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Method of purifying composition comprising group b adenovirus

A technology of adenovirus and composition, applied in the field of purifying compositions comprising group B adenovirus

Pending Publication Date: 2022-02-22
PSIOXUS HOUSE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, while ion-exchange chromatography is currently the gold standard for adenovirus purification, it is ineffective against group B viruses (e.g., Ad11-type viruses such as EnAd) because these viruses behave differently from group C viruses such as Ad5

Method used

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  • Method of purifying composition comprising group b adenovirus
  • Method of purifying composition comprising group b adenovirus
  • Method of purifying composition comprising group b adenovirus

Examples

Experimental program
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example

[0268] Example 1 - Evaluation of the Standard Purification Process for Purification of Group B Adenoviruses

[0269] Figure 2A A standard known purification procedure for adenoviral vectors is shown. EnAd virus was subjected to this standard purification procedure.

[0270] Ad11 / 3 vector Carrier #1 Carrier #1 Carrier #1 Carrier #2 Drug substance (ng HCP / 2x10 12 vp)

[0271] Significant amounts of host cell proteins remain in the final product even after purification using standard procedures.

example 2-B

[0272] The improved purification process of example 2-B group adenovirus

[0273] Figure 2B The improved purification process of the present disclosure for an EnAd encoding a transgene between the L5 and E4 regions is shown. After step 4 of the known procedure, a new step 5 is added. New step 5 is a diafiltration step using a diafiltration buffer with a high salt content. image 3 listed Figure 2B technical details of the process.

[0274] Step 1 Lyse virus-infected HEK293 using lysis buffer:

[0275] Benzonase (a low-salt buffer is required during Benzonase treatment because high concentrations of salt inactivate the enzyme);

[0276] Then use inactivation buffer to inactivate benzonase: 4.3M NaCl, 0.05M HEPES, pH 7.5;

[0277]Step 2 clarifies using two depth filters. Filter 1 was a pod depth filter CE35 (4 to 2 μm) from Merck Millipore, then filtered through a pod depth filter CE50 (1-0.4 μm) also from Merck Millipore. The final stage of clarification is to use the...

example 3-B

[0285] The one-step purification process of example 3-B group adenovirus

[0286] The possibility of completely omitting the chromatography step was investigated. Figure 4 The design of the purification process is shown, which after steps 1 and 2 (lysis, endonuclease treatment, inactivation and clarification) has only a diafiltration step. The technical details of this process are as follows Figure 5 shown. This process is carried out using EnAd viruses encoding transgenes.

[0287] Steps 1 and 2 were as detailed in Example 2 above. Step 2 is then followed by diafiltration as described in Step 5 above. The results are shown in Table 2 below.

[0288] table 3

[0289] process stage vp / mL total vp Recycle% HCP (ng / ml) HCP (ng / 2E12 vp) after step 2 6.39E+10 3.26E+14 na 2.31E+04 7.23E+05 final formulation 4.69E+11 9.85E+13 30 Below LOQ na

[0290] It can be seen that the host cell protein levels are below quantitative levels ...

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Abstract

A method of purifying a composition comprising a group B adenovirus, for example comprising a purification step of: subjecting a composition comprising said group B adenovirus to diafiltration employing a diafiltration-buffer with a conductivity of at least 180 mScm-1, for example a conductivity of 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, or 290 mScm-1. Also provided is a composition obtained using the purification method disclosed herein.

Description

[0001] The present disclosure relates to methods of purifying compositions comprising group B adenoviruses, and purified compositions obtainable therefrom. Background technique [0002] Currently, the field of medicine is close to realizing the potential of viruses as therapeutic agents for human use. Viruses derived from ONXY-15 (ONYX Pharmaceuticals, and obtained from Shanghai Sunway Biotech) have so far been approved for head and neck cancer in a limited number of countries. However, many viruses currently exist in the clinic, which should lead to some of these viruses being registered for use in humans. [0003] One or more therapies are based on group B adenovirus EnAd (formerly known as ColoAd1), a chimeric oncolytic adenovirus derived from Ad11 (WO 2005 / 118825 and its armed version is disclosed in WO2015 / 059303 and WO2016 / 174200 , each of which is incorporated herein by reference). EnAd is currently in clinical trials for the treatment of colorectal cancer. As part o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02C12R1/93
CPCC12N7/00C12N2710/10351C12N2710/10332A61K35/761C12N7/02C07K14/075C12N2710/10051
Inventor P·克拉克S·阿尔维斯
Owner PSIOXUS HOUSE
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