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Application of reference technology of Tofflon HEK293 cell culture-medium family in production of different products

2023-03-09
News
790

1. Tofflon HEK293 cell culture medium family


image.png



Serial numberModel numberApplicationAvailable formProduct description
1

HEK293 CDM15


Protein expression, AAV, AdV


Dry Powder/Liquid


Chemical composition limited, suitable for suspension culture of HEK293,HEK293F,293T and transfection of protein, AAV, AdV

2

HEK293 CDM26


Protein expression, AAV, AdV



Dry Powder/Liquid


Suitable for suspension culture of HEK293,HEK293F,293T and transfection of protein, AAV, AdV and LV

3

HEK293 CDM30


Protein expression, AAV, AdV


Dry Powder/Liquid


Suitable for suspension culture of HEK293,HEK293F,293T and transfection of protein, AAV, AdV and LV

4

HEK293 SFM28


Protein expression, AAV, AdV


Liquid

No serum, suitable for 293T cell adherence maintenance and expression of transfection products

5


Supplement A

(Tobitec FA)



Universal type 

(HEK293, CHO)



Dry Powder/Liquid


No serum, no protein and protein hydrolysate, chemical composition restriction

6


Supplement B 

(Tobitec FB)



Universal type

 (HEK293, CHO)



Dry Powder/Liquid


No serum, no protein and protein hydrolysate, chemical composition restriction


2. Tofflon HEK293 is applied in different product transient reference process


2.1 Instantaneous reference process for the production of Dongfulon HEK293 protein

Basal mediumInstructionsRefillInstructions
HEK293 CDM15

Contains glutamine

Supplement A (Tobitec FA)

Glutamine-free

HEK293 CDM26

Supplement B (Tobitec FB)

Glutamine and glucose free

HEK293 CDM30
Protein instant feed processAt about 24 and 96 h after the instantaneous transfer of plasmid, 7% FA, 0.7% FB, 4mM glutamine, glucose control to 5g/L(excluding sugar in FA) were added.
Transfection process with PEI and other chemical reagents

1) For cells with a growth density of more than 10 e6 cells/mL in the host cell, the cell VCD was adjusted during transfection: 4.5 E6 cells/mL;DNA: 1.8mg/L; PEI:DNA=3

2) cells with host cell growth density less than 10 e6 cells/mL: adjust cell VCD during transfection: 2 E6 cells/mL;DNA: 1mg/L; PEI:DNA=3

Other parameters suggestedInitial culture temperature 36.5℃, no cooling, pH:7.1±0.2, DO: 40%.(Recommended speed in shaker: 120rpm, amplitude: 50mm, 5-8%CO2)



2.2 Tofflon HEK293 Virus production transient reference technology -- suspension system

 

Take the 1mL transfection system of AAV virus as an example:


Base mediumDNA dosageFectoVIR-AAV volumeThe amount of medium used for the preparation of the complexSupplement



HEK293 CDM26



1.5 ug

(1.5 ~ 2.0 ug)



2.25 uL

(1.5 ~ 4.0 ul)


0.1 mL

Tobitec FA&Tobitec FB


Instantaneous refill process



At about 24 and 96 h after instantaneous virus transfer, 4% FA, 0.4% FB and 4mM valley were added respectivelyAmmonia, glucose controlled to 5g/L(except sugar in FA)


Carrier ratios are recommended as follows (take 1.5ug/mL DNA as an example)


Expression vectorMolar ratio

Recommended carrier molecule Amount (Kbp)


ug DNA/mL culture volume
Rep/Cap plasmid2
90.95
Helper plasmid0.5110.29
AAV-ITR plasmid150.26


2.3. Instantaneous Reference technology for production of HEK293 virus in Dongfulon -- Adherent system



Basic mediumCategoriesCraft
HEK293 SFM28Lentivirus Instantaneous Reference Process

1) HEK293T adherent culture was carried out with medium (generally basic medium + serum) according to the original process, and the cell growth and fusion degree reached 80% after standing culture;

2) Viral transfection was performed using the original medium or HEK293 SFM28;

3) The serum-containing medium was removed and cleaned 4 to 6h later, and HEK293 SFM28 was added for further culture. Scandalous operation was performed 48 to 72 h after transfection.

Suggestions for other parametersThe initial culture temperature is 37℃, and the temperature is cooled to 32℃ after transfection


3. Reference process for stability of Tofflon HEK293


The recommended process is as follows:


Base mediumStatingRefillInstructions
HEK293 CDM26/30Already contains glutamineSupplement A (Tobitec FA)Glutamine-free 
Supplement B (Tobitec FB)Glutamine and glucose free 
Feeding processWhen the cell density is ≥4 e6cells/mL to start the feeding, it is recommended to add 5% per 48 hfA, 0.5% FB, 4mM glutamine (no need to add in GS system), glucose control to 6-8g/L
Other parameters recommended

Initial culture temperature 36.5℃, day5 cooling to 33℃, pH:7.1±0.2, DO: 40%.

(Recommended speed in shaker: 120rpm, amplitude: 50mm, 5-8%CO2)


Remarks80mM Gln can be added directly to FA when mixing according to process requirements


4. Tofflon HEK293 medium supporting

 

4.1. Provide overall solutions and application support for key reagents according to different applications and types of biological drug R&D and production:


微信图片_20240424100017.png

4.2. On the basis of medium application technology, combined with Tofflon's core equipment, support the scale and industrialization of biological drug projects;



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4.3. Starting from drug application and marketing, complete product documents and registration records for supporting products are provided, as well as product compliance documents and application support.



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