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The critical process technology for gene recombinant virus

manufacturing which we mastered

Cell culture techniques

We have excellent cell culture techniques. We are really good at culturing conventional animal cells in the different culture container, such as T25 flasks, T225 flasks, cell factories , and bioreactors. We can accomplish the more difficult cell related experiment, such as cell domestication.

Adherent cell culture

Adherent cells are mainly statically cultured in the DMEM medium containing 10% serum, at 37℃, 5.0%CO2 in the incubator. The adherent cells include HEK293, Hela, Vero, 293T, BHK-21, A549, etc. We can skillfully operate the conventional experiment, such as recovery, passage, frozen storage, etc.

Suspension cell culture

The suspension cells are mainly cultured in the complete serum free culture medium, at 37℃, 5.0-8.0%CO2 condition, through static or shaking culture. When they grow to a certain density, the cells can be cultured with perfusion by continuously addition of serum free mediumto carry out continuous culture. The conventional suspension cells include HER096, Hela-F, 293F, etc. Among these, Hela-F cells and 293F cells were domesticated by our company.

Cell domestication

Our company has successfully domesticated a number of cell lines. We are able to domesticate the target cells by screening varieties of serum free mediums , find out the most suitable medium for the cell culture and optimize the culture process.

Hela cells(Before domestication, adherent)

Hela-F cells(After domestication, suspension)

Virus amplification techniques

small scale amplification

Virus Type Cell Vessel Harvest
Adenovirus HEK293 cells (adherent),HER096
cells (suspension) or the other cells
customers provide
225cm2 flask/
10-fold cell factory
The cell suspension, loose cells and medium,
were collected in 50 ml Falcon tubes, centrifugation,
the cell pellets were resuspended altogether with solution
Lentivirus 293T 225cm2 flask Virus particles released to extracellular medium,
collected supernatant
Vaccinia virus Amplification with Hela cells 、
(adherent, suspension),
BHK-21 cells or the other cells customers provide.
175cm2 or 225cm2 flask
The adherent cells with virus in them were collected by scraping the bottom of the flask with scrapers.
The cell suspension, loose cells and medium, were collected in 50 ml Falcon tubes,
centrifugation,the cell pellets were resuspended altogether with solution

The NBS bioreactor perfusion culture techniques

The NBS bioreactor equips with 5 L cells tanks, 7.5 L cells tanks and 14 L tanks, mainly for the 293 cell adherent culture, amplifying adenovirus.

The main technical characteristics:

Flake carrier (FibraCel) culture: large specific surface area, so high cell yield in the per unit volume culture; advantages of both the suspension culture and adherent culture; simplify the detection and control of various environmental factors in cell growth, good reproducibility; medium utilization rate is higher; cell harvesting process is not complicated; labor intensity is small; culture systems takes up little space.

Perfusion culture: continuously add fresh medium and constantly take consumption culture medium containing cell metabolic waste, allow cells to grow in a relatively stable environment, save time and effort, and reduce the chance of a cells produce pollution; can improve the density of cells more than 10 times; when the cells to a certain density, infect with virus, eventually can reach 1015 virus particles.

WAVE bioreactor serum-free suspension culture techniques

Our company’s WAVE bioreactor can support 100 mL– 25L culture volume, mainly be used in serum-free suspension cell culture, amplify adenovirus/vaccinia virus.

The main technical characteristics:

WAVE bioreactor system utilizes an inflated disposable plastic bag as the cell cultivation chamber. These specially designed chambers are made of pharmaceutical grade material and are certified sterile by radiation, eliminating the need for cleaning and verification, shortening the period of technology development and production. The cellbag can improve the success rate of cell, and the closed system cultivation can avoid direct contact of material liquid and operating personnel, to ensure the safe operation. Oxygen transfer and mixing are accomplished by wave-induced agitation,to result in low-shear conditions and highly efficient oxygen transfer for cell cultures. It can improve cell state, increase cell density and yield. Based on typical cell respiration rates, the cell density is sufficient to grow up to 1×107cells/ml. WAVE bioreactor has flexible volume range, simple operation, reliable control precision. It is easy to scale-up.

Serum free medium has high limit composition, the physical environment is easy to control, so it is conducive to the development of the downstream purification process. There are obvious advantages in the suspension cells culture to product virus. We can get 1015 virus particles by the 20L cell bag.

The serum free culture technology meet the development trend of the technology of cell culture in biological pharmaceutical. It promotes the product quality and shortens the production cycle, and the serum free medium components can be safe controled.

Virus purification techniques

Our company has multiple virus purification platforms, and we can purify various types of adenovirus, lentivirus and poxvirus (vaccinia virus). Indeed, we research new purification method for herpesvirus and adeno-associated virus, in order to further application.

Our company has mature adenovirus purification platform. It is used for three wide used adenovirus vector (Ad2, Ad5 and Ad35), which can be cultured by the NBS –bioreactor or WAVE bioreactor . The purified virus product would have high viral activity, high purity, high concentration and in accordance with the requirements of drug quality specification.

Our company also established a well-used lentivirus purification platform, and it is able to purify HIV and SIV lentivirus which were cultured by culture flasks. The purified lentivirus products apply to basic research, pre-clinical and clinical studies.


Centrifugation is a process which involves the use of the centrifugal force for the sedimentation of heterogeneous mixtures with a centrifuge, and it always used by high-speed refrigerated centrifuge.

In virus purification process, our company uses the centrifuge, Beckman Avanti J20XPI, to remove cell debris and other large particulate impurities. The centrifuge is able to run a continuous flow mode centrifugation in the pilot scale production, which is able to deal with more than 100 liters solution. In another side, the centrifuge also can run ultracentrifugation in lentivirus purification.


Filtration is an ideal choice for pre-filtering applications in the biomedical field, which is able to remove small particles and reduce bioburden. Filtration can be performed at room temperature with high capacity and high efficiency. It does not apply morphological or chemical changes for sample. Therefore, it can maintain high viral activity.

Also, we use disposable filter cartridge with low non-specific adsorption and high recovery yield that provide high biological safety and avoid residues between batches.


Ultrafiltration is a variety of membrane filtration in which forces like pressure or concentration gradients lead to a separation through a semipermeable membrane. Suspended solids and solutes of high molecular weight are retained in the so-called retentate, while water and low molecular weight solutes pass through the membrane in the permeate, to the result of partial purification for macromolecules. In virus purification, ultrafiltration can remove small molecular impurities and concentrate crude virus solution, to prepare for the next step purification.

Compared to conventional ultrafiltration, our company uses tangential flow ultrafiltration cartridge, which is higher capacity, higher efficiency and high recovery yield

Column chromatography

Our company has two ÄKTA purification systems for column chromatography, ÄKTA explorer and ÄKTA prime. ÄKTA explorer contains three main parts, a high efficient dual-channel gradient pump with velocity range 0.01-100 ml / min and pressures up to 10Mpa, a high sensitivity multi-wavelength UV-Vis and a combined monitor for accurate, online measurement of pH, conductivity, and temperature. ÄKTA explorer systems are entirely controlled by UNICORN software, that easy to operate and provide high accuracy result. It is used for method development and application, as well as pilot scale production.

Our company adopts the virus purification method of column chromatography, including ion-exchange chromatography and size-exclusion chromatography. The column chromatography method is advantaged in no heat production, no external force, high recovery yield, high resolution, high automation, and a large number of success stories.

Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. It is often used in protein purification. In the adenovirus purification, depending on the virus type, different types of medium would be applied respectively, such as Source 30Q, Q Sepharose XL, and Fractogel DEAE. In ion exchange chromatography, the loading sample must be low salt and the harvest solution would be high salt, which maintains high biological activity of the virus during the purification process. However, salt concentration of the result solution should be decreased as soon as possible.

Virus preservation solution formulation and preparation of packing technology

Virus preservation solution formula

According to different type of virus, have different preservation solution formula, including adenovirus preservation solution formula, slow virus preservation solution formula, pox virus preservation solution formula, etc.

Partial shipments

According to customer request packaging different specifications of small capacity liquid injection preparation (in line with the GMP production requirements).

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