for a certain time. Based on this, there is provided a method capable of effectively producing a high quality or equivalency antibody population comprising a desired ratio of isomeric antibodies.
  • a population of antibodies refers to an antibody group comprising a main active antibody and an isomeric antibody.
  • the antibody population is intended for a main active antibody and an isomeric antibody. It means the antibody group included in the ratio to do.
  • the antibody population includes only one type of antibody, or includes a group of antibodies containing both main active and isomeric antibodies.
  • said antibody population preferably means an antibody population comprising a desired amount of isomeric antibodies.
  • the antibody population has a difference in quality such as efficacy when the content of the isomeric antibody, which is considered to be important in the biosimilar antibody medicine for the purpose of the present invention, is different from the reference drug, thereby causing problems in evaluating equivalence.
  • it can mean an antibody population comprising a content of isomeric antibodies within the same or similar range as the biosimilar counterpart.
  • control means a drug that is a subject of replication of the biosimilar drug, but is not limited thereto and may mean a drug of a target that is intended to ensure equivalence.
  • antibody populations comprising the main active antibody and the isomeric antibody can be prepared in the same or corresponding composition as the reference drug.
  • the term “antibody” refers to a substance produced by stimulation of an antigen in the immune system, and specifically binds to a specific antigen, causing a substance to float on lymph and blood to generate an antigen-antibody reaction.
  • the antibody is one of proteins for high quality purification, and can be efficiently prepared and purified by the method according to the present invention.
  • the antibody to be purified in the present invention is not limited thereto, but may preferably be an antibody having an isoelectric point of 7 to 11, more preferably 8 to 10.
  • the antibody of the present invention is not limited thereto, but may preferably include all therapeutic antibodies commonly used in the art, and more preferably targets Human Epidermal Growth Factor Receptor 2 (HER-2).
  • the antibody may be trastuzumab or pertuzumab, and most preferably trastuzumab.
  • trastuzumab also known as Herceptin, is a humanized antibody against HER2 developed by Genentech in the United States, which is known as an antibody therapeutic against HER2 / neu mainly expressed in breast cancer cells.
  • the term "mainly active antibody” is a major component included in the antibody population of the present invention, wherein some amino acids in the antibody are modified by deamine or oxidation, so that the biological activity is not lowered, That is, it means an antibody that is not an acidic or basic isomeric antibody.
  • the main active antibody is the most important component for controlling the quality of the desired antibody population, the antibody with the highest biological activity among the components of the antibody.
  • the term “isomer antibody” refers to an antibody in which some amino acids of the main active antibody are modified by deamine or oxidation, and include acidic isomers and basic isomeric antibodies. Examples include isomers in which asparagine is deamined in amino acids to form aspatate, and isomers in which methionine is oxidized to become methionine sulfate.
  • glutamate when glutamate is present at the N-terminus of the heavy chain, the glutamate forms an pentagonal ring structure and includes an isomeric antibody modified with pyruglutamate.
  • the isomeric antibody When isomeric antibodies are included in the host cell culture at a high rate when the antibody is produced in host cells, such as CHO cells, some of the isomeric antibodies must be removed through a process such as chromatography to be included in the antibody population at the desired rate.
  • the isomeric antibody may be an acidic isomeric antibody, but is not limited thereto. Since there are many isomeric antibodies in the antibody product expressed through the host cell, it is important to demonstrate homogeneity to make the quality most similar to the reference drug to prepare the antibody biosimilar.
  • the isomeric antibodies are a modified form of several amino acids of the main active antibody, with slight differences in charge between the main active antibody and the acidic isomeric antibody.
  • isomeric antibodies are modified by deamine or oxidation of some amino acids in the antibody, it is known that there is a difference in biological activity for each isomeric antibody, it is consistent quality to bring a constant content distribution of the isomeric antibodies Is important to maintain.
  • the present invention has been developed a method for producing an antibody population, which can adjust the content of the isomeric antibody according to the desired content. For example, when preparing an antibody drug such as trastuzumab, the content of the isomeric antibodies may be relatively lower than that of the control drug depending on the culture conditions.
  • the mixed solution of the antibody of step (a) may be a culture solution for producing an antibody or a purification solution obtained in various purification steps, and the culture solution may be a culture solution containing cells or a culture supernatant from which cells have been removed.
  • the cell removal method may be a method commonly used in the art, but is not limited thereto.
  • a filter more preferably a filtration filter or an ultrafiltration may be used.
  • the term “desired content of isomeric antibody” means a desired amount of isomeric antibody, depending on the antibody applied to the method of the invention.
  • this may mean an antibody that is the same or similar to the reference in the manufacture of the biosimilar antibody drug product, for example, within the range of ⁇ 15% of the content of isomeric antibody in the reference drug, ⁇ 10% It may be in the range, and based on the AM (main acidic peak)%, it may be in the range of ⁇ 17%, within the range of ⁇ 20% and AM of the reference drug, but is not limited thereto, and the desired content It can be variously changed according to the isomeric antibody.
  • the pH used in the stationary may be pH 6.0 or more pH 9.0 or less, or may be pH 7.0 or more pH 9.0 or less, preferably pH 7.5 or more pH 9.0 or less, or pH 7.0 or more pH 7.5 or less, or pH 8.0 or more.
  • the pH may be 8.2 or less, but is not limited thereto, and the pH used for standing may be selected according to the content of the desired isomeric antibody.
  • the temperature used for standing within the selected pH may be 4 ° C to which the culture or purified liquid is exposed in various purification steps, preferably 4 ° C or more and 40 ° C or less, more preferably 15 ° C at room temperature.
  • the temperature may be 30 ° C. or lower, but is not limited thereto. If the pH is left at room temperature, there is no need to raise or lower the temperature, which may be advantageous in terms of process.
  • AM% increases with each increase in temperature, and furthermore, it was confirmed that AM% increased specifically during pH standing at room temperature (FIGS. 6A and 6B, 7A and 7b).
  • the pH settling time may be 1 hour or more and 48 hours or less, preferably 24 hours or less, but is not limited thereto and may be performed by a time selected according to conditions.
  • the pH and time selection step of step (a) may be performed by the following method.
  • AM% in the desired AM% range is selected as AM% after the pH stationary, Selecting a pH and AM rise rate to be used in the process;
  • Rate of AM rise (% after pH settling-AM% before pH settling) ⁇ Settling time (hr)
  • pH Settling Time (hr) (Target AM%-AM% Before pH Settling) ⁇ AM Ascent Rate Selected in Step (i) above.
  • Increasing the pH at the same time may be variously determined according to experimental conditions.
  • the pH may be increased to 0.01, 0.05, 0.1, 0.5, etc., but is not limited thereto.
  • the rate of AM rise of each sample is obtained, and then the AM% within the desired AM% range is selected as AM% after pH settling.
  • the rate of increase can be selected.
  • the “desired AM%” may be used interchangeably with the “purpose AM%” in the present invention.
  • the isomeric antibody content pattern of the initial sample it was confirmed that by selecting the appropriate pH and the appropriate time can adjust the content of the isomeric antibody, moreover, based on the AM% content It was confirmed that the settling time and pH can be determined when setting.
  • the pH settling step is to adjust the content of the isomeric antibody in the antibody mixture to the desired content, (i) the mixture of the antibody is purified using cation exchange chromatography (Cation Exchange Chromatography, CEX); (ii) the mixed solution of the antibody was purified by the Virus Inactivation (VI) step; (iii) the mixture of antibodies was purified using Hydrophobic Interaction Chromatography (HIC); (iv) the mixed solution of the antibody was purified by ultrafiltration (Ultrafiltation and Diafiltration, UF / DF); Or (v) a mixture of antibodies can be applied to a variety of antibody purification or preparation steps, such as those purified using Anion Exchange Chromatography, and can be carried out in one or more of each of the above steps.
  • each step is not a sequential step and a combination of each step depending on the type of antibody population to be produced. Can be selected.
  • the pH settling step can be performed on the eluate purified in each step.
  • the pH settling step may be performed after the mixed solution of the antibody is purified by the virus inactivation step.
  • cationic exchange chromatography refers to a column filled with a cation exchange resin, and may be subjected to cation exchange chromatography to remove isomeric antibodies and impurities, preferably host cell proteins.
  • the cation exchange chromatography is a synthetic resin that serves to exchange cations in an aqueous solution with its own cation. Since antibodies have high isoelectric point, they have a cation in a pH buffer below the isoelectric point value. Therefore, the quality of the antibody population can be improved by using cation exchange chromatography capable of adsorbing the cationized antibody.
  • the cation exchange chromatography may be used that is commonly used in the art, but is not limited to this may be preferably a column having a functional group of COO - or SO 3 , more preferably carboxymethyl (CM ), Fructogel, sulfoethyl (SE), sulfopropyl (SP), phosphate (P) or sulfonate (S) and the like, and more preferably carboxymethyl sepharose (CM sepharose).
  • CM sepharose carboxymethyl sepharose
  • fructogel COO ⁇ may be used.
  • virus inactivation includes making a virus contained in a culture or purified liquid non-functional or removing the virus from the culture or purified liquid.
  • Methods for making the virus nonfunctional or removing the virus include a thermal inactivation, pH inactivation, or chemical inactivation method, and the like, but preferably, a pH inactivation method is not limited thereto.
  • the pH inactivation method is a method in which the virus is treated at a pH such that the non-functionality is sufficient, and the method of pH inactivation includes a low pH virus inactivation method.
  • the antibody eluate eluted in the previous chromatography step may be carried out by titration at a pH in the range of 3.0 to 4.0, preferably at pH 3.8, but is not limited thereto.
  • hydrophobic reaction chromatography means a column filled with a hydrophobic interaction resin, and in this step, a column capable of removing impurities, preferably a host cell protein, may be subjected to hydrophobic interaction chromatography. it means. Proteins are hydrophilic in general but have hydrophobic regions with hydrophilicity, and the hydrophobic nature of these regions is not expressed under strong electrostatic interaction conditions, but the relative weakness of the electrostatic interactions is increased by increasing the ionic strength or dielectric constant of the solvent. It is characterized by strong expression.
  • hydrophobic ligands long hydrocarbon chains or aromatic rings
  • hydrophilic chromatography substrates agarose gel-pies, organic polymer supports, etc.
  • a strong salt concentration to adsorb various proteins.
  • the host cell protein using the hydrophobic interaction column can be removed. have.
  • the hydrophobic interaction resin may be used that is commonly used in the art, but is not limited thereto, and preferably, phenyl column, butyl column, phenyl sepharose or fructogel EMD phenyl column may be used. More preferably, phenyl sepharose may be used.
  • Ultrafiltration in the present invention may be a step for buffer replacement or concentration in the antibody mixture.
  • anion exchange chromatography refers to a column filled with an anion exchange resin, and in this step, a column capable of removing an impurity, preferably a host cell protein, is performed by performing anion exchange chromatography.
  • the anion exchange resin is a synthetic resin that serves to exchange its own anion with a specific anion in an aqueous solution, the anion exchange column may adsorb a protein having an anion above the isoelectric point.
  • the antibody since the isoelectric point is high, when the neutral pH buffer is used, the antibody does not adhere to the anion exchange resin, but the impurities including the host cell protein may be adsorbed and removed by the anion exchange resin because the isoelectric point is low. Can be used for the preparation of high purity antibody populations.
  • the anion exchange resin may be used that is commonly used in the art, but is not limited thereto, preferably Q sepharose, quaternary aminoethyl or quaternary amine (Q) and the like. More preferably, Q Fast Flow may be used.
  • the anion exchange column is an efficient column for removing endotoxin as well as the host cell protein, the desired antibody population with high purity can be purified by removing endotoxin along with the host cell protein in the final purification step.
  • the method for producing an antibody population comprising the desired content of isomeric antibodies comprises the steps of: (a) subjecting a sample comprising a mixture of antibodies to Cation Exchange Chromatography (CEX) for purification; (b) virus inactivation (VI) of the mixed solution of the antibody purified in step (a) to an acidic pH; (c) purifying the mixed solution of the virus-inactivated antibody of step (b) by applying hydrophobic reaction chromatography (HIC); (d) purifying the antibody mixture purified in step (c) by primary ultrafiltration (1st Ultrafiltation and Diafiltration, UF / DF I); And (e) subjecting the antibody mixture of step (d) to purification by applying anion exchange chromatography, wherein the pH setting is at least one of steps (a) to (e).
  • step (c) may be carried out, preferably, at least two, at least three, at least four, or all five, and most preferably, the mixed solution of the purified antibody of step (b) at an acidic pH
  • step (c) may be performed before step (c), but is not limited thereto.
  • the antibody mixture solution from each purification step was left at a constant pH to compare the effect of adjusting the content of the isomeric antibody, It was confirmed that the content of the acidic isomeric antibody, which was lower than the control drug, was increased by the pH standing over the entire process, thereby having a content similar to that of the control drug (Example 3).
  • the antibody mixture after virus inactivation it was determined that it was advantageous in terms of control and process efficiency of isomeric antibodies than in other processes.
  • the invention provides an antibody population comprising the desired amount of isomeric antibodies prepared by the above method.
  • the method, antibody population and desired amount of isomeric antibodies are as described above.
  • the present invention provides a method for preparing an antibody, the method comprising the steps of (a) selecting a pH and a time at which a desired amount of isomeric antibody is prepared; And (b) allowing the sample containing the mixed solution of the antibody to stand for a predetermined time within the pH selected in step (a). To provide.
  • step of selecting the pH and time of step (a) raises the pH used in the stationary reaction for the same time to obtain the rate of AM increase according to the following formula at each pH, and then the AM within the desired AM% range. Selecting% as AM% after pH settling, and then selecting pH and AM rise rates to be used for settling; And
  • Rate of AM rise (% after pH settling-AM% before pH settling) ⁇ Settling time (hr)
  • pH settling time (hr) (target AM%-AM% before pH settling) ⁇ The AM rise rate selected in step (i), wherein the method, antibody population, isomer antibody, AM rise rate, settling time, etc. As described above.
  • the present invention provides a method for preparing an antibody, the method comprising the steps of (a) selecting a pH at which a desired amount of isomeric antibody can be prepared; And (b) allowing the sample containing the mixed solution of the antibody to remain within the pH selected in step (a), to control the content of the isoform in the antibody population or to a desired content.
  • a method for producing an antibody population comprising isomeric antibodies a method is provided for determining pH settling time using an hourly rate of increase of a certain main acidic peak (AM).
  • the pH selection and settling time determination is performed by (i) raising the pH used for the stationary reaction for the same time to obtain an AM rising rate according to the following formula at each pH, and then adjusting the AM% within the desired AM% range to pH Selecting the percentage of AM after standing, and then selecting the pH and AM rising rate to be used for standing; And
  • Rate of AM rise (% after pH settling-AM% before pH settling) ⁇ Settling time (hr)
  • pH Settling Time (hr) (Target AM%-AM% Before pH Settling) ⁇ AM Ascent Rate Selected in Step (i) above.
  • the method, antibody population and isomeric antibody, AM rise rate, settling time and the like are as described above.
  • the AM growth rate (% / hr), that is, the AM increase rate, was calculated using the increase rate of the main acidic isomer (Main Acidic peak, AM) increased by pH standing. Even if the content of specific isomer antibody in the culture or purified liquid was produced low by using the AM rise rate, a method was developed to determine the settling time by substituting the AM rise rate. The process time was simplified to complete the pH settling process. Using the above results, the antibody production process including the actual pH settling was carried out on the 1000 liter production scale, and when the trastuzumab manufacturing process including the pH settling was carried out through the actual applied process, the content of the isomeric antibody was sufficiently controlled on the production scale. It was confirmed that it was possible (Examples 4 and 5).
  • the change in the isoform of the antibody is confirmed in the antibody mixture from pH 6.0 to sequentially 8.0.
  • a sample having a lower acidic isoform (charge isoform) of 2% compared to the reference drug was used.
  • charge isoform charge isoform
  • Table 1 to raise the pH to more than 7.0, using 25mM TrisHCl buffer was diluted with buffer to pH 7.0, 7.2, 7.5, 7.7, 8.0.
  • the initial antibody mixture was 25mg / ml, and diluted 5 times with dilution buffer for each pH to 5mg / ml.
  • the target pH was raised to 2M Tris, and pH 6.0 diluted with 5 mM histidine buffer pH 6.0 was compared as a control.
  • the temperature was allowed to stand at 25 ° C., and 0.5 ml of 0hr, 4hr, 8hr, and 24hr, respectively, were stored at ⁇ 20 ° C. or lower until just before analysis.
  • Changes of the isomeric antibodies, including acidic isomers, were confirmed using CEX HPLC (Cation Exchange High Performance Liquid Chromatography) analysis, and the conditions are shown in Table 2 below.
  • Table 1 Increase confirming acid isomer antibody (pH 7.0 ⁇ 8.0 condition) pH influence experiment pH Buffer Vol (ml) Conc. (Mg / mL) Temp °C Remarks PC pH 6.0 5 mM HistidineHCl pH 6.0 5 5 25 Sampling Time0 hr4 hr8 hr24 hr P-1 pH 7.0 25 mM TrisHCl pH 7.0 5 5 25 P-2 pH 7.2 25 mM TrisHCl pH 7.2 5 5 25 P-3 pH 7.5 25 mM TrisHCl pH 7.5 5 5 25 P-4 pH 7.7 25 mM TrisHCl pH 7.7 5 5 25 P-5 pH 8.0 25 mM Tris-HCl pH 8.0 5 5 25
  • CEX-HPLC Cation Exchange High Performance Liquid Chromatography
  • the present inventors confirmed that, through Example 1.1, the acidic peak was increased by pH maturation from pH 6.0 to pH 8.0 using the trastuzumab process solution, thereby increasing the equivalence with the reference drug.
  • the AM (main acidic peak) content of the initial 0 hour sample and the total content of the acidic portion were 7.59% and 16.08%, respectively, and the AM and acidic portion contents of the reference drug were 12.45% and 20.25%, respectively.
  • the AM and acidic portion contents of the reference drug were 12.45% and 20.25%, respectively.
  • the AM% which was lower than the reference drug increased over all the pH ranges, and peaks other than the AM peak similar to the reference drug were increased. They could confirm that there is no big change.
  • This experiment was carried out to determine whether the content of the isomeric antibody can be controlled by pH maturation in the culture. As such, when pH maturation is performed in the culture medium, the pH is increased in the incubator or harvest tank, and no additional equipment is required in the process equipment. It has the advantage of being sufficiently removable in the purification process.
  • the experiment proceeded to the procedure shown in Table 5.
  • the culture medium was removed using a depth filter, the pH was raised to 7.5 using 1N NaOH.
  • 200 ml was dispensed into a 250 ml bottle, and the temperature was pH maturated by static reaction in 25 ° C., 30 ° C. and 37 ° C. incubator, respectively.
  • the samples were purified by rProtein A for desalting and analyzed for analysis.
  • FIG. 8 shows a step candidate group that can be applied by inserting the pH maturation in the existing Trastuzumab purification process of the present inventors.
  • pH maturation is applied after each of CEX (Cation Exchange Chromatography), VI (Virus Inactivation), HIC (Hydrophobic Interaction Chromatography), UF / DF (Ultrafiltation and Diafiltration), and AEX (Anion Exchange Chromatography).
  • CEX Cation Exchange Chromatography
  • VI Virus Inactivation
  • HIC Hydrophilic Interaction Chromatography
  • UF / DF Ultrafiltration and Diafiltration
  • AEX Application Exchange Chromatography
  • PH maturation was applied to the purified liquid from the CEX column and VI (virus inactivation) process.
  • the temperature was carried out at 4 °C, 25 °C the temperature encountered in the process (Tables 7 and 8).
  • CEX (Cation Exchange chromatography) Step 1) 1 liter of culture was removed using a Depth filter. 2) The culture supernatant from which the cells were removed was lowered to pH 5.0 using 10% acetic acid and held for 1hr. 3) Using the same Depth filter, precipitation was performed. 4) The conductivity was adjusted to 6mS / cm for loading on the CEX column, and then sterilized and filtered. 5) pH was adjusted to 2M Tris using elute purified using CEX column (Fractogel COO (M)). Raised to 8.0, pH maturation test was performed. Maturation temperature: 4 ° C, 25 ° C.
  • PH maturation was performed in the procedure of Table 10 in the HIC purification step of the purification process.
  • the content was reduced at 25 °C, after 24 hours was confirmed to decrease by about 2% compared to the control.
  • the basic portion was reduced by about 1% compared to the control, and no difference was observed in all conditions.
  • the pH maturation in HIC resulted in lower effect than CEX or VI.
  • the content was similar to that of the reference drug after 24 hours based on AM (main acidic peak), but the pH maturation of HIC samples was 1% less than that of the reference drug.
  • the acidic base line is elevated (Fig. 12). This is because HIC elute contains high salt and 2M Tris is not able to raise pH to 8.0 due to weak buffering when raising the pH, and it may have a negative effect on the product because the pH should be increased by adding 1N NaOH. Because.
  • pH maturation in HIC step is more efficient in pH maturation with longer time and higher temperature, but it has disadvantage of high pH with strong base because it contains high salt. It has been confirmed that there may be disadvantages in terms of aspects.
  • Table 13 Isomer antibody content% with time by pH maturation in UF / DF1 step Step Number AM Acidic Main Basic UF / DF1 UF / DF1 Control 9.22 21.05 72.77 6.18 UF / DF1 4 °C (6h) 9.67 20.72 73.10 6.19 UF / DF1 4 °C (24h) 10.11 21.30 72.72 5.98 UF / DF1 25 °C (6h) 10.45 21.64 72.33 6.03 UF / DF1 25 °C (24h) 11.71 23.37 70.63 6.00 Ref Ref.H0713 12.43 21.11 71.91 6.98
  • HIC is slightly disadvantageous in terms of charge isoform quality control and similar quality control is performed in the remaining steps.
  • AM main acidic peak
  • basic base line were observed to rise together.
  • HIC gave a low score due to the problem of excessive 1M NaOH in raising the pH
  • CEX gave a slightly lower score due to the process unnecessary to increase the pH and lower it again for VI.
  • HIC contains a lot of salt
  • NaOH also contains a lot of high risk of aggregation occurred that gave a low score.
  • UF / DF1 and AEX were penalized as they are after the HIC process where most of the aggregation can be removed.
  • CEX has a conventional pH down process in VI process, and after pH maturation in VI, 2X buffer (pH 6.0) is mixed for HIC Load prep. In order to lower the progression without further processing gave a high score. The final score was therefore assessed as VI> CEX> AEX> UF / DF1 >> HIC (Table 14).
  • the percentage of main acidic peak (AM) was 12.45% on average, 21.8% for acidic portion, 71.7% for main portion, and 6.49% for basic portion (Table 15).
  • the main acidic peak (AM) rises depending on the pH and time as a result of the above embodiments, it can be used as a time determination criterion for pH maturation using AM%.
  • AM growth rate was calculated, which means the amount (%) of the AM rising time.
  • the cultures (7 days) were collected, and the samples purified on CEX column (Fractogel COO (M)) were lowered to pH 3.8 for VI, and then gradually increased to pH 7.8 to 8.6 after 1hr inactivation, respectively.
  • Samples were collected in ml. Samples sampled at 30 ml for each pH were maturated at room temperature (18 ⁇ 23 °C) for 24 hours, and the AM growth rate (% / hr) was divided by the reaction time (24 hours) of the AM difference after maturation and before maturation. ) was calculated (Table 16).
  • the present pH maturation method was introduced on a 1000 L culture scale.
  • Trastuzumab Purification processes for the preparation of Trastuzumab include Cation Exchange Chromatography (CEX), Virus Inactivation (VI) by low pH, Hydrodrophobic Interaction Chromatography (HIC), and Ultrafiltration. Consists of 1st Ultrafiltation and Diafiltration (UF / DF I), Anion Exchange Chromatography, 2nd Ultrafiltration and Diafiltration II (UF / DF II), VF (Virus Filtration), Final Formulation It is.
  • Cation Exchange Chromatography CEX
  • VI Virus Inactivation
  • HIC Hydrodrophobic Interaction Chromatography
  • Ultrafiltration Consists of 1st Ultrafiltation and Diafiltration (UF / DF I), Anion Exchange Chromatography, 2nd Ultrafiltration and Diafiltration II (UF / DF II), VF (Virus Filtration), Final Formulation It is.
  • the maturation time was calculated and maturation proceeded as follows for pH maturation during VI process at 1000l production scale.
  • the initial AM% criterion was based on the CEX elute sample, and the target AM% was applied to 10.5%.
  • the reason why the target AM (main acid form) content% is 10.5% is because the UF / DF1 and AEX processes operate at pH 7.5, which results in an average AM increase of more than 1% due to the high pH. Therefore, we gave a safety margin to reflect the rise of the process after pH maturation, so the target in VI was determined to be 10.5%.
  • AM growth factor This factor calculates the rate of increase of AM (main acidic peak), which is a target peak for pH maturation, per hour. This growth factor was determined through data analysis through the scale down experiment, and the conditions were designed to complete pH maturation within 24hr. This factor is a factor dependent on the maturation pH. At pH 8.1, 0.17% / hr meets Target AM% (10.5%) in 24hr.
  • # Margin ⁇ 2hr is the range set by 2 times considering the time that it takes more than 1hr to add 2X HIC load prep buffer to stop the pH maturation reaction.
  • the pH maturation method of the present invention is sufficiently applicable to the production scale, and that the present method not only can produce high-quality antibodies, but also has the same quality as that of the reference drug in terms of biosimilar production. It supports the production of antibodies.

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Abstract

La présente invention concerne un procédé de préparation d'une population d'anticorps de haute pureté et de haute qualité par ajustement à un niveau souhaité de la quantité d'anticorps hétérogènes dans une population d'anticorps, une population d'anticorps préparée par ce procédé, un procédé d'ajustement de la quantité d'anticorps hétérogènes dans la population d'anticorps, et un procédé pour déterminer un temps de stabilisation du pH pour ajuster la quantité d'anticorps hétérogènes. Par utilisation du procédé de la présente invention, la quantité d'anticorps hétérogènes dans la population d'anticorps est ajustée à une valeur souhaitée, en préparant de ce fait d'une manière constante une population d'anticorps dont la haute qualité ou l'équivalence sont assurées.
PCT/KR2013/011236 2013-12-05 2013-12-05 Procédé de préparation d'un anticorps par ajustement de la quantité d'anticorps hétérogènes WO2015083853A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023249892A1 (fr) * 2022-06-20 2023-12-28 Amgen Inc. Clairance d'agrégats à partir de bains uf/df dans une purification d'anticorps en aval

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WO2011009623A1 (fr) * 2009-07-24 2011-01-27 F. Hoffmann-La Roche Ag Optimisation de la production d’anticorps
WO2012084829A1 (fr) * 2010-12-21 2012-06-28 F. Hoffmann-La Roche Ag Préparation d'anticorps enrichie en isoformes et son procédé d'obtention
US20120178910A1 (en) * 2009-09-23 2012-07-12 Medarex, Inc. Cation exchange chromatography (methods)
KR20130069515A (ko) * 2011-12-15 2013-06-26 한화케미칼 주식회사 항체의 정제 방법

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WO2011009623A1 (fr) * 2009-07-24 2011-01-27 F. Hoffmann-La Roche Ag Optimisation de la production d’anticorps
US20120178910A1 (en) * 2009-09-23 2012-07-12 Medarex, Inc. Cation exchange chromatography (methods)
WO2012084829A1 (fr) * 2010-12-21 2012-06-28 F. Hoffmann-La Roche Ag Préparation d'anticorps enrichie en isoformes et son procédé d'obtention
KR20130069515A (ko) * 2011-12-15 2013-06-26 한화케미칼 주식회사 항체의 정제 방법

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* Cited by examiner, † Cited by third party
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WO2023249892A1 (fr) * 2022-06-20 2023-12-28 Amgen Inc. Clairance d'agrégats à partir de bains uf/df dans une purification d'anticorps en aval

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