CN111304176A - A large-scale purification method of adenovirus vector based on Q-6XL and 4FF - Google Patents

Abstract

A large-scale adenovirus vector purification method based on Q-6XL and 4FF relates to the field of adenovirus vector purification, and particularly designs a large-scale adenovirus purification scheme based on Q-6XL and 4FF series connection, aiming at improving the preparation efficiency and purity of adenovirus vectors and finally improving the yield and quality of adenovirus preparations. After virus fermentation liquor is processed by the processes of centrifugation, freeze thawing, PEG concentration, buffer solution replacement and the like, host protein and host DNA in the virus fermentation liquor are removed by Q-6XL anion exchange chromatography to obtain adenovirus crude pure liquid. And then, purifying the virus crude pure solution by adopting 4FF gel filtration chromatography (molecular sieve) to finally obtain the high-purity adenovirus vector. In particular, the method is suitable for large-scale purification of adenovirus vectors. Meets the increasing demand of high-purity adenovirus vector dosage in clinical stage.

Description

A large-scale purification method of adenovirus vector based on Q-6XL and 4FF

Technical field:

The present invention relates to the field of adenovirus vector purification, and particularly designs a large-scale adenovirus purification scheme based on the tandem of Q-6XL and 4FF. It aims to improve the efficiency and purity of adenoviral vector preparation, and ultimately improve the yield and quality of adenoviral preparations.

Background technique:

Adenovirus has great potential in gene therapy, and its purity, efficacy, and safety have strict standards. Unlike traditional vaccine preparations, adenoviruses have different physicochemical properties than proteins, and it is necessary to maintain viral activity as much as possible during the purification process. Therefore, it is a key problem to be solved to prepare a high-purity adenovirus that meets the dosage and to optimize the downstream processing technology.

Downstream processing of adenoviral vectors includes a series of steps aimed at increasing the efficiency and purity of adenoviral vector preparation. Among them, the purification process of adenovirus is the key to improve the yield and quality of the final virus preparation. In the past 10 years, considerable progress has been made in the field of downstream processing of viral vectors. However, most of the research work has focused on the purification process of widely used herpes virus, lentivirus, etc. However, there are few studies on the purification process of adenovirus, especially the large-scale purification process that meets the dosage requirements. The traditional purification method of adenovirus is CsCl (cesium chloride) density gradient centrifugation. The technique uses high-speed centrifugation to enhance the density gradient of a solution, separating viruses from other molecules based on their differences in size, shape, and density from contaminants. During the centrifugation process, the virus with smaller molecular weight is located in the upper layer of the solution, and other contaminants are located in the lower layer of the solution. However, this purification method generally requires 2-3 rounds of repeated centrifugation to obtain relatively pure adenovirus particles, and it is difficult to achieve complete separation of the upper and lower layers. The yield of adenovirus obtained by this method is therefore low and very time consuming. Therefore, a purification scheme that can produce high-purity adenoviral vectors on a large scale is urgently needed.

Q-6XL Anion Exchange Chromatography (AEX) is a scalable and efficient purification protocol for viral vectors. Adenoviral vectors have an isoelectric point below 7.4 and are negatively charged under physiological pH conditions. Compared with protein contaminants in fermentation broth, adenoviruses bind more strongly to anion exchange chromatography columns. The binding capacity of nucleic acid pollutants exceeds that of viruses, and competes with viruses for binding sites during the chromatography process. Therefore, different salt concentrations are used for subsequent elution to achieve separation and purification of adenovirus.

The separation range of 4FF gel filtration chromatography is 60KD-200MD, and the adenovirus molecule is about 700KD. During the 4FF chromatography process, the molecular weight of the adenovirus and other contaminants is different, resulting in different partition coefficients, and the virus is purified by successive elution during the chromatography process. Moreover, the solution desalting and impurity removal can be carried out directly during the chromatography process. However, the general characteristics of molecular sieves are non-adsorption chromatography, low loading capacity, and it is difficult to achieve industrial scale-up. The 4FF selected in this patent is a highly coupled agarose filler with high mechanical strength, which can solve the bottleneck problem that the adenovirus purification process is difficult to amplify. Therefore, it is selected as the purification process of the last step of virus purification.

SUMMARY OF THE INVENTION

The present invention proposes a large-scale purification scheme of adenovirus based on the tandem of Q-6XL and 4FF. Adenovirus-infected host cells were used to form virus plaques after culturing for 48 hours, and the adenovirus fermentation broth was harvested. The fermentation broth is pre-treated by freezing and thawing, centrifugation, PEG (polyethylene glycol) precipitation and concentration, and buffer replacement. First, the crude adenovirus solution was obtained by purification by Q-6XL anion exchange chromatography, and further purified by the chromatographic scheme of 4FF molecular sieve, and finally a high-purity adenovirus vector was obtained.

The present invention aims to establish a purification process scheme capable of large-scale production of high-purity adenovirus, which is mainly divided into the following steps:

(1) lysing the host cell from the cell fermentation broth to release the adenovirus vector;

(2) removing cells and cell debris from the harvested cell lysate by centrifugation to obtain a preliminary clarified adenovirus fermentation broth;

(3) adopt PEG precipitation method to concentrate fermentation broth;

(4) replacing the fermentation broth into the Q-6XL chromatographic sample loading buffer;

(5) using Q-6XL ion-exchange chromatography column to carry out crude purification of adenovirus to obtain a crude adenovirus solution;

(6) Purify the crude adenovirus liquid by using 4FF molecular sieve to obtain a high-purity adenovirus vector.

The present invention will describe each part in detail:

1. The method for lysing host cells by freeze-thaw method provided by the present invention mainly includes:

1. The adenovirus of the present invention is enriched both inside the cell and in the cell culture supernatant. Collect all virus fermentation broth

2. Use batch centrifugation to separate the supernatant from the cell pellet

3. Freeze and thaw the pellet at -80°C and 25°C for 3 times to obtain a cell lysate.

2. The method for removing host cell debris provided by the present invention mainly includes:

1. Centrifuge the harvested cell lysate and collect only the entire supernatant fraction.

Three, the PEG precipitation concentration provided by the invention, the replacement fermentation broth method mainly includes:

2. Add PEG and adenovirus fermentation broth 1:7 (volume ratio) to PEG solution (500ml H20, 500gPEG-8000)

3. Add 10% (volume of fermentation broth) 1M NaCl (sodium chloride) (1L H20, 58.5gNaCl)

1. Stir for 20min, let stand at 4℃ overnight

2. Centrifuge, remove the supernatant, and resuspend the pellet in the chromatographic loading buffer Buffer A.

Fourth, the present invention provides the Q-6XL anion exchange chromatography method mainly including:

1. Equilibrate Q-6XL with chromatography loading buffer Buffer A, 5-10CV (CV column volume)

2. Load the sample (substituted adenovirus fermentation broth) and collect the flow-through.

3. Wash the column with chromatography loading buffer Buffer A for 5-10CV

4. 30% chromatographic loading eluent Buffer B was eluted for 5 CV, and eluent 1 was collected.

5.50% chromatographic loading eluent Buffer B was eluted for 5CV, and the eluent 2 (crude adenovirus solution) was collected

Five, the present invention provides 4FF anion exchange chromatography method mainly including:

1. Equilibrate the column with Buffer A for 2CV

2. Loading (eluent 1 collected in the first step of purification)

3. Elute 2CV and collect the peaks of each component

Description of drawings

Figure 1: Distribution gradient elution chromatography results of Q-6XL

Figure 2: 4FF chromatographic results

Figure 3: Determination of protein content in feed solution at different stages of chromatography by SDS-PAGE

Note: 1: marker 2: after centrifugation 3: after PEG precipitation concentration replacement (10-fold dilution) 4: protein peak 5: Q-6XL virus crude peak 6: virus peak after 4FF

Detailed ways

The present invention will be described below in conjunction with specific examples

Example 1:

Adopting the method of the present invention, the specific steps of adenovirus large-scale purification based on the Q-6XL and 4FF two-step method include:

1. Adenovirus pretreatment part:

1. Use HEK293-A host cells to culture adenovirus, and harvest 3L of virus fermentation broth 48 hours after inoculation.

2. The harvested virus fermentation broth was centrifuged at 4°C, 3500rpm for 15min.

3. Repeat freeze-thaw at -80°C and 25°C for 3 times, centrifuge at 4°C, 2000rpm for 10min, and collect the supernatant, a total of 2.9L.

4. PEG and adenovirus fermentation broth were added in 1:7 (volume ratio) PEG solution (500ml H20, 500gPEG-8000) 414ml, 10% (fermentation broth volume) 1M NaCL solution (1L H20, 58.5gNaCl) 290ml.

5. Stir for 20 minutes, and stand at 4°C for 8h-12h.

6.4°C, 8000rpm, centrifuged for 40min, discard the supernatant, resuspend the pellet with 80ml of Q-6XL chromatography loading buffer Buffer A, and sample (after replacement).

2. Adenovirus purification process:

1. The buffer configuration used by Q-6XL is as follows:

Chromatography buffer Buffer A and chromatographic eluent Buffer B involve Tris (tris(trihydroxymethylaminomethane)) as the buffer component, NaCl as the elution component, and specially added 5% (accounting for the prepared Buffer A and B solutions). Volume) glycerol is used as a virus protective agent component, NaOH (sodium hydroxide) involved in Buffer C is a cleaning component of the chromatography column (to ensure repeated use of the chromatography column), and the ethanol solution involved in Buffer D is a storage component of the chromatography column . The solutions used are all filtered at 0.45μm before use. The specific proportions of the solutions are shown in the following table:

4. The distribution gradient elution experiment process of Q-6XL

Based on the above elution steps, a preliminary separation of host protein and adenovirus was achieved. The chromatographic results are shown in Figure 1. Under the 30% chromatographic eluent Buffer B, the host protein was eluted, and the eluate 1 was collected. Under the 50% chromatographic eluent Buffer B, the adenovirus was eluted, The eluate 2 (crude adenovirus, after Q-6XL) was collected, and the host DNA was completely bound to the chromatography column and eluted during the washing process.

5.4FF uses the same buffer configuration as Q-6XL

6.4 FF chromatographic elution experiment process

Based on the above elution steps, the adenovirus fermentation broth was purified, and the chromatographic results were shown in accompanying drawing 2. The virus peak was eluted under the conditions of the elution solution. Eluent 3 (adenovirus semen, after 4FF) was collected.

3. Adenovirus purification results:

1. In all purification steps, the collected eluates are host protein, virus crude liquid, and virus pure liquid. Western-blot was used to determine the protein content in the feed solution at different stages of the chromatography, and the SDS-PAGE results were shown in Figure 3.

2. TCID50 was used to measure the amount of virus at each stage, and the results are shown in Table 1.

(1) Prepare a 96-well plate, inoculate 8000 cells per well, and culture overnight. The cells used are HEK293A.

(2) The adenovirus was sequentially diluted 10 times (gradient of 10 ^-3 -10 ^-10 ), and mixed well.

(3) Aspirate the medium in the 96-well plate and wash once with PBS.

(4) 100 μl of adenovirus dilution solution was inoculated repeatedly at each concentration, and cultured in a CO2 incubator at 37°C.

(5) After 10 days, the cytopathic changes were observed under a microscope, and the lesions were recorded.

Table 1. Recovery of adenovirus purification method based on Q-6XL and 4FF two-step method

Claims (5)

1. based on the adenovirus vector large-scale purification method of Q-6XL and 4FF, it is characterized in that, adopt adenovirus to infect the host cell cultivated under 100rpm rotating speed condition, obtain virus culture liquid; The adenovirus was further purified by Q-6XL anion exchange chromatography and 4FF gel filtration chromatography after precipitation concentration and buffer replacement. 2 . The method according to claim 1 , wherein the adenovirus-infected host cell is HEK293-A, and after culturing for 48 hours, viral plaques are formed, and the adenovirus culture solution is harvested. 3 . 3. the method as claimed in claim 1 is characterized in that, adding the glycerol of 5% of the total volume of the buffer as a virus protective agent in the chromatography loading buffer, and finally adjusting the pH of the buffer to under the condition of 8.0 Purify. 4. method as claimed in claim 1, is characterized in that, adopts Q-6XL packing column to load sample with 4ml/min flow velocity, carries out crude purification to adenovirus, when monitoring wavelength 280nm and 254nm go out peak, collect eluate , which is the crude adenovirus solution. 5. the method as claimed in claim 1 is characterized in that, the 4FF packing column that adopts is sampled with 1ml/min flow velocity, carries out purification to adenovirus, when monitoring wavelength 280nm and 254nm go out peak, collects eluate, That is adenovirus semen.

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