WO2020258704A1 - Procédé chromatographique à écoulement continu sans interruption - Google Patents

Procédé chromatographique à écoulement continu sans interruption Download PDF

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Publication number
WO2020258704A1
WO2020258704A1 PCT/CN2019/120924 CN2019120924W WO2020258704A1 WO 2020258704 A1 WO2020258704 A1 WO 2020258704A1 CN 2019120924 W CN2019120924 W CN 2019120924W WO 2020258704 A1 WO2020258704 A1 WO 2020258704A1
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chromatography
type
continuous flow
seamless
devices
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PCT/CN2019/120924
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English (en)
Chinese (zh)
Inventor
蔺智勇
杨忠华
王亚东
周凯松
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信达生物制药(苏州)有限公司
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Priority to CN201980097866.XA priority Critical patent/CN114026108A/zh
Publication of WO2020258704A1 publication Critical patent/WO2020258704A1/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
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes

Definitions

  • the present invention relates to the technical field of separation and purification, in particular to the purification of biological agents, in particular to a method for chromatography using a chromatography device, and in particular to a seamless continuous flow chromatography method.
  • the traditional chromatography process needs to collect and mix the intermediate product produced after the previous step of chromatography is completed, and then load the intermediate product for the next step of chromatography.
  • intermediate products need to undergo processing such as filtration, low temperature storage, pH adjustment, etc., occupying equipment resources; and multiple chromatography processes need to be performed one by one, which requires a long process time.
  • three work areas respectively perform cell juice harvesting, chromatography and product Pr acquisition.
  • the cell culture harvest liquid obtained in the first work area P1 is collected in the storage container T1 for temporary storage.
  • the harvested liquid in the storage container T1 is subjected to the first chromatography through the chromatography column C1.
  • the captured intermediate product is temporarily stored in the storage container T2.
  • the intermediate product in the storage container T2 is subjected to the second chromatography through the chromatography column C2.
  • the captured intermediate product is temporarily stored in the storage container T3.
  • the intermediate product in the storage container T3 is subjected to the third chromatography through the chromatography column C3.
  • the captured intermediate product is temporarily stored in the storage container T4. This not only requires the use of multiple large-capacity temporary storage containers, but also takes up additional temporary storage time.
  • Patent publication WO2014/180852A1 discloses a continuous flow chromatography method, which can realize the simultaneous operation of multiple chromatography processes, but the use efficiency of chromatography packing is not improved, the overall process time is long (more than 30 hours), and the production cost The reduction of production efficiency and the improvement of production efficiency are limited.
  • the purpose of the present invention is to overcome or at least alleviate the above-mentioned shortcomings of the prior art, and to provide a seamless continuous flow chromatography method with a partial continuous flow seamlessly connecting the whole.
  • the present invention provides a seamless continuous flow chromatography method, which uses at least three chromatography devices, wherein at least two chromatography devices constitute a first-class chromatography device, and at least one chromatography device constitutes a second-class chromatography device,
  • the first type of chromatography device is used to realize continuous flow chromatography
  • the first-type chromatography device and the second-type chromatography device jointly realize seamless connection chromatography.
  • no storage container is used between the first-type chromatography device and the second-type chromatography device.
  • the preparation to be chromatographed prior to the continuous flow chromatography, is subjected to virus inactivation, and the virus inactivation method is preferably an organic solvent detergent method.
  • the preparation to be chromatographed is subjected to depth filtration before the continuous flow chromatography.
  • the start time and end time of sample loading of each of the first type of chromatography devices are different; each of the first type of chromatography devices can work simultaneously; one of the first type of chromatography devices The sample flow-through product of the device can be captured by another chromatographic device of the first type; the chromatographic device of the first type continuously or discontinuously loads samples, and collects continuously or discontinuously.
  • the first type of chromatography device is used to capture the target product, and the purity of the target product in the capture collection liquid of the first type of chromatography device is greater than 80%.
  • the second-class chromatography device is used for purification, and the purity of the target product in the collected liquid of the second-class chromatography device is greater than 90%.
  • the second type of chromatographic device completes equilibrium and waits for the sample to flow in before the first type of chromatographic device collects products, and the first type of chromatographic device starts the process of chromatography.
  • the chromatography process of the second type of chromatography device is described.
  • the chromatography device is a chromatography column, each of the first type of chromatography device has the same filler, each of the second type of chromatography device has different fillers, and each of the two The packing of the type of chromatography device is different from the packing of the type of chromatography device; or
  • the chromatography device is a membrane chromatography device, the chromatographic membrane of each of the first-type chromatography devices is the same, the chromatographic membrane of each second-class chromatography device is different, and each of the second-class layers
  • the chromatographic membrane of the analysis device is different from the chromatographic membrane of the first type of chromatographic device.
  • the first type of chromatography device is an affinity chromatography device
  • the ligand is protein A
  • the method is used to purify a biological agent, which is preferably an antibody.
  • the method includes:
  • the first type of chromatography device performs affinity chromatography, and when the affinity chromatography is eluted, the pH is controlled to be 3.2-4.0, and the conductivity is controlled to be ⁇ 30 mS/cm.
  • the second-class chromatography device performs cation chromatography, and when the cation chromatography is eluted, the pH is controlled to be 5.0-8.0.
  • the chromatographic product can be directly obtained without processing intermediate products, and the process time is shortened.
  • the local continuous flow improves the use efficiency of the chromatography packing, reduces the packing cost, and can indirectly reduce the equipment scale of the chromatography column and the buffer storage tank.
  • the method improves production efficiency and reduces production costs as a whole.
  • Figure 1 is a schematic diagram of a traditional multi-chromatography process in the prior art.
  • FIG. 2 is a schematic diagram of the process flow of the seamless continuous flow chromatography method according to the first embodiment of the present invention.
  • the seamless continuous flow chromatography method according to the present invention uses at least three chromatography columns, of which at least two chromatography columns (also called a type of chromatography device) are used to achieve partial continuous flow chromatography.
  • the packing is the same (for example, used to capture the same first component); at least another chromatography column (also called the second type of chromatography device, which is different from the first type of chromatography device, for example, used to capture the second Components or for purification) are connected in series with the local continuous flow chromatography system to realize the overall seamless connection chromatography.
  • each type of chromatography device of the partial continuous flow chromatography system at least two of the chromatography devices have different chromatography processes, that is, the start and end times (start and end times) of each process are not completely the same; preferably, The start and end times of sample loading for each type of chromatography device are not exactly the same.
  • Each type of chromatography device of the local continuous flow chromatography system is equivalent to multiple small chromatography columns, and multiple small chromatography columns can perform chromatographic operations without affecting each other, so that the local continuous flow chromatography can be completed in a unit of time
  • the purification amount of is greater than that of a large chromatography column composed of the sum of the fillers of multiple small chromatography columns.
  • the chromatographic products of each first-class chromatography device are transported to the downstream second-class chromatography devices.
  • the first-class chromatography device and the second-class chromatography device can be No storage container is needed to temporarily store the formulation.
  • the process of virus inactivation by organic solvent/detergent method needs to be advanced before the chromatography process.
  • the harvesting process before the chromatography process also includes depth filtration.
  • the harvesting process also includes centrifugation, flocculation or sedimentation.
  • the above-mentioned chromatographic device (type 1 chromatographic device and type 2 chromatographic device) can be a chromatography column or a membrane chromatography device.
  • the chromatography packing can be, for example, affinity packing, cationic packing, anionic packing, hydrophobic packing or mixed mechanism packing.
  • the chromatography device adopts a membrane chromatography device, depending on the membrane medium, for example, affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography or mixed mechanism chromatography can be realized.
  • the cell culture harvest liquid obtained is a bevacizumab with an expression amount of 8 g/L.
  • step S12 centrifugation step S13 precipitation or flocculation (especially acid precipitation) and step S14 to obtain the cell supernatant after deep filtration.
  • the virus inactivation collection solution is then subjected to 6-column seamless continuous flow chromatography in the second operation area P2.
  • the first type of chromatography device is 4 affinity columns
  • the second type of chromatography device is 1 cation column and 1 An anion column.
  • the affinity filler is PrismA (product of GE, article number 17549803)
  • the cation chromatography filler is Capto S Impact (product of GE, article number 17371705)
  • the anionic filler is POROS XQ (product of Thermo, article number 4467818).
  • the four affinity columns constitute a local affinity continuous flow chromatography system CC1, and the preparation uses the affinity continuous flow chromatography system CC1 for continuous flow chromatography in step S21. Then, in step S22, the cation column C02 and the anion column C03 are used for seamless connection chromatography. Among them, the sample eluted by affinity chromatography is loaded and bound to the cation column CO2, the cation column CO2 is eluted with an eluent of pH 6.5 to pH 8.0; the eluent of the cation column CO2 directly enters the anion column C03.
  • step S31 virus removal filtration, step S32 nanofiltration and ultrafiltration, step S33 is performed to obtain the product Pr.
  • Table 1 is the chromatographic process sequence of the affinity continuous flow chromatography system CC1. Columns 1 to 4 in the table correspond to 4 affinity columns, and the table shows that each affinity column completes two loadings The start and end time of each process corresponding to the process.
  • Table 2(a) and Table 2(b) respectively show the process time required to purify a batch of monoclonal antibodies of this embodiment using the traditional process and the seamless continuous flow chromatography process according to this embodiment.
  • affinity chromatography needs about 8 hours
  • cation chromatography needs to be performed after the batch of preparations have completed affinity chromatography
  • anion chromatography needs to be performed after the batch of preparations have completed cation chromatography.
  • the seamless continuous flow chromatography process it only takes about 5 hours for the same batch of monoclonal antibodies to perform affinity continuous flow chromatography, and cation chromatography and anion chromatography can start with affinity chromatography. After a period of time and capture the sample, it is performed without waiting for the batch of preparation to complete the affinity chromatography. It should be understood that since the chromatography process includes non-loading times such as equilibrium, affinity chromatography, cation chromatography, and anion chromatography can even be started at the same time.
  • Using the seamless continuous flow chromatography process according to the present embodiment saves 12 hours (two shifts) of working time compared to the chromatography of the traditional process.
  • the cell culture harvest liquid is bevacizumab, and two affinity columns are used for partial continuous flow chromatography, and then one cation column and one hydrophobic column are used for seamless connection chromatography.
  • Affinity filler Mabselect SuRe (GE Products, Catalog No. 17543803)
  • cationic filler fractogel EMD SO 3 - (M) Merck Products, Cat. No. 1.16882.5000
  • the hydrophobic filler is Toyopearl Phenyl-600M (TOSOH Products, Catalog 0021890).
  • the cell culture harvest liquid is centrifuged, acid precipitation, and deep filtration to obtain the cell supernatant.
  • S/D virus inactivation is carried out before the start of chromatography, and the virus inactivation collection solution is then subjected to 4 columns of seamless continuous flow chromatography.
  • Two affinity columns are used as continuous flow capture products, and the affinity chromatography eluted sample is loaded and bound to the cation column.
  • the cation column uses salt-containing eluent for linear gradient elution, and the conductivity during elution is ⁇ 23mS/cm.
  • the collection fluid directly enters the hydrophobic chromatography column, and the sample flows directly through and is collected in a storage container. After that, the hydrophobic collection liquid continues to undergo nanofiltration, ultrafiltration, and preparation of the original solution to complete the entire process.
  • the process parameters in the affinity chromatography process include,
  • Affinity balance solution 20mmol/L Tris+150mmol/L NaCl, pH 7.2 balance 3CV, loading capacity 50g/L
  • affinity wash solution 100mmol/L citric acid-sodium citrate buffer solution, pH 5.
  • affinity eluent 100mmol/L citric acid-sodium citrate buffer, pH 3.2 ⁇ 4.0 elution
  • affinity regeneration solution 100mmol/L citric acid solution to regenerate 3CV, 0.1mmol/ L NaOH CIP 3CV.
  • the process parameters in the cation chromatography process include,
  • Cation balance solution 20mmol/L citric acid-sodium citrate buffer solution, pH5.0 balance 5CV, loading capacity 20 ⁇ 80g/L
  • cation eluent B 20mmol/L citric acid-citric acid Sodium buffer+0.5mol/L NaCl, pH5.0, 0 ⁇ 100B pull linear gradient elution 10CV, conductivity ⁇ 8mS/cm.
  • the process parameters in the hydrophobic chromatography process include,
  • Hydrophobic balance solution 20mmol/L citrate-sodium citrate buffer solution, pH 5.0 balance 5CV. Load ⁇ 150g/L.
  • Table 3(a) and Table 3(b) respectively show the process time required to purify a batch of bevacizumab using the traditional process and the seamless continuous flow chromatography process according to this embodiment.
  • Using the seamless continuous flow chromatography process according to the present embodiment saves 12 hours (two shifts) of working time compared to the chromatography of the traditional process.
  • Table 4 shows the comparison between the quality data of the seamless connection chromatography product according to this embodiment and the quality data of the traditional single-column system chromatography product. It can be seen from the table that the seamless connection chromatography method according to this embodiment is used There is no significant difference between the prepared sample and the sample prepared by the traditional single-column chromatography process in terms of quality (such as purity and impurities) and process performance (such as yield).
  • the cell culture harvest liquid is a recombinant anti-interleukin 23p19 subunit antibody injection, and the expression amount is 5 g/L.
  • the cell culture harvest liquid is centrifuged, acid precipitation, and deep filtration to obtain the cell supernatant.
  • S/D virus inactivation is performed before the start of chromatography, and the virus inactivation collection solution is then subjected to 5 columns of seamless continuous flow chromatography.
  • Affinity filler is Mabselect SuRe LX (product of GE company, article number 17547404), cation chromatography filler is POROS HS (product of Thermo company, article number 1-3359-09), and anionic filler is POROS XQ (product of Thermo company, article number 4467818).
  • affinity chromatography eluted sample is loaded and bound to the cation column.
  • the cation column is eluted with an eluent from pH 6.5 to pH 8.0.
  • the eluent enters the anion chromatography column directly, and the sample flows directly through and is collected in a storage container. After nanofiltration, ultrafiltration, and original solution preparation, the entire process is completed.
  • the process parameters in the affinity chromatography process include,
  • Affinity balance solution 20mmol/L Tris+150mmol/L NaCl, pH7.2 balance 3CV, loading capacity 60g/L
  • affinity wash solution 20mmol/L Tris-HCl+1mol/L sodium chloride, pH 7.2 Wash 3CV
  • affinity eluent 50mmol/L citric acid-sodium citrate buffer, pH 3.2 ⁇ 4.0 elution
  • affinity regeneration solution 3% acetic acid buffer to regenerate 3CV, 0.2mmol/L NaOH CIP 3CV.
  • the process parameters in the cation chromatography process include,
  • Cation balance solution 20mmol/L citric acid-sodium citrate buffer solution, pH5.0 balance 5CV, loading capacity 20 ⁇ 80g/L, cation eluent: 20mmol/L Tris+50mmol/L NaCl, pH6 .5 ⁇ 8.0.
  • the process parameters in the anion chromatography process include,
  • Anion balance solution 20mmol/L Tris-HCl, pH 7.5 balance 5CV. Load ⁇ 150g/L.
  • Table 5(a) and Table 5(b) respectively show the purification of a batch of recombinant anti-interleukin 23p19 subunit antibody injection using the traditional process and the seamless continuous flow chromatography process according to this embodiment. The required process time.
  • Using the seamless continuous flow chromatography process according to the present embodiment saves 12 hours (two shifts) of working time compared to the chromatography of the traditional process.
  • virus inactivation by the detergent method is carried out before the start of the chromatography step to realize the overall seamless connection chromatography.
  • SD method detergent method
  • the present invention integrates partial continuous flow chromatography technology in the overall seamless connection chromatography technology, which improves the utilization rate of the packing of the chromatography column, directly reduces the packing cost, and indirectly reduces the chromatography equipment and The scale of other storage tanks.
  • the present invention is suitable for, but not limited to, the purification of biological agents.
  • the biological agents mentioned above especially include antibodies, fusion proteins and cytokines.
  • antibody includes references to glycosylation and glycosylation of any isotype or subclass.
  • Non-glycosylated immunoglobulins or their antigen-binding regions that compete with intact antibodies for specific binding, including human antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies, monoclonal antibodies, polyclonal antibodies, and oligomeric antibodies Object or antigen-binding fragment.
  • proteins with antigen-binding fragments or antigen-binding regions such as Fab, Fab', F(ab')2, Fv, bifunctional antibodies, Fd, dAb, single-chain antibody molecules, complementarity determining region (CDR) fragments, scFv , Trifunctional antibodies, tetrafunctional antibodies, and polypeptides containing at least a part of immunoglobulins sufficient to confer specific antigen binding effects on the target polypeptide.
  • antibodies produced using the method of the present invention include, but are not limited to, Sintilimab, adalimumab, bevacizumab, Aflibercept, Inflix Riximab (infliximab), nimotuzumab (nimotuzumab), pertuzumab (pertuzumab), ranibizumab (ranibizumab), rituximab (rituximab) and trastuzumab (trastuzumab) ).

Abstract

L'invention concerne un procédé chromatographique à écoulement continu sans interruption. Au moins trois dispositifs de chromatographie sont utilisés dans le procédé, au moins deux des dispositifs de chromatographie constituant un premier type de dispositif de chromatographie, et au moins l'un des dispositifs de chromatographie constitue un second type de dispositif de chromatographie, le premier type de dispositif de chromatographie étant utilisé pour mettre en œuvre une chromatographie à écoulement continu, et le premier type de dispositif de chromatographie et le second type de dispositif de chromatographie réalisent ensemble une chromatographie reliée sans interruption. Un produit ayant subi une chromatographie selon ledit procédé de chromatographie à écoulement continu sans interruption peut être obtenu directement sans avoir besoin de traiter des produits intermédiaires, ce qui permet de raccourcir le temps de traitement, d'améliorer l'efficacité de production, et de réduire le coût de production.
PCT/CN2019/120924 2019-06-28 2019-11-26 Procédé chromatographique à écoulement continu sans interruption WO2020258704A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113880907A (zh) * 2021-10-12 2022-01-04 郑州创迈生物科技有限公司 一种可用于Protein A亲和层析连续流工艺开发的研究方法

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CN105358572A (zh) * 2013-05-06 2016-02-24 赛诺菲 用于纯化抗体的连续多步骤方法

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AR108800A1 (es) * 2016-06-17 2018-09-26 Genentech Inc Purificación de anticuerpos multiespecíficos
CN107485891B (zh) * 2017-07-20 2020-04-21 上海药明生物技术有限公司 改良的层析装置及其用于连续流层析的方法

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CN105358572A (zh) * 2013-05-06 2016-02-24 赛诺菲 用于纯化抗体的连续多步骤方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113880907A (zh) * 2021-10-12 2022-01-04 郑州创迈生物科技有限公司 一种可用于Protein A亲和层析连续流工艺开发的研究方法

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