DESC:FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION: A METHOD OF DEVELOPING FREE FLOATING CONDITION FOR TRANSFECTION AND INFECTION OF CELLS
2. APPLICANT:
(a) NAME : OMNIBRX BIOTECHNOLOGIES PRIVATE
LIMITED
(b) NATIONALITY : India
(c) ADDRESS : B-202, Royal Residency, Nr. Shukan,
Opp. Vandematram Arcade, New S.G. Road,
Gota, Ahmedabad - 382481,
Gujarat, India
3. PREAMBLE TO THE DESCRIPTION
PROVISIONAL
The following specification describes the invention. þ COMPLETE
The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION
The present invention relates to a method of production of biological products from the cells and more particularly it relates to method of developing free floating condition of the adherent cells for transfection and infection of cells. The present invention leads to an increase of process efficiency. Further, it is not limited to production of viral vector, vaccine, therapeutic protein, etc.

BACKGROUND OF INVENTION

There are varieties of products produced from bioprocessing such as viral vector, vaccine, monoclonal antibodies, etc. Viral vectors are the material which encompasses the copy of desired genes. These viral vectors are used as tools in gene therapy to deliver a copy of “healthy gene” into the human where these genes are integrated into the human body in-place of “unhealthy gene”. The manufacturing process for these viral vectors with desired gene is a combination of few process steps such as expression and production of viral vectors from the host cells (upstream), purification of produced vectors (downstream) and formulation of purified vectors before injecting into the human.

Biologics production is a complex and highly regulated process that involves multiple stages, from research and development to manufacturing and quality control. The process begins with identifying a target biological component, cloning it into a suitable host organism, and optimizing its expression. The manufacturing phase includes upstream processing in bioreactors and downstream purification and formulation. Rigorous quality control measures are implemented throughout to ensure product safety and efficacy.

In consultation of same process flow, vaccines are produced by host cells such as african green monkey kidney cells (VERO), MRC-5 etc. The host cells are grown in specified container such as bioreactor for large scale production of vaccine followed by infection of these grown cells by the virus of the particular disease. Infected host cells produce multiple copies of the virus. Other bioprocess also follows similar phases of process flow such as growth phase and production phase.

In particular, key factors influencing this process include host organism selection, bioreactor design and operation, culture media optimization, process control and monitoring, and addressing emerging challenges. By optimizing these elements, researchers and manufacturers can enhance the efficiency, scalability, and cost-effectiveness of biologics production. Further, the production of viral vector, vaccine, therapeutic proteins, etc is very critical as it shared majority of the complexity, resources & time of the whole end-to-end manufacturing process to produce the desired biologic. In upstream process, host cells human embryonic kidney 293 cells (HEK 293), Vero, etc are grown in large numbers. These cells are transfected with the mixture of nucleic acid mixture. This nucleic acid integrates its gene responsible for production of viral vectors into the host cell. The host cells start to produce multiple copies of viral vectors and excrete them into the culture media or keep these virus copies inside the cells.

Adherent cell culture bioprocesses are essential for producing vaccines, as they provide a suitable environment for cells to express complex proteins accurately. However, infecting adherent cells in bioreactors presents unique challenges. The present invention investigates the effect of cell detachment during the cell infection phase on overall bioprocess yield. By understanding these factors, one can optimize vaccine production in adherent cell culture bioprocesses.

Transfection and infection are crucial for viral vector production and vaccine production in adherent cell cultures. The present invention showcases the effect of cell detachment during the cell transfection and infection phase on overall bioprocess yield. By understanding these factors, the present invention can optimize viral vector production and vaccine production in adherent cell cultures.
Majority of host cells used for the viral vector production are adherent cells such as HEK 293. These cells are required to grow in large numbers to generate large quantity of product (viral vectors). Conventionally, a bioreactor system is required to generate a lot of viral vectors at larger scale. Host cells are grown in bioreactor and other type of bioreactor can develop icells from pall, fibracell disc from Eppendorf, etc. Further, after reaching the sufficient cell density, cells are transfected or infected with the desired DNA mixture or virus. This process is termed as transfection or infection. Duration of transfection and infection remains variable i.e. 4 hours to 24 hours. After the transfection and infection, the production phase starts which lasts upto 25 days. The user can execute multiple harvests from the bioreactor.

Once, the cells are grown in large numbers, more than 50% surface area of the cells remain unexposed as the cells use their half surface area to remain attached with the surface of the support platform. Therefore, this unexposed surface area remains unusable for the transfection and infection. The availability of lesser exposed surface area leads to decreased transfection or infection efficiency. The single use bioreactor technologies such as packed bed, fixed bed, and dynamic bed supports high density cell culture however the issue of exposure of usable surface i.e. unexposed surface area of the cells remains the same.

OBJECT OF THE INVENTION

The principal object of present invention is a method of developing free floating condition for transfection and infection of cells.

Yet another object of the present invention is to dislodge the cells from the cell carrier matrix.

Another object of the present invention is to increase the transfection and infection efficiency.

Still another object of the present invention is to enhance efficiency, scalability and cost effectiveness of biologics production.

Yet another object of the present invention is to increase the transfection and infection efficiency for the production of viral vector, vaccine and therapeutic proteins.

SUMMARY OF THE INVENTION

In light of the foregoing background, the following presents a simplified summary of the present disclosure in order to provide a basic understanding of some aspects of the disclosure. This summary is not an extensive overview of the disclosure. It is not intended to delineate the scope of the disclosure. The following summary merely presents some concepts of the disclosure in a simplified form as a prelude to the more detailed description provided below.

The present invention relates to a method of transfection and infection in free floating condition. Particularly, a bioreactor system is required to generate vaccine, viral vectors, biotherapeutics in large scale. The host cells are grown in the bioreactor to attain sufficient cell density, after reaching the sufficient cell density into the bioreactor, cells are dislodged from the surface by some mechanical (vibration), chemical means (enzyme) and combination of vibration and enzyme before transfection and infection. As the cells are in free floating condition, the entire surface of the cells is exposed (open) and available for the DNA mixture (transfection mix) to act and also increase the chance of tranfection by the virus particle. The process of transfection and infection is faster than cell re-attachment. Therefore, majority of the cells which are re-attached would be transfected or infected. The whole event of cell detachment, transfection/infection and cell re-attachment is unique and ultimately leads to the increment of the transfection and infection efficiency significantly.

BRIEF DESCRIPTION OF THE DRAWING

Objects and advantages of the invention will be apparent from the following detailed description taken in conjunction with the accompanying figure of the drawing wherein:

Fig. 1 illustrates a general practice of viral vectors production in prior art.
Fig. 2 illustrates a method of vaccine production.
Fig. 3 illustrates a method of viral vector production.
Fig. 4 illustrates cell growth profile of VERO cells through vibration method.
Fig. 5 illustrates cell growth profile of HEK cells through vibration method.
Fig.6 illustrates impact of vibration on process yield (titer/ml) through vibration method for VERO and HEK cells.
Fig.7 illustrates impact of vibration on process yield (total titer) through vibration method for VERO and HEK cells.

Fig.8 illustrates impact of vibration on process yield (transfection unit) of HEK cells.
Fig. 9 illustrates cell growth profile of VERO cells through enzymatic method.
Fig.10 illustrates cell growth profile of HEK cells through enzymatic method.
Fig.11 illustrates impact of enzyme treatment on process yield (titer/ml) for VERO and HEK cells.
Fig.12 illustrates impact of enzyme treatment on process yield (total titer) for VERO and HEK cells.
Fig.13 illustrates impact of enzyme treatment on process yield (transfection unit) of HEK cells.
Fig.14 illustrates cell growth profile of VERO cells through combination of vibration and enzymatic method.
Fig.15 illustrates cell growth profile of HEK cells through combination of vibration and enzymatic method.
Fig.16 illustrates impact of enzyme treatment and vibration on process yield (titer/ml) for VERO and HEK cells.
Fig.17 illustrates impact of enzyme treatment and vibration on process yield (total titer) for VERO and HEK cells.
Fig.18 illustrates impact of enzyme treatment and vibration on process yield (transfection unit) for HEK cells.

DETAILED DESCRIPTION OF THE INVENTION

Before explaining the present invention in detail, it is to be understood that the invention is not limited in its application to the details of the construction and arrangement of parts illustrated in the accompany drawings. The invention is capable of other embodiments, as depicted in different figures as described above and of being practiced or carried out in a variety of ways. It is to be understood that the phraseology and terminology employed herein is for the purpose of description and not of limitation. The invention may have various embodiments and they may be performed as described in the following pages of the complete specification.

The terms and words used in the following description are not limited to the bibliographical meanings, but, are merely used by the inventors to enable a clear and consistent understanding of the invention. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present invention are provided for illustration purpose only and not for the purpose of limiting the scope of the invention.

Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and/or in combination with or instead of the features of the other embodiments.

It is to be understood that the term “vector" is used in reference to a vehicle used to introduce a nucleic acid sequence into a cell.

It is to be understood that the term “viral vectors” is used in reference to a virus that has been modified to allow recombinant DNA sequences to be introduced into the host cells or cell organelles.

It is to be understood that the term “transfection” refers to the introduction of a nucleic acid into the cell. The nucleic acid may be in the form of naked DNA or RNA, associated with various proteins or the nucleic acid was incorporated into a vector.
It is to be understood that the term “infection” refers to the introduction of a virus into the cell. The virus may be in the form of whole virus, individual or group of viruses, part of virus such as genetic material of the virus.

It is to be understood that the biological method of transfection based on viral vectors was termed as “transduction”.

The present invention investigates a broad range of bioprocesses, encompassing vaccine and viral vector production, as well as adherent cell culture-based processes.

It is to be understood that in order to successfully achieve the idea of free floating condition of cells i.e. to utilize the fully exposed area of the cells through vibration, enzyme and combination of vibration and enzymatic method to increase the transfection and infection of cells, the inventors of the present invention has undergone viral vector production and vaccine production to prove the above mentioned. Therefore, it should be noted that present invention is not limited to viral vector production and vaccine production.

In order to prove that the transfection and infection efficiency gets increased when the grown cells are in free floating condition i.e. their surface area is fully exposed and not adhered to the surface. The present invention has undergone vaccine production through VERO cells and viral vector production through HEK 293 cells. Thus, the present invention has provided detailed description of the procedure involved in each production method including but not limited to seed expansion, cell culture conditions in bioreactor, infection and transfection protocols and titer/yield. These production methods serve as benchmarks for evaluating the performance of the invented bioprocesses.

The host cells used for vaccine and viral vectors production are generally adherent cells which were grown in a bioreactor to generate the viral vectors at a large scale. After attaining the sufficient cell density into the bioreactor, the cells are dislodged from the surface by some mechanical (vibration), chemical means (enzyme) or combination of vibration and enzyme before transfection and infection. As the cells are in free floating condition the entire surface of the cells is exposed (open) and available for the virus or DNA mixture (transfection mix) to act. The process of transfection is faster than cell re-attachment. Therefore, majority of the cells which are re-attached would be transfected or infected. The whole event of cell detachment, transfection and infection and cell re-attachment is unique and ultimately leads to increase the transfection efficiency significantly.

It is to be understood that “infection” is employed for vaccine production and “transfection” is employed for viral vector production.

The present invention has employed the method of developing free floating condition for transfection and infection of cells through vibration, enzyme and combination of vibration and enzymatic method.

Three variables; only vibration, only enzyme and combination of vibration and enzymatic method were studied to evaluate the effect of cell detachment on efficiency of transfection and infection which are described in table below:

Table 1: Details of Variables and its process step

Sr. No Variable name Pre-infection/Pre-transfection Process step
01 Only Vibration After cell growth phase, remove spent growth media, wash the grown cells with PBS (If required), Add fresh media for transfection and Infection Vibration Low Moderate number (Approx. 30-50%) of cells get detached and enter in suspension. Infection and Transfection executed post vibration. Infection and transfection followed by harvest.
Vibration High
02 Only Enzyme After cell growth phase, remove spent growth media, wash the grown cells with PBS (If required), Add Enzyme & incubate for pre-defined time TrypLE Enzyme Remove enzyme. Add fresh media for infection and transfection. Moderate number (Approx. 30-50%) of cells get detached and enter in suspension. Add fresh media for Infection and transfection.
Accutase Enzyme
03 Vibration + Enzyme After cell growth phase, remove spent growth media, wash the grown cells with PBS wash (If required), Add Enzyme & incubate for pre-defined time TrypLE Enzyme Remove enzyme, Add fresh media for transfection & infection Vibration Low Higher number of cells gets detached and enter in suspension proportionally. Perform transfection and infection Post vibration.

Accutase Enzyme Vibration High

Table 1 depicts majorly three aspects; mechanical in the form of vibration, chemical in the form of different enzymes and combination of vibration and enzymatic method were employed as cell dissociation means. Post cell growth till expected sufficient cell density, cells were detached and infected and transfected followed by cell re-attachment. Mechanism of cell detachment was studied in depth and few process variables of cell detachment were evaluated for the efficiency of transfection and infection and ultimately process yield. Details related to the process variables are described in below table,

Table 2: Detailed description of process variables

Sr. No. Variable name Detail of case studies
A Only Vibration 1) Vero cells? Growth phase? Vibration Low?Infection
2) Vero cells? Growth phase? Vibration High?Infection
3) HEK Cells ? Growth phase? Vibration Low? Transfection
4) HEK Cells ? Growth phase? Vibration High? Transfection
B Only Enzyme 5) Vero cells? Growth phase? TrypLE Enzyme?Infection
6) Vero cells? Growth phase? Accutase Enzyme ?Infection
7) HEK Cells ? Growth phase? TrypLE Enzyme ? Transfection
8) HEK Cells ? Growth phase? Accutase Enzyme ? Transfection
C Vibration + Enzyme 9) Vero cells? Growth phase? TrypLE Enzyme? Vibration Low ? Infection
10) Vero cells? Growth phase? Accutase Enzyme ? Vibration Low ? Infection
11) Vero cells? Growth phase? TrypLE Enzyme? Vibration High ? Infection
12) Vero cells? Growth phase? Accutase Enzyme ? Vibration High ? Infection
13) HEK Cells ? Growth phase? TrypLE Enzyme ? Vibration Low? Transfection
14) HEK Cells ? Growth phase? Accutase Enzyme ? Vibration Low ? Transfection
15) HEK Cells ? Growth phase? TrypLE Enzyme ? Vibration High ? Transfection
16) HEK Cells ? Growth phase? Accutase Enzyme ? Vibration High ? Transfection

Table 2 depicts process results in terms of cell growth and titer. Cell growth was measured by executing the cell count by sampling of the cell carrier followed by its follow up treatment for the cell counting. Inside the biosafety cabinet, a cell carrier from the SUB (Single use bioreactor) was picked up by using sterile forcep. The sampled cell carrier was subjected to counting of nuclei by nuclei counting method. The yield from both the processes; vaccine production and viral vector production were estimated through different methods.

Two process performance indicators were tested to evaluate the effect of variable (i.e.: vibration) on the productivity of biologics (i.e. vaccine and viral vector) which are yield of vaccine production with VERO cells and yield of viral vector production with HEK cells. Vaccine production from VERO cells was expressed in IU/ml (International unit per ml). It represents the biological activity of a vaccine and is determined by standardized ELISA assays that measure the vaccine's ability to induce an immune response.

Further, viral vector production from HEK cells was expressed in VP/ml and TU/ml. It should be noted that while viral particles (VP) represent the physical structure of the virus, transducing/transfection units (TU/ml and total TU) represent the functional capacity of the virus to deliver its genome. Both concepts are essential for understanding and characterizing viral vectors.

Moreover, in order to achieve desired result the present invention has been worked on HEK 293 cells and VERO cells however it is not limited to the same and provides identical result on african green monkey kidney cells (VERO), human embryonic kidney 293 cells (HEK 293), chinese hamster ovary cells (CHO-K1), medical research council cell strain (MRC-5), T-lymphocytes (T-cells), chicken embryo fibroblast cells (CEF), madin darby canine kidney cells (MDCK), baby hamster kidney cells (BHK), adipose tissue derived stem cells (ADSC), bone marrow derived stem cells (BMSC).

EXAMPLE:
The invention has been described with reference to specific embodiment which is merely illustrative and not intended to limit the scope of the invention as defined in the complete specification.

Upstream bioprocessing for vaccines and viral vectors involves the cultivation of host cells for generation of sufficient numbers of cells and to provide this generated seed culture to initiate the production scale bioreactors to produce the desired viral antigen. Key factors for upstream process include seed culture expansion, bioreactor process parameter and operation, process parameters such as growth phase, infection phase and transfection phase and production phase, at last batch harvest.

A) MEDIA PREPARATION FOR VIRAL VECTOR AND VACCINE PRODUCTION PRODUCTION:

Media preparation is crucial for viral vector production and vaccine production. The chosen medium provides essential nutrients, growth factors, a suitable pH, environment for optimal cell growth and virus replication. Key components of the media include basal media, serum, sodium bicarbonate, sodium pyruvate, glucose, dulbecco’s modified eagle medium [DMEM powder], fetal bovine serum [FBS], optipro liquid media and WFI [water for injection].

i) PREPARATION OF SODIUM BICARBONATE:
Step 1 : A suitable container was filled with 90% of the final volume with the help of WFI water at an ambient temperature.

Step 2 : The container was placed under stirring conditions and while gently stirring, 70 g of sodium bicarbonate powder was added.

Step 3 : The stirring was continued for approximately 5 minutes until the powder gets completely dissolved.

Step 4 : Upon complete dissolution the mixing of the contents were stopped.

Step 5 : In order to bring the final volume 1 L, WFI water was added followed by sterilization of the solution by autoclaving at 121º C and 15 lbs pressure for 20 minutes or filter-sterilized aseptically using 0.22 µM polyether sulfone (PES) capsule filters.

It is to be noted that the composition outlined in the above mentioned steps could be scaled up or down as required for preparation.

ii) PREPARATION OF GLUCOSE:
Step 1 : A suitable container under stirring conditions was taken and filled with 60% of the total volume with the help of WFI.
Step 2 : 200 g of (D+)-Glucose anhydrous powder was added while gently stirring.
Step 3 : The stirring was continued till the powder gets completely dissolved for approximately 10 minutes.
Step 4 : The stirring was stopped upon the complete dissolution of the powder, followed by the addition of WFI to bring the final volume upto 1.0 L.
Step 5 : The solution was sterilized by autoclaving at 121° C and 15 lbs pressure for 20 minutes or filter-sterilized aseptically through 0.22 µM polyether sulfone (PES) capsule filters.

It is to be noted that the composition outlined in the above mentioned steps could be scaled up or down as required for preparation.

iii) FINAL PREPARATION OF MEDIA:
Table 3: Details of raw materials for growth media preparation
Sr No. Media ingredient Quantity (unit / L) Unit (g or mL)
1 DMEM powder 13.37 g
2 Na-bicarbonate 3.7 g
3 NA- Pyruvate 0.11 g
4 FBS 100 mL
5 WFI Q.S. 1000 mL

Step 1 : A suitable container as per the required total volume was taken and filled up to 90% of the final volume with WFI at an ambient temperature.
Step 2 : The container was placed under stirring conditions, and while gently stirring, 13.37 g/L of DMEM powder was added till the powder gets completely dissolved.
Step 3 : Addition of 110 mg/L of sodium pyruvate was added to the solution followed by 100 mL of FBS and 3.7 g/L of sodium bicarbonate, as mentioned in the media composition table 3.
Step 4 : The stirring was continued until the powder gets completely dissolved for approximately 30 minutes.
Step 5 : The final volume of 1 L was made by adding WFI. Further, the pH was measured by taking the 5 ml sample of the medium aliquoted from the container. The acceptance criterion of pH was 6.60 to 7.20.
Step 6 : The solution was filter-sterilized aseptically through 0.22 µM polyether sulfone (PES) capsule filters or a bottle-top filter.

It is to be noted that the composition outlined in the above mentioned steps could be scaled up or down as required for preparation.

B. PREPARATION OF PRODUCTION MEDIA FOR VACCINE PRODUCTION:

i) Table 4: Details of raw materials for production media preparation
Sr No. Media ingredient Quantity (unit / L) Unit (g or mL)
1 DMEM powder 13.37 g
2 Na-bicarbonate 3.7 g
3 NA- Pyruvate 0.11 g
4 FBS 20 mL
5 WFI Q.S. 1000 mL

Step 1 : A suitable container as per the requirement was taken and filled upto 90% of the final volume with WFI at an ambient temperature.
Step 2 : The container was placed under stirring conditions and while gently stirring, 13.37g/L of DMEM powder was added.
Step 3 : The stirring was continued till the powder gets completely dissolved.
Step 4 : 3.7 g/L of sodium pyruvate, 20 mL of FBS and 0.11 g/L of sodium bicarbonate was added to the solution.
Step 5 : The stirring was continued until the powder completely dissolved for approximately 20 minutes.
Step 6 : The final volume of 1 L was made by adding WFI. Further, 5 ml sample of the medium was aliquoted from the container and the pH was measured. The pH acceptance criterion was 6.90 to 7.50.
Step 7 : The solution was filter sterilized aseptically through 0.22 µM polyether sulfone (PES) capsule filters or a bottle-top filter.

It is to be noted that the composition outlined in the above mentioned steps could be scaled up or down as required for preparation.

ii) PRODUCTION MEDIA FOR VIRAL VECTOR PRODUCTION:

Table 5: Details of production media
Sr No. Media ingredient Quantity taken Unit (g or mL)
1 OptiPro liquid media 5000 mL

Step 1 : The OptiPro media was liquid formulation and available in prepacked, pre sterilized ready to use form.

C. GENERAL METHOD FOR DEVELOPING FREE FLOATING CONDITION OF CELLS FOR TRANSFECTION OF CELLS [VIRAL VECTOR PRODUCTION]:

Step 1 : For the initial stages of cell propagation the surface treated tissue culture flasks were taken. Further, the cell bank vial was removed from liquid nitrogen container and thawed as per standard procedure followed by reviving the cell bank vial in treated tissue culture flask and incubating at 37°C with 5% CO2 in static incubator for 3 to 5 days.
Step 2 : Media is added into bioreactor for further stage.
Step 3 : The cell expansion (seeding density) is carried out as per the number of cells required to inoculate 1L bioreactor scale. Further, the installation, media fill and other bioreactor related operations were executed as per the standard procedure.
Step 4 : The 1L bioreactor was inoculated with number of cells required to meet defined cell density per unit surface area. Further, the inoculation criteria for cell growth study experiments (HEK) are 0.025 to 0.035 million cells per cm2 surface area. The seeding density may vary depending upon type of cell line, planned day of infection and transfection and ranging from 0.005 to 0.9 million cells/cm2.
Step 5 : The parameters were maintained and monitored such as temperature at 36 ± 1.5 °C throughout batch execution, pH at 7.1 ± 0.4 with 7% sterilized bicarbonate or CO2 gas, dissolved oxygen concentration at 1 to 80% through agitation 1 to 5000 RPM depending on the type of bioreactor or media recirculation from 5 ml/minute to 25 ml/minute.
Step 6 : The sterile sampling of carrier was performed by picking the cell carrier from the SUB [single use bioreactor]. The cell growth was measured by nuclei count method. Further, the spent media was sampled through a sampling port located at bottom of the SUB. The spent media was used to analyze pH, concentration of glucose, lactate and other metabolites.
Step 7 : The residual glucose concentration was maintained by the addition of 20% sterilized glucose solution at a value of atleast 2.0 g/L, and the media perfusion or recirculation with perfusion rate of 0 to 30 RV/day (reservoir volume per day).
Step 8 : The cells were detached through mechanical stress including vibration or shaking, through chemical including enzymes and combination thereof.
Step 9 : Upon detachment, the cells were observed in free floating condition in which a polyethylenimine-to-deoxyribonucleic acid [PEI-to-DNA] ratio was 1:0.5 to 5:1 was considered for the transfection procedure, with a DNA concentration of 0.5 to 10 micrograms per million cells which was a production phase.
Step 10 : The cells are allowed to reattach followed by initiation of production phase. The harvest from the bioreactor batch was connected to a bottle. Further, the sample for titer estimation was collected and estimated through qPCR method.
It is to be noted that different variable parameters were considered during the transfection to check the titer value.

It is to be noted that the bioreactors devoid of agitation involves media recirculation and perfusion, i.e. the mixing mechanism is employed without agitation to generate mixed cultural environment into the bioreactor.

Moreover, the RPM [revolutions per minute] range may vary upon the different configuration of impeller, operation scale, type of impeller. The significance of RPM and media flow rate is to keep the content of the bioreactor in a well mixed condition. It varies with the employed technology of bioreactor.

D. METHOD FOR DEVELOPING FREE FLOATING CONDITION OF CELLS FOR INFECTION OF CELLS [VACCINE PRODUCTION]:

Step 1 : For the initial stages of cell propagation the surface treated tissue culture flasks are taken. Further, the cell bank vial are removed from liquid nitrogen container and thawed as per standard procedure followed by reviving the cell bank vial in treated tissue culture flask and incubating at 37°C with 5% CO2 in static incubator for 3 to 5 days.
Step 2 : Media is added into bioreactor for further stage.
Step 3 : The cell expansion (seeding density) is carried out as per the number of cells required to inoculate bioreactor scale. Further, the installation, media fill and other bioreactor related operations are executed as per the standard procedure.
Step 4 : The bioreactor is inoculated with number of cells required to meet defined cell density per unit surface area. Further, the inoculation criteria for cell growth study experiments (VERO) are 0.025 to 0.035 million cells per cm2 surface area. The seeding density may vary depending upon type of cell line, planned day of infection and transfection and ranging from 0.005 to 0.9 million cells/cm2.
Step 5 : The parameters are maintained and monitored such as temperature at 36 ± 1.5 °C throughout batches execution, pH at 7.1 ± 0.4 with 7% sterilized bicarbonate or CO2 gas, the dissolved oxygen concentration at 1 to 80% through agitation 1 to 5000 RPM and media recirculation from 5 ml/minute to 25 ml/minute depending upon the bioreactor.
Step 6 : The sterile sampling of carrier is performed by picking the cell carrier from the SUB [single use bioreactor]. The cell growth is measured by nuclei count method. Further, the spent media is sampled through a sampling port located at bottom of the SUB. The spent media is used to analyze offline pH, concentration of glucose, lactate and other metabolites.
Step 7 : The residual glucose concentration is estimated and maintained by the addition of 20% sterilized glucose solution at value of at least 2.0 g/L and the media perfusion or recirculation with perfusion rate of 0 to 30 RV/day (reservoir volume per day).
Step 8 : The cells are detached through mechanical stress including vibration or shaking, through chemical including enzymes and combination thereof.
Step 9 : Upon detachment, the cells are observed in a free floating condition which are infected with 0.1 to 50 MOI [MOI: multiplicity of infection] virus which is a production phase.
Step 10 : The free floating cells are allowed to reattach followed by initiation of production phase. The harvest from the bioreactor batch is collected in the connected bottle. The sample for titer estimation is collected and estimated through TCID50 method.

It is to be noted that different variable parameters as per the above mentioned are considered during infection phase to check the titer value.

Further, it is to be understood that the day for detachment of cells dependent on the requirements of the cell growth.

DETACHMENT OF CELLS THROUGH VIBRATION METHOD:

The procedure as mentioned in the above mentioned segment “C and D” of production is performed till step 7 which is the growth phase. It is to be noted that the major difference between the different variables are during the phase of infection and transfection.

The present invention has employed process variable for cell detachment through vibration method. The frequency of vibration is studied and their output is evaluated.

Step 1 : Upon completion of growth phase, the spent media is removed and phosphate-buffered saline (PBS) is optionally washed in a bioreactor.
Step 2 : After step 1) addition of fresh infection/fresh transfection media to the bioreactor is done.
Step 3 : Vibration at 10 to 3000 RPM and amplitude of rotation from 0.5 mm to 20 mm for 0.1 to 30 minutes or with 0.1 to 5 minutes of interval is applied to the cell carrier shaft leading to partial detachment of the grown cells from the support cell carrier matrix i.e. the cells are observed in a free floating condition into the bioreactor vessel.
Step 4 : Upon completion of step 3, infection/transfection process is initiated as mentioned in steps 9 to 10 of the above mentioned segment “C and D” which is a production phase.
It is to be noted that the RPM range is based on the bioreactor with different arrangement of cell carrier matrix and mechanism of generating mechanical stress for cell detachment such as shaking and vibration in any direction i.e. vertical or horizontal. Moreover, the mechanical stress impact from internal or external stress generation means is also covered by the scope of the present invention. The purpose of the above mentioned is to generate and transfer mechanical stress to the cells in order to dislodge from the surface.

DETACHMENT OF CELLS THROUGH ENZYMATIC METHOD:

The procedure as mentioned in the above mentioned segment “C and D” of production is performed till step 7 which is the growth phase.

The present invention has employed process variable for cell detachment through enzymatic method. The enzyme is selected but not limited to recombinant TrypLE, trypsine,Versene and accutase for cell detachment purpose. Further, while TrypLE is more specific for cleaving peptide bonds after lysine or arginine residues, accutase has a broader range of proteolytic activity.

It is to be noted that the major difference between the different variables will be during the phase of infection and transfection. Further, the growth phase and production phase process as mentioned above in “C and D” for production remains constant for across different enzyme choices.

Step 1 : Upon completion of growth phase, the spent media is removed and the cell carrier matrix is optionally washed with phosphate-buffered saline (PBS).
Step 2 : The enzyme solution is added to the single use bioreactor (SUB) whilst the cell carrier matrix is incubated for 0.1 to 40 minutes.
Step 3 : All the process parameters are maintained at OFF mode during the incubation period. Further, the enzyme is removed, and fresh infection /transfection media is added to the SUB whilst keeping all the process parameters such as temperature, pH in an OFF mode.
Step 4 : After the completion of step 3, all the process parameters of cell growth phase such as RPM, temperature, pH are set in an auto mode which leads to partial detachment of the grown cells from the support cell carrier matrix.
Step 5 : Followed by step 4, infection/transfection procedure is initiated and the production phase steps 9 and 10 are performed as shown in “C and D”.
It is to be understood that the washing of the grown cells is an optional procedure, and it depends upon type of cell and type of enzyme used. If the porcine origin trypsine is used for cell detachment which is a very potent enzyme, use of PBS is required. In case of accutase enzyme, PBS washing step can be avoided.

DETACHMENT OF CELLS THROUGH COMBINATION OF VIBRATION AND ENZYMATIC METHOD:
The present invention has employed cell detachment through the combined approach of vibration and enzymatic method in bioreactors. Further, by systematically evaluating various vibration parameters (high vibration and low vibration) and comparing the effectiveness of different enzymes recombinant TrypLE, Collagenase, Versene, trypsin and Accutase. The aim of the present invention is to evaluate the synchronized effect of both process inputs on infection and transfection efficiency.

It is to be noted that the major difference between the different variables will be during the phase of infection and transfection. In particular, Vero cells with low vibration and TrypLE enzyme, HEK cells with high vibration and Accutase enzyme and vice versa were studied. Moreover, the growth phase and production phase process as mentioned above in ‘C and D” remains constant for entire process.

The procedure as mentioned in the above mentioned segment “C and D” for production is performed till step 7 which is the growth phase.
Step 1: Upon completion of growth phase, the spent media is removed, and the cell carrier matrix is optionally washed with phosphate-buffered saline (PBS) in a bioreactor.
Step 2: Enzymes solution is added to the SUB, and the cell carrier matrix is incubated for 0.1 to 40 minutes. Further, all the process parameters are maintained at OFF mode during the incubation period.
Step 3: The enzymes are removed and fresh infection/transfection media is added to the SUB after which physical vibration is applied to the cell carrier shaft at 10 to 3000 RPM for 0.1 to 30 minutes or with 0.1 to 5 minutes of interval in addition to that different amplitude of rotation ranging from 0.5 mm to 20 mm is also provided.
Step 4: Upon completion of step 3, major detachment of the grown cells from the support cell carrier matrix is observed in a free floating condition into the bioreactor.
Step 5: After step 4, transfection/infection procedure is initiated as mentioned in steps 9 to 10 of the above mentioned segment “C and D” which is a production phase.

EXPERIMENTAL EXAMPLE 1:

I METHOD FOR VIRAL VECTOR PRODUCTION:

Step 1 : The preparation of media for seed development and batch execution was prepared. Further, the media was taken for seed development and 13.37 g/L DMEM high glucose with 10% FBS is added in a bioreactor.
Step 2 : For the initial stages of cell propagation the surface treated tissue culture flasks were taken. Further, the cell bank vial was removed from liquid nitrogen container and thawed as per standard procedure followed by reviving the cell bank vial in treated tissue culture flask and incubating at 37°C with 5% CO2 in static incubator for 3 to 5 days.
Step 3 : The cell expansion (seed development) was carried out as per the number of cells required to inoculate 1L bioreactor scale. Further, the installation, media fill and other bioreactor related operations were executed as per the standard procedure.
Step 4 : The 1L bioreactor was inoculated with number of cells required to meet defined cell density per unit surface area. Further, the inoculation criteria for cell growth study experiments (HEK) were 0.03 cells per cm2 surface area.
Step 5 : The parameters were maintained and monitored such as temperature at 36 ± 1.0 °C throughout batch execution, pH at 7.2 ± 0.2 with 7% sterilized bicarbonate or CO2 gas, dissolved oxygen concentration at 40% through agitation 40 RPM.
Step 6 : The sterile sampling of carrier was performed by picking the cell carrier from the SUB [single use bioreactor]. The cell growth was measured by nuclei count method. Further, the spent media was sampled through a sampling port located at bottom of the SUB. The spent media was used to analyze offline pH, concentration of glucose, lactate and other metabolites.
Step 7 : The residual glucose concentration was maintained by the addition of 20% sterilized glucose solution at a value of at least 2.0 g/L, and the media perfusion or recirculation with perfusion rate of 5 RV/day (reservoir volume per day).
Step 8 : The cells were detached through vibration, enzyme and combination of vibration and enzymatic method.
Step 9 : Upon detachment, the cells were observed on 4th day in free floating condition in which a polyethylenimine-to-deoxyribonucleic acid [PEI-to-DNA] ratio is 1:1 was considered for the transfection procedure, with a DNA concentration of 3 micrograms per million cells which was a production phase.
Step 10 : The free floating cells were allowed to reattach followed by initiation of production phase with optiPro liquid media. The harvest from the bioreactor batch was collected in the connected bottle. The sample for titer estimation was collected and estimated through qPCR method.

II. METHOD FOR VACCINE PRODUCTION:
Step 1 : The preparation of media for seed development and batch execution was prepared. Further, the media was taken for seed development and 13.37 g/L DMEM high glucose with 10% FBS is added in a bioreactor.
Step 2 : For the initial stages of cell propagation the surface treated tissue culture flasks were taken. Further, the cell bank vial was removed from liquid nitrogen container and thawed as per standard procedure followed by reviving the cell bank vial in treated tissue culture flask and incubating at 37°C with 5% CO2 in static incubator for 3 to 5 days.
Step 3 : The cell expansion (seed development) was carried out as per the number of cells required to inoculate 1L bioreactor scale. Further, the installation, media fill and other bioreactor related operations were executed as per the standard procedure.
Step 4 : The bioreactor was inoculated with number of cells required to meet defined cell density per unit surface area. Further, the inoculation criteria for cell growth study experiments (VERO) were 0.03 million cells per cm2 surface area.
Step 5 : The parameters were maintained and monitored such as temperature at 36 ± 1.0 °C throughout batch execution, pH at 7.2 ± 0.2 with 7% sterilized bicarbonate or CO2 gas, the dissolved oxygen concentration at 40% through agitation 40 RPM.
Step 6 : The sterile sampling of carrier was performed by picking the cell carrier from the SUB [single use bioreactor]. The cell growth was measured by nuclei count method. Further, the spent media was sampled through a sampling port located at bottom of the SUB. The spent media was used to analyze pH, concentration of glucose, lactate and other metabolites.
Step 7 : The residual glucose concentration was estimated and maintained by the addition of 20% sterilized glucose solution at value of at least 2.0 g/L and the media perfusion or recirculation with perfusion rate of 5 RV/day (reservoir volume per day).
Step 8 : The cells were detached through vibration, enzyme and combination of vibration and enzymatic method.
Step 9 : Upon detachment, the cells were observed on 4th day in a free floating condition which were infected with 1:30 MOI [MOI: multiplicity of infection] virus which was a production phase.
Step 10 The free floating cells were allowed to reattach followed by initiation of production phase with DMEM powder 13.37 g/L, Na-bicarbonate 3.7 g/L, Na-pyruvate 0.11 g/L, FBS 20 ml/L and WFI 1000 ml/L. The harvest from the bioreactor batch was collected in the connected bottle. The sample for titer estimation was collected and estimated through TCID50 method.

EVALUATING EFFICIENCY OF TRANSFECTION AND INFECTION OF CELLS FOR VIRAL VECTOR PRODUCTION AND VACCINE PRODUCTION IN A FREE FLOATING CONDITION

1.1. VIBRATION METHOD FOR VIRAL VECTOR PRODUCTION:
The procedure as mentioned in the above mentioned segment “II” of viral vector production was performed till step 7 which is the growth phase. The host cells were HEK 293 cell line.

Step 1 : Upon completion of growth phase, the spent media was removed.
Step 2 : addition of fresh transfection media in the bioreactor was done.
Step 3 : Vibration at lower (800 RPM) and higher speed (1500 RPM) in addition to that different amplitude of rotation ranging from 5 mm was also provided for 10 minutes to the cell carrier matrix leading to partial detachment of the grown cells from the support cell carrier matrix i.e. the cells were observed in a free floating condition into the bioreactor vessel.
Step 4 : Upon completion of step 3, viral transfection process was initiated as mentioned in steps 9 to 10 of the above mentioned segment “I” which is a production phase.
It is to be noted that annotation B#01 is for lower vibration and B#02 is for higher vibration employed for viral vector production. Figure no.5, 6, 7 and 8 shows growth of the HEK 293 cells and impact of vibration on the same.

1.2. VIBRATION METHOD FOR VACCINE PRODUCTION:
The procedure as mentioned in the above mentioned segment “II” of vaccine production was performed till step 7 which is the growth phase. The host cells were VERO cell line.

Step 1 : Upon completion of growth phase, the spent media was removed.
Step 2 : addition of fresh infection media to the bioreactor was done.
Step 3 : Vibration at lower speed (800 RPM) and higher speed (1500 RPM) and amplitude of rotation is 5 mm for 10 minutes was applied to the cell carrier shaft leading to partial detachment of the grown cells from the support cell carrier matrix i.e. the cells were observed in a free floating condition into the bioreactor vessel.
Step 4 : Upon completion of step 3, viral infection process was initiated as mentioned in steps 9 to 10 of the above mentioned segment “I” which is a production phase.

It is to be noted that annotation B#01 is for lower vibration and B#02 is for higher vibration employed for vaccine production. Figure no. 4, 6 and 7 shows growth of the VERO cells and impact of vibration on the same.

Table 6: Cell growth data of vaccine production [VERO cells] and viral vector production [HEK cells] through vibration method

Cell Growth profile
VERO cells [million cells] HEK cells [million cells]
Hrs of growth B#9 control B#1 Lower vibration B#2 higher vibration B#9 control B#1 Lower vibration B#2 higher vibration
0 290 281 314 292 279 310
24 322 341 376 374 386 422
48 722 668 745 703 728 790
72 1289 1346 1388 1548 1511 1610
96 2153 2108 2190 2420 2370 2460

From the above table 6 cell growth profile of VERO and HEK 293 cells at variable hours of growth i.e. 0, 24, 48, 72 and 96 were seen when the cells were provided with lower and higher vibration.

Table 7: Titer data of vaccine production [VERO cells] and viral vector production [HEK cells] (per ml)

Titer data (per ml)
VERO (IU/mL) HEK (VP/mL)
Titer B#9 (Control) B#1 Lower vibration B#2 Higher vibration B#9 (Control) B#1 Lower vibration B#2 Higher vibration
Titer for vaccine (IU/mL) 0.92 1.09 1.21 NA NA NA
Titer for viral vector (VP/mL) NA NA NA 1.22E+09 1.76E+09 1.86E+09
Harvest volume(mL) 3000 3000 3000 5000 5000 5000

From the above table 7 it was seen that IU/ml i.e. International unit per ml was evaluated for VERO cells and VP/ml i.e. viral particle per ml was evaluated for HEK cells by providing lower and higher vibration was provided. At the end, harvest collection for each cell line was done.

Table 8: Titer data of vaccine production [VERO cells] and viral vector production HEK cells (Total)

Titer data (total)
VERO (IU) HEK (VP)
Titer B#9 (Control) B#1 Lower vibration B#2 Higher vibration B#9 (Control) B#1 Lower vibration B#2 Higher vibration
Viral titer (IU) 2760 3270 3630 NA NA NA
Viral titer (VP) NA NA NA 6.11E+12 8.78E+12 9.32E+12

From the above table 8 it was seen that total titer for VERO cells and HEK cells were evaluated by providing lower and higher vibration.

Table 9: Titer data of viral vector production [HEK cells] (Transfection units)

Titer data of Transfection with HEK cells (Transducing unit, TU)
Titer B#9 (Control) B#1 Lower vibration B#2 Higher vibration
Viral titer (TU/mL) 1.7E+06 2.6E+06 2.9E+06
Viral titer (Total TU) 8.7E+09 1.3E+10 1.5E+10

From the above table 9 it was seen that the transfection on the HEK cells for viral vector production was evaluated and showcases the increment in TU/ml and in total volume when the vibration was applied on lower frequency and higher frequency.

CONCLUSION:

The above shown vibration method was conducted in a 1L bioreactor through a 3D non-woven support matrix for cell adherence and growth. The primary variable investigated was the effect of vibration on infection and transfection efficiency. Vibration was applied post-cell growth, and the results demonstrated that cell growth trends remained consistent, indicating process robustness and phenotypic stability.

Further, vibration played a crucial role in facilitating the transfer of genetic material or whole virions into the grown cells. Different vibration frequencies (800 and 1500 RPM) led to partial cell detachment in both VERO and HEK cell lines, resulting in an increase in infection and transfection efficiency, which ultimately increased the titer.

Moreover, VERO cells with vibration variable exhibit superior titer, compared to control (0.92 IU/ml), lower vibration (1.09 IU/ml) and higher vibration (1.21 IU /ml) which delivered higher titer. HEK cells with vibration variable exhibit superior titer, compared to control (1.22x10e9 VP/ml), lower vibration (1.76x10e9 VP/ml) and higher vibration (1.86x10e9 VP/ml) which delivered higher titer.

Furthermore, the data presented in the above shown tables 6-9 and figures 4 to 8 illustrate the superior titer (per ml and total titer) achieved with different cell lines (HEK and VERO) and vibration frequencies (low and high) compared to the control with variable of vibration in the bioprocess. The evaluation of transducing units in HEK cells further supports the conclusion that vibration enhances viral vector yield.

EXPERIMENTAL EXAMPLE 2:

EVALUATING EFFICIENCY OF TRANSFECTION AND INFECTION OF CELLS FOR VIRAL VECTOR AND VACCINE PRODUCTION IN A FREE FLOATING CONDITION THROUGH ENZYMATIC METHOD
1.3 EVALUATING ENZYMATIC ACTIVITY FOR VIRAL VECTOR PRODUCTION [HEK CELLS]:

The procedure as mentioned in the above mentioned segment “I” of viral vector production was performed till step 7 which is the growth phase. The cells were taken from HEK cell line.

Step 1 : Upon completion of growth phase, the spent media was removed and the cell carrier matrix was optionally washed with phosphate-buffered saline 1L (PBS).
Step 2 : 1 L TrypLE express enzyme or 1 L accutase solution was added to the SUB and the cell carrier matrix was incubated for 10 minutes whilst all the process parameters were maintained at OFF mode during the incubation period.
Step 3 : Upon completion of step 2, the enzyme was removed and fresh transfection media was added to the SUB whilst keeping all the process parameters of cell growth phase such as RPM, temperature, pH in an OFF mode.
Step 4 : After the completion of step 3, all the process parameters of cell growth phase such as RPM, temperature, pH were set in an auto mode which leads to partial detachment of the grown cells from the support cell carrier matrix.
Step 5 : Followed by step 4, the viral transfection procedure was initiated and the production phase steps 9 and 10 were performed as shown in “I”.

It is to be noted that annotation B#03 TrypLE and B#04 Accutase was used for enzymatic method for viral vector production. Figure no. 10, 11, 12 and 13 shows growth of the HEK cells and impact of enzymes on the same.
1.4 EVALUATING ENZYMATIC ACTIVITY FOR VACCINE PRODUCTION [VERO CELLS]:

The procedure as mentioned in the above mentioned segment “II” of vaccine production was performed till step 7 which is the growth phase. The cells were taken from VERO cell line.

Step 1 : Upon completion of growth phase, the spent media was removed and the cell carrier matrix was optionally washed with 1 L phosphate-buffered saline (PBS).
Step 2 : The enzyme 1 L TrypLE express enzyme or 1 L accutase solution was added to the single use bioreactor (SUB) whilst the cell carrier matrix was incubated for 10 minutes.
Step 3 : All the process parameters were maintained at OFF mode during the incubation period. Further, the enzyme was removed, and fresh incubation media was added to the SUB whilst keeping all the process parameters such as temperature, pH in an OFF mode.
Step 4 : After the completion of step 3, all the process parameters of cell growth phase such as RPM, temperature, pH were set in an auto mode which leads to partial detachment of the grown cells from the support cell carrier matrix.
Step 5 : Followed by step 4, the viral infection procedure was initiated and the production phase steps 9 and 10 were performed as shown in “II”.
It is to be noted that annotation B#03 TrypLE and B#04 Accutase was used for enzymatic method for vaccine production. Figure no.9, 11 and 12 shows growth of the VERO cells and impact of enzymes on the same.

Table 10: Cell growth data of VERO cells and HEK cells through enzymatic method
Cell Growth profile
VERO cells [million cells] HEK cells [million cells]
Hrs of growth B#9 control B#3 Tryple B#4 Accutase B#9 control B#3 Tryple B#4 Accutase
0 314 281 290 290 290 330
24 386 322 314 344 386 362
48 668 775 722 720 810 780
72 1388 1346 1289 1560 1710 1490
96 2190 2153 2108 2420 2580 2390

From the above table 10, it was seen that cell growth profile of VERO cells and HEK cells at variable hours of growth i.e. 0, 24, 48, 72 and 96 hours were seen when the cells were provided with different enzymatic treatment.

Table 11: Titer data of VERO cells & HEK cells (per ml)
Titer data (per ml)
VERO (IU/mL) HEK (VP/mL)
Titer B#9 (Control) B#3 Tryple B#4 Accutase B#9 (Control) B#3 Tryple B#4 Accutase
Viral titer (IU/mL) 0.92 1.35 1.24 NA NA NA
Viral titer (VP/mL) NA NA NA 1.22E+09 2.26E+09 2.48E+09
Harvest volume(mL) 3000 3000 3000 5000 5000 5000

From the above table 11, it was seen that IU/ml i.e. international unit per ml was evaluated for VERO cells and VP/ml i.e. viral particle per ml was evaluated for HEK cells by providing different enzymatic treatment and at the end harvest collection for each cell line was done.

Table 12: Titer data of VERO cells and HEK cells (Total)
Titer data (Total)
VERO (IU) HEK (VP)
Titer B#9 (Control) B#3 Tryple B#4 Accutase B#9 (Control) B#3 Tryple B#4 Accutase
Viral titer (IU) 2760 4050 3720 NA NA NA
Viral titer (VP) NA NA NA 6.11E+12 1.13E+13 1.24E+13

From the above table 12, it was seen that total titer for VERO cells and HEK cells were evaluated.

Table 13: Titer data of HEK cells (Transfection units)
Titer data of Transfection with HEK cells (Transducing unit, TU)
Titer B#9 (Control) B#3 Tryple B#4 Accutase
Viral titer (TU/mL) 1.74E+06 9.08E+06 1.03E+07
Viral titer (Total TU) 8.71E+09 4.54E+ 10 5.14E+ 10

From the above table 13, it was seen that the transfection on the HEK cells for viral vector production was evaluated and has showcased the increment in total volume and in TU per ml of the titer when different enzymatic treatment was provided.

CONCLUSION:

The above shown enzymatic method was conducted in a 1L bioreactor with the help of a 3D non-woven support matrix for cell adherence and growth. The primary variable investigated was the effect of different cell dislodgment means such as enzymes on infection and transfection efficiency. Enzymes were provided post-cell growth, and the results demonstrated that cell growth trends remained consistent, indicating process robustness and phenotypic stability.

Further, enzyme treatment played a crucial role in facilitating the transfer of genetic material or whole virions into the grown cells because of cell dislodgment. Different enzymes (TrypLE and accutase) led to partial cell detachment in both VERO and HEK cell lines, resulting in a significant increase in infection and transfection efficiency, which ultimately increased the titer.

Moreover, VERO cells with enzymatic treatment variable exhibit superior titer, compared to control (0.92 IU/ml), TrypLE (1.35 IU/ml) and accutase (1.24 IU /ml) which delivered higher titer. HEK cells with enzymatic treatment variable exhibit superior titer, compared to control (1.22x10 e9 VP/ml), TrypLE (2.26x10e9 VP/ml) and accutase (2.48x10e9 VP/ml) which delivered higher titer.

Furthermore, the data presented in the above shown tables 10-13 and figures 9 to 13 illustrate the superior titer (per ml and total titer) achieved with different cell lines (HEK and VERO) and different enzymes compared to the control in the bioprocess. The evaluation of transducing units in HEK cells further supports the conclusion that enzymatic method enhances viral vector yield.

EXPERIMENTAL EXAMPLE 3:

EVALUATING EFFICIENCY OF INFECTION AND TRANSFECTION OF CELLS FOR VIRAL VECTOR AND VACCINE PRODUCTION IN A FREE FLOATING CONDITION THROUGH COMBINATION OF VIBRATION AND ENZYMATIC METHOD

1.5. COMBINATION OF VIBRATION AND ENZYMATIC METHOD FOR VIRAL VECTOR PRODUCTION:
The procedure as mentioned in the above mentioned segment “I” of viral vector production was performed till step 7 which is the growth phase. The cells were taken from HEK cell line.

Step 1: Upon completion of growth phase, the spent media was removed, and the cell carrier matrix was optionally washed with 1L phosphate-buffered saline (PBS) in a bioreactor.
Step 2: TrypLE express enzyme and accutase solution is added to the SUB, and the cell carrier matrix was incubated for 10 minutes. Further, all the process parameters were maintained at OFF mode during the incubation period.
Step 3: The enzyme was removed and fresh production/ transfection media was added to the SUB after which physical vibration was applied to the cell carrier shaft at lower speed 800 RPM and higher speed 1500 RPM for 10 minutes in addition to that different amplitude of rotation ranging from 5 mm was also provided.
Step 4: Upon completion of step 3, major detachment of the grown cells from the support cell carrier matrix was observed in a free floating condition into the bioreactor.
Step 5: After step 4, viral transfection procedure was initiated as mentioned in steps 9 to 10 of the above mentioned segment “II” which was a production phase.
It should be noted that annotation B#5 Lower vibration + TrypLE, B#6 Lower vibration + Accutase, B#7 High vibration + TrypLE and B#8 High vibration + Accutase is employed for viral vector production. Figure no.15, 16, 17 and 18 shows growth of the HEK 293 cells and impact of combination of vibration and enzymatic method.

1.6. COMBINATION OF VIBRATION AND ENZYMATIC METHOD FOR VACCINE PRODUCTION:
The procedure as mentioned in the above mentioned segment “II” of vaccine production was performed till step 7 which is the growth phase. The cells are taken from VERO cell line.

Step 1: Upon completion of growth phase, the spent media was removed and the cell carrier matrix was optionally washed with phosphate-buffered saline 1L (PBS) in a bioreactor.
Step 2: 1 L TrypLE Express Enzyme and 1L Accutase solution was added to the SUB, and the cell carrier matrix was incubated for 10 minutes. Further, all the process parameters were maintained at OFF mode during the incubation period.
Step 3: The enzyme was removed and fresh 1L production /transfection media is added to the SUB after which physical vibration was applied to the cell carrier shaft at lower speed for 800 RPM and higher speed of 1500 RPM for 10 minutes in addition to amplitude of rotation is 5 mm was also provided.
Step 4: Upon completion of step 3, major detachment of the grown cells from the support cell carrier matrix was observed in a free floating condition into the bioreactor vessel.
Step 5: After step 4, viral infection procedure was initiated as mentioned in steps 9 to 10 of the above mentioned segment “I” which is a production phase.
It is to be noted that annotation B#05 Lower vibration + TrypLE, B#06 Lower vibration + Accutase, B#07 High vibration + TrypLE and B#08 High vibration + Accutase is employed for vaccine production. Figure no.14, 16 and 17 shows growth of the VERO cells and impact of combination of vibration and enzymatic method.

Table 14: Cell growth data of VERO cells and HEK cells

Cell Growth profile
VERO cells [million cells] HEK cells [million cells]
Titer B#9 control B#5 Low vibrat+ Tryple B#6 Low vibrat+ Accutase B#7 High vibrat+ Tryple B#8 High vibrat+ Accutase B#9 control B#5 Low vibrat+ Tryple B#6 Low vibrat+ Accutase B#7 High vibrat+ Tryple B#8 High vibrat+ Accutase
0 294 255 322 284 283 312 260 320 300 270
24 362 312 393 330 341 350 330 380 350 320
48 703 613 693 646 712 770 650 750 770 720
72 1371 1308 1377 1392 1380 1490 1410 1520 1540 1510
96 2157 2289 2139 2073 2189 2420 2450 2356 2503 2210

From the above table 14 it was seen that cell growth profile of VERO cells and HEK cells at variable hours of growth i.e. 0, 24, 48, 72 and 96 hours were seen when the cells were provided with combination of vibration and enzymatic method.

Table 15: Titer data of VERO cells and HEK cells (per ml)
Titer data (per ml)
VERO (IU/mL) HEK (VP/mL)
Titer B#9 (Control) B#5 Low vibrat+ Tryple B#6 Low vibrat+ Accutase B#7 High vibrat+ Tryple B#8 High vibrat+ Accutase B#9 (Control) B#5 Low vibrat+ Tryple B#6 Low vibrat+ Accutase B#7 High vibrat+ Tryple B#8 High vibrat+ Accutase
Viral titer (IU/mL) 0.92 1.54 1.61 1.74 1.81 NA NA NA NA NA
Viral titer (VP/mL) NA NA NA NA NA 1.22E+09 9.10E+09 1.22E+10 1.49E+10 1.54E+10
Harvest volume (mL) 3000 3000 3000 3000 3000 5000 5000 5000 5000 5000

From the above table 15 it was seen that the titer value in per ml volume was evaluated by providing different frequency of vibration in combination of variable enzymes on VERO cells and HEK cells. At the end harvest collection for each cell line was done.

Table 16: Titer data of VERO cells and HEK cells (Total)
Titer data (Total)
VERO (IU) HEK (VP)
Titer B#9 (Control) B#5 Low vibrat+ Tryple B#6 Low vibrat+ Accutase B#7 High vibrat+ Tryple B#8 High vibrat+ Accutase B#9 (Control) B#5 Low vibrat+ Tryple B#6 Low vibrat+ Accutase B#7 High vibrat+ Tryple B#8 High vibrat+ Accutase
Viral titer (IU) 2760 4620 4830 5220 5430 NA NA NA NA NA
Viral titer (VP) NA NA NA NA NA 6.11E+12 4.55E+13 6.11E+13 7.47E+13 7.70E+13

From the above table 16, total titer for VERO cells and HEK cells were evaluated by providing the combination of different frequency of vibration and variable enzymes.

Table 17: Titer data of HEK cells (Transfection units)
Titer data of Transfection with HEK cells (Transducing unit, TU)
Titer B#9 (Control) B#5 Low vibrat+ Tryple B#6 Low vibrat+ Accutase B#7 High vibrat+ Tryple B#8 High vibrat+ Accutase
Viral titer (TU/mL) 1.74E+06 1.60E+07 1.68E+07 1.84E+07 1.96E+07
Viral titer (Total TU) 8.71E+09 8.01E+10 8.40E+10 9.20E+10 9.80E+10

From the above table 17 it was seen that the transfection on the HEK cells for viral vector production was evaluated and has showcased the increment in total volume and in per ml when applied in combination of different frequency of vibration and variable enzymes.

It is to be noted that the RPM range is based on the bioreactor with different arrangement of cell carrier matrix and mechanism of generating mechanical stress for cell detachment such as shaking and vibration in any direction i.e. vertical or horizontal. Moreover, the mechanical stress impact from internal or external stress generation which is also covered by the scope of the present invention. The purpose of the above mentioned is to generate and transfer mechanical stress to the cells in order to dislodge from the surface.

CONCLUSION:

The above shown combination of vibration and enzymatic method was conducted in a 1L bioreactor with the help of 3D non-woven support matrix for cell adherence and growth. The primary variable investigated was the effect of different cell dislodgment means in combination of vibration and enzymatic method on infection and transfection efficiency. Further, enzyme was provided post-cell growth followed by vibration for efficient cell dislodgment. The results demonstrated that cell growth trends remained consistent, indicating process robustness and phenotypic stability.

Moreover, the synergistic effect of dual mechanism of cell dislodgement (enzyme treatment with vibration) dislodge the adherently grown cells and which ultimately played a crucial role in facilitating the transfer of genetic material or whole virions into the grown cells. Different enzymes (TrypLE and accutase) combined with vibration of the cell carrier bed led to thorough cell detachment in both VERO and HEK cell lines, resulting in a significant increase in free floating cells before addition of the infection and transfection mix which significantly increased infection and transfection efficiency, and ultimately increased the titer.

Furthermore, VERO cells in combination with enzymatic treatment and vibration exhibited superior titer, compared to control (0.92 IU/ml), Low vibration + TrypLE (1.54 IU/ml), low vibration + accutase (1.61 IU/ml), high vibration + TrypLE (1.74 IU/ml), high vibration + accutase (1.81 IU/ml) which delivered higher titer.

In addition to that, HEK cells with enzymatic treatment with vibration variable exhibit superior titer, compared to control (1.22x10e9 VP/ml), Low vibration + TrypLE (9.1x10e9 VP/ml), Low vibration + Accutase (1.22x10e10 VP/ml), High vibration + TrypLE (1.49x10e10 VP/ml), High vibration + Accutase (1.54x10e10 VP/ml) which delivered higher titer.

The data presented in the above shown tables 14-17 and figures 14 to 18 illustrate the superior titer (per ml and total titer) achieved with different cell lines (HEK and VERO) and different enzymes with different vibration speed compared to the control in the bioprocess. The evaluation of transducing units in HEK cells further supports the conclusion that enzyme with vibration enhances viral vector yield.

The present invention has been described with reference to specific embodiment which is merely illustrative and not intended to limit the scope of the invention as defined in the present complete specification.
,CLAIMS:We claim:

1. A method of developing free floating condition for transfection and infection of cells comprises the following steps:
a) reviving the cell bank vial in treated tissue culture flask and incubating at 37° C temperature with 5% CO2 in static incubator for 3 to 5 days;
b) preparing media and adding into bioreactor;
c) expanding cells or developing seed culture density and inoculating the test bioreactor from the cells generated from the seed bioreactor;
d) studying the cell growth and maintaining the temperature, pH, CO2 gas, dissolved oxygen concentration, glucose concentration, agitation, media recirculation and perfusion rate in the test bioreactor;
e) detaching the cells through mechanical stress including vibration or shaking, through chemical including enzymes and combination thereof;
f) infecting the cells with 0.1 to 50 multiplicity of infection (MOI) with virus and transfecting the cells with polyethylenimine-to-deoxyribonucleic acid (PEI-to-DNA) ratio of 1:0.5 to 5:1;
g) allowing the cells to reattach followed by production phase; and
h) collecting the harvest and estimating the titer.

2. The method as claimed in claim 1, wherein the method is selected but not limited to production of viral vector, vaccine and therapeutic proteins.

3. The method as claimed in claim 1 step a), wherein the cells are selected from african green monkey kidney cells (VERO), human embryonic kidney 293 cells (HEK 293), chinese hamster ovary cells (CHO-K1), medical research council cell strain (MRC-5), T-lymphocytes (T-cells), chicken embryo fibroblast cells (CEF), madin darby canine kidney cells (MDCK), baby hamster kidney cells (BHK), adipose tissue derived stem cells (ADSC), bone marrow derived stem cells (BMSC).

4. The method as claimed in claim 1 step c), wherein the seed culture density is depend upon type of cell line, planned day of infection and transfection and ranging from 0.005 to 0.9 million cells/cm2.

5. The method as claimed in claim 1 step d), wherein the temperature is maintained at 36 ± 1.5° C, pH at 7.1 ± 0.4 with 7% sterilized bicarbonate or CO2 gas, dissolved oxygen concentration at 1 to 80%, at least 2 g/L of 20% sterilized glucose solution, agitation at 1 to 5000 RPM, media recirculation from 5 ml/minute to 25 ml/minute and perfusion rate at 0 to 30 RV/day.

6. The method as claimed in claim 1 step e), wherein detaching the cells through vibration further comprises the following steps:
i) removing the spent media after completion of growth phase steps a) to d);
ii) adding fresh infection media and fresh transfection media in the bioreactor;
iii) applying physical vibration from 10 to 3000 RPM and amplitude of rotation from 0.5 mm to 20 mm for 0.1 to 30 minutes or with 0.1 to 5 minutes of interval to cell carrier;
iv) detaching the grown cells from the support cell carrier matrix; and
v) performing the viral infection and viral transfection process.

7. The method as claimed in claim 1 step e), wherein detaching the cells through enzymes further comprises the following steps:
i) removing the spent media after completion of growth phase steps a) to d);
ii) optionally washing the cell carrier matrix with phosphate buffer saline (PBS) and removing it;
iii) adding enzymes in the bioreactor and incubating the cell carrier matrix for 0.1 to 40 minutes;
iv) removing the enzymes;
v) adding fresh infection media and fresh transfection media into the bioreactor;
vi) detaching the grown cells from the support cell carrier matrix with maintaining the parameters of cell growth phase steps a) to d); and
vii) performing the infection and viral transfection process.

8. The method as claimed in claim 1 step e), wherein the enzymes are selected but not limited to recombinant trypLE, collagenase, versene, trypsin and accutase enzyme.

9. The method as claimed in claim 1 step e), wherein detaching the cells through combination of vibration and enzymes comprises the following steps:
i) removing the spent media after completion of growth phase steps a) to d);
ii) optionally washing the cell carrier matrix with phosphate buffer saline (PBS) and adding enzymes to the bioreactor;
iii) incubating the cell carrier matrix for 0.1 to 40 minutes and adding fresh infection media and fresh transfection media in the bioreactor;
iv) applying physical vibration to the cell carrier at 10 to 3000 RPM and amplitude of rotation from 0.5 mm to 20 mm for 0.1 to 30 minutes or with 0.1 to 5 minutes of interval to cell carrier;
v) detaching the grown cells from the support cell carrier matrix with maintaining the parameters of cell growth phase steps a) to d); and
vi) performing the infection and viral transfection process.

10. The method as claimed in claim 1 step f), wherein the DNA concentration is 0.5 to 10 micrograms per million cells for growth.

Dated this on October 14th, 2024