METHOD FOR LARGE-SCALE PREPARATION OF PURIFIED PREPARATION OF RECOMBINANT LENTIVIRAL VECTOR AT GMP GRADE

Abstract

Provided is a method for large-scale preparation of a purified preparation of a recombinant lentiviral vector at the GMP grade. The method comprises: (a) providing raw material feed liquid to be purified that comprises recombinant viral vectors; (b) carrying out a microfiltration treatment on the feed liquid to obtain a microfiltered filtrate comprising the recombinant viral vectors; (c) optionally concentrating the filtrate to obtain a concentrated filtrate; (d) purifying the filtrate obtained in the previous step by means of chromatography to obtain a crude pure product comprising the recombinant viral vectors; and (e) subjecting the crude pure product obtained in the previous step to liquid exchange and elaborate purification to obtain the purified recombinant viral vectors.

Claims

1. A method for large-scale purification of recombinant viral vector preparation, wherein the method comprises the following steps: (a) providing a raw material comprising recombinant viral vectors to be purified, wherein the raw material is a feed liquid, and the volume of the feed liquid is Va; (b) carrying out microfiltration treatment on the feed liquid to obtain a microfiltered filtrate comprising the recombinant viral vectors, and the volume of the filtrate is Vb; (c) optionally concentrating the filtrate to obtain a concentrated filtrate, wherein the volume of the concentrated filtrate is Vc; (d) purifying the filtrate obtained in the previous step by means of chromatography to obtain a crude pure product comprising the recombinant viral vectors; and (e) subjecting the crude pure product obtained in the previous step to liquid exchange and elaborate purification, thereby obtaining purified recombinant viral vectors; wherein the chromatography is selected from the group consisting of anion chromatography, size exclusion chromatography, multi-mode composite resin chromatography, and combinations thereof.

2. The method of claim 1, wherein the anion resin is selected from the group consisting of Capto Q, Capto ImpRes, and Capto DEAE.

3. The method of claim 1, wherein the chromatography purification is to perform anion chromatography for crude purification firstly, and then perform multi-mode composite chromatography for elaborate purification.

4. The method of claim 1, wherein in step (d), the purified recombinant lentiviral vectors have one or more features selected from the following group: (p1) the biological titer of recombinant lentiviral vectors is 1.0.sup.610.sup.9 Tu/mL; (p2) the residue of BSA is <50 ng/mL; (p3) the content of endotoxin is <1 EU/mL.

5. The method of claim 1, wherein in step (c), the concentration is performed by ultrafiltration.

6. A purified recombinant lentivirus prepared by the method of claim 1.

7. (canceled)

8. A purification device for performing the method of claim 1, which comprises: (S1) an optional first container, wherein the first container is used for holding raw material of recombinant lentivirus to be purified; (S2) a microfiltration unit, wherein the microfiltration unit is used to perform microfiltration on the recombinant lentivirus to be purified to obtain a microfiltration filtrate; (S3) an optional concentration unit, wherein the concentration unit is used to concentrate the filtrate to obtain a concentrated filtrate; (S4) a chromatography purification unit, wherein the chromatography purification unit is used to perform chromatography purification on the filtrate from the microfiltration unit or the concentration unit to obtain a purified recombinant lentiviral vector; and (S5) a collection unit, wherein the collection unit is used to collect the purified recombinant lentiviral vector.

9. The purification device of claim 8, wherein the chromatography purification unit comprises a size exclusion chromatography unit and an anion chromatography unit.

10. The purification device of claim 8, wherein the purification device further comprises: (S6) a nuclease treatment unit, wherein the nuclease treatment unit comprises an adding device for adding nuclease.

11. The method of claim 1, wherein after step (e), the method further comprises: (f) subjecting the purified recombinant viral vectors to liquid exchange to obtain a virus freezing solution comprising the recombinant viral vectors; and (g) sterilizing the virus after liquid exchange by filtration to obtain sterilized recombinant viral vectors.

12. The method of claim 1, wherein the recombinant viral vectors comprise lentiviral vectors.

13. The method of claim 1, wherein the multi-mode composite chromatography resin is selected from the group consisting of Capto adhere ImpRes and Capto core 700.

14. The method of claim 1, wherein the filtrate obtained from step (b), or the optionally concentrated filtrate obtained from step (c), is subjected to nuclease treatment before being purified by chromatography.

15. The method of claim 1, wherein in step (b), the microfiltration treatment is carried out with a microfiltration hollow fiber column.

Description

EMBODIMENTS FOR CARRYING OUT THE INVENTION

[0068] Based on extensive and intensive research, the inventors have firstly found a method for large-scale purification of recombinant lentivirus at GMP grade, which is fast and simple and has excellent purification effect. In the method of present invention, the raw material comprising recombinant lentivirus can be purified efficiently, rapidly and at large scale by using specific purification medium and specific purification steps and conditions, so as to prepare recombinant lentivirus preparation with high purity, less impurities and no endotoxin. On this basis, the invention is completed.

[0069] Terms

[0070] As used herein, the term composite filler resin refers to Capto Q, Capto ImpRes, Capto DEAE.

[0071] As used herein, the term composite filler resin chromatography refers to chromatography performed by using a composite filler resin.

[0072] As used herein, the terms recombinant lentivirus, lentiviral vector can be used interchangeably, and refer to lentiviral vector produced by introducing specific plasmids into specific packaging cells. Typically, the lentiviral vectors can be used to transfect predetermined cells (comprising human and non-human mammalian cells) for subsequent therapeutic or non-therapeutic purposes.

[0073] The method described in the present invention can rapidly obtain high-purity lentivirus preparation by using (but not limited to) the combination of a new generation of Capto Core700 and Capto adhere ImpRes resin.

[0074] The purified preparation of the recombinant lentiviral vector prepared by the method of present invention can be used for the production of cells or gene medicines.

[0075] The main advantages of the present invention include:

[0076] High-purity lentivirus preparation can be quickly obtained by combining Capto Core700 and Capto adhere ImpRes for lentivirus purification.

[0077] The present invention will be further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally based on conventional conditions, such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Unless stated otherwise, percentages and parts are by weight.

EXAMPLE 1

[0078] (1) Harvesting feed liquid: lentivirus feed liquid was collected.

[0079] (2) Microfiltering and cleaning: [0080] a) 0.45-0.8 M microfiltration hollow fiber column and AKTA Flux 6 system were connected, and the integrity was tested; [0081] b) AKTA Flux 6 system was sterilized by using 1M NaOH online; [0082] c) AKTA Flux 6 system was cleaned with injection water; [0083] d) AKTA Flux 6 system was cleaned by sterile 1PBS; [0084] e) 20 L recombinant lentivirus feed liquid was divided into two aliquots and poured into feed liquid bucket. Microfiltration was performed, and the filtrate was harvested.

[0085] (3) Ultrafiltration and concentration: [0086] a) 300-800K ultrafiltration hollow fiber column was connected to AKTA Flux 6 system and the integrity was tested; [0087] b) AKTA Flux 6 system was sterilized by using 1M NaOH online; [0088] c) AKTA Flux 6 system was cleaned with injection water; [0089] d) AKTA Flux 6 system was cleaned by sterile 1PBS; [0090] e) the microfiltered lentivirus feed liquid was ultrafiltered and concentrated by using 300-800K ultrafiltration column and AKTA Flux 6 system, and the filtrate was discard; [0091] f) the lentivirus feed liquid was concentrated from 20 L to 12 L.

[0092] (4) Nuclease treatment: [0093] a) nuclease was added into said 12 L lentivirus feed liquid at a ratio of 101000 U/mL and mixed completely; [0094] b) incubating overnight at 2-8 C.

[0095] (5) Capto Core700 and Capto adhere ImpRes connected in series were used to remove impurities and to capture viruses: [0096] a) 500 mL Capto Core700 and 500 mL Capto adhere ImpRes were connected in series and installed on the AKTA pure 150 chromatography system; [0097] b) AKTA Pure 150 system was sterilized by using 1M NaOH online; [0098] c) AKTA Pure 150 system was cleaned with injection water; [0099] d) sterile lentivirus freezing solution was loaded and passed through AKTA Pure 150 system; [0100] e) balancing; [0101] f) 12 L of feed liquid was loaded. After loading, 2050 mM Tris-Cl/11.5M NaCl was used to elute, and elution peak was collected.

[0102] (6) Fluid exchange by Ultrafiltration [0103] a) 300-800K ultrafiltration hollow fiber column and AKTA Flux 6 system were connected and the integrity was tested; [0104] b) AKTA Flux 6 system was sterilized by using 1M NaOH online; [0105] c) AKTA Flux 6 system was cleaned with injection water; [0106] d) AKTA Flux 6 system was cleaned with sterile lentivirus freezing solution; [0107] e) the microfiltered lentivirus feed liquid was ultrafiltered for liquid exchange by using 300-800K ultrafiltration column and AKTA Flux 6 system, and the filtrate was discard; [0108] f) recombinant lentiviral vectors were harvested and volume thereof was 100300 mL.

[0109] (7) Sterilization by filtration, packaging and frozen storage: [0110] a) the purified lentivirus feed liquid was filtered with 0.2 M filter; [0111] b) the final product was packaged as 1 ml/tube preparation; [0112] c) the lentiviral preparations were stored at ultra-low temperature refrigerator (70 C.).

[0113] 1. Results:

[0114] (1) The lentivirus final product had a concentration of 210.sup.9 to 410.sup.9 lentivirus/mL;

[0115] (2) BSA was <50 ng/mL;

[0116] (3) HCP was <1 ng/mL;

[0117] (4) the residue of nucleic acid was <5 pg/mL;

[0118] (5) RCL was negative.

[0119] 2. Conclusion

[0120] 0.45-0.8 M microfiltration hollow fiber column, 300-800K hollow fiber column and Capto Core700+Capto adhere ImpRes composite filler were used stepwise for cleaning filtration, concentration, liquid exchange and removal of impurities and could quickly and effectively obtain high-purity lentivirus preparation.

[0121] All literatures mentioned in the present application are incorporated herein by reference, as though each is individually incorporated by reference. Additionally, it should be understood that after reading the above teachings, those skilled in the art can make various changes and modifications to the present invention. These equivalents also fall within the scope defined by the appended claims.