CN-121991904-A - Eluent for improving lentivirus anion exchange chromatography recovery rate and application thereof

Abstract

The invention discloses an eluent for improving the recovery rate of lentivirus anion exchange chromatography and application thereof. The eluent comprises basic buffer, 50-500 mM of NaCl and 0.2-2 of M of L-arginine, and the pH value of the eluent is 6.5-8.0. Aiming at the problems of high damage to the activity of the slow virus and low recovery rate caused by high-salt elution in the prior art, the method can effectively elute the slow virus and avoid the damage of a high-salt environment to the virus envelope structure by compounding the main eluting component NaCl in the eluent with L-arginine after reducing the concentration, and improves the eluting efficiency by utilizing the interaction characteristic of the L-arginine inhibin. The eluent provided by the invention is used for chromatographic purification, so that the recovery rate and activity of the lentivirus can be obviously improved, impurities such as host cell proteins and the like can be effectively removed, and a key purification solution is provided for preparing the high-purity and high-activity lentivirus vector for gene therapy.

Inventors

• HE RONGHUA
• YANG LINLIN
• ZHANG CHANGJIANG

Assignees

• 无锡生基医药科技有限公司

Dates

Publication Date

20260508

Application Date

20260210

Claims (10)

1. 1. An eluent for improving the recovery rate of lentivirus anion exchange chromatography, which is characterized in that the eluent comprises a basic buffer, 50-500 mM NaCl and 0.2-2M L-arginine, and the pH value of the eluent is 6.5-8.0.
2. 2. The eluent as claimed in claim 1, wherein the concentration of L-arginine is from 0.5M to 1M, preferably from 0.6M to 0.75M.
3. 3. The eluent of claim 1 wherein the concentration of NaCl is 150 mM.
4. 4. The eluent as claimed in claim 1, wherein the basic buffer is selected from phosphate buffer or Tris-HCl buffer, and the pH value of the eluent is 6.5 to 7.5.
5. 5. The eluent as claimed in claim 1, wherein the eluent further comprises a protective agent selected from sucrose or lactose at a concentration of 2% -4%m/V, preferably 2%m/V sucrose.
6. 6. The eluent of claim 1, wherein said eluent further comprises 2mM MgCl 2 .
7. 7. The eluent of claim 1 consisting essentially of 20mM phosphate buffer, 150mM NaCl, 0.5M to 0.75M L-arginine, 2% m/V sucrose and 2mM MgCl 2 , at a pH of 6.5.
8. 8. The eluent of claim 7 consisting essentially of 20mM phosphate buffer, 150mM NaCl, 0.75M L-arginine, 2% m/V sucrose and 2mM MgCl 2 , at pH 6.5.
9. 9. Use of an eluent as claimed in any one of claims 1 to 8 in lentivirus anion exchange chromatography comprising the steps of: S1, loading, namely loading a slow virus-containing feed liquid to an anion exchange chromatographic column balanced by a balancing liquid; s2, leaching, namely flushing the anion exchange chromatographic column by using the balance liquid to remove impurities; s3, eluting, namely eluting the slow virus combined on the anion exchange chromatographic column by using the eluent and collecting an eluting peak.
10. 10. The use according to claim 9, wherein the turbidity of the lentivirus-containing feed solution is below 20NTU, the packing ligand of the anion exchange chromatography column is DEAE, the equilibration solution consists of 20mM phosphate buffer, 150mM NaCl, 2%m/V sucrose and 2mM MgCl 2 , the pH of which is 6.5, the loading retention time is 3-6 min, the elution retention time is 3-10 min; the application further comprises: S4, regulating the osmotic pressure, namely diluting the eluent by using 20mM phosphate buffer with pH of 6.5, and regulating the osmotic pressure to 280-350 Osm/kg.

Description

Eluent for improving lentivirus anion exchange chromatography recovery rate and application thereof Technical Field The invention relates to the technical field of biological medicines, in particular to an eluent for improving the recovery rate of lentivirus anion exchange chromatography and application thereof. Background Lentiviral vectors are critical tools in gene therapy for their unique advantages. Lentiviruses belong to the family of retroviruses and are capable of stably integrating foreign genes into the genome of host cells, enabling long-term expression of the gene of interest. Based on lentiviral delivery technology, the U.S. FDA has approved the market for ten additional products, including widely known CAR-T therapies such as YESCARTA, TECARTUS, KYMRIAH, CARVYKTI, etc. These products, which are successfully marketed, not only bring new therapeutic options to patients, but also fully demonstrate the great potential and important value of lentiviral vectors in gene therapy. In recent years, the number of clinical trials related to lentiviruses in vivo has grown 35% worldwide. In vivo applications put forward standards for lentiviruses for purity, activity, and safety far beyond in vitro applications. For example, in vivo injection formulations require stringent control of Host Cell Protein (HCP) residues below 100ng/mg and the viral particles need to maintain a complete envelope structure to avoid in vivo immunogenic reactions. This presents a completely new challenge to lentiviral purification technology. Whether it is the basic research conducted by the scientific research institutions or the clinical trials and drug development conducted by pharmaceutical enterprises, a large number of high-quality lentiviral vectors are required. According to the predictions of market research institutions, the global lentiviral vector market will continue to expand at a higher growth rate for the next few years, and its market size will continue to expand. Under the market background, how to efficiently and stably produce high-quality lentiviral vectors meets the increasing market demands and becomes a key problem to be solved in the field of gene therapy. The traditional lentiviral purification technology has the dilemma that the purification of lentiviral vectors is a key step for obtaining high-activity and high-purity products, and the safety and effectiveness of gene therapy are directly affected. However, the conventional lentivirus purification technology currently faces many challenges in practical application, and it is difficult to meet the increasing market demands and strict quality standards. Ultracentrifugation (one) limitations of precipitation The ultracentrifugation precipitation method is one of the more commonly used lentivirus purification methods at present, and the principle is that lentivirus is separated from complex systems such as cell culture solution by utilizing the sedimentation speed difference of different substances in a high-speed centrifugal force field. In practice, the lentiviral particle is precipitated at the bottom of the centrifuge tube by centrifuging the lentiviral-containing supernatant at an ultra high rotational speed for several hours. Although this method can achieve lentiviruses of a certain purity in laboratory small scale preparations, its limitations are also apparent. On the one hand, the ultracentrifugation precipitation method requires very high equipment requirements, requires expensive ultracentrifuges, and is expensive in maintenance and operation, which greatly increases the economic burden of production and limits its application in mass production. On the other hand, the method is complex to operate and requires a professional technician to operate, and lentiviral particles are easily influenced by high shearing force and high temperature in the centrifugation process, so that viruses are inactivated, and the infection activity and titer of the viruses are reduced. In addition, the ultracentrifugation precipitation method is difficult to realize continuous production, has lower production efficiency and cannot meet the requirement of large-scale industrial production. According to the related research, when the ultracentrifugation precipitation method is adopted for lentivirus purification, the recovery rate of the virus can only reach 30% -50% generally, and the activity loss is larger. This is a significant waste of precious lentiviral resources. (II) deficiencies of conventional ion exchange chromatography Ion exchange chromatography is another commonly used lentivirus purification technique, which is based on electrostatic interaction between lentivirus surface charge and ion exchange medium, and realizes separation of lentivirus and impurities by adjusting pH value and ionic strength of buffer solution. In conventional ion exchange chromatography, lentiviruses bound to the medium are eluted, typically by high salt elution. However, this metho