Mammalian-ST™ Stable Cell Line / Stable Pool Development Service

Mammalian-ST™ is the stable mammalian cell line development service provided by Bio Bench. Bio Bench has achieved more than 600 stable cell line projects since 2013, including more than 50 antibody drug expressing cell lines. The table below lists the cell types that have been successfully generated into stable cell line, do not hesitate to request a quote from us!

Stable mammalian cell line is one of the most important tools in drug discovery, toxicity test, biological research and bio-pharma industry. In order to establish stable cell line capable of long-term stably expressing the GOI (gene of interest), the operation is commonly achieved by transfection of a reconstructed plasmid containing the GOI together with a selection marker (antibiotic or fluorescent protein), through either liposome-mediated transfection or virus infection (or called viral transduction).

Service and Timeline	Service Description (To be determined according to your specific project)	Deliverables	Price
P1521 – Stable pool Mammalian stable pool generation service 8 – 10 Weeks	1. Positive control protein/antibody characterization. 2. Codon optimization, gene synthesis, cloning and plasmid preparation. 3. Transient transfection test and antibiotic concentration determination. 4. Transfection and stable pool generation. 5. Stable pool characterization: expression level, cell viability, growth curve, etc. More characteristics optional! 6. SDS-PAGE, WB, ELISA, and/or FACS analysis. 7. Protein sample expression and purification. 8. QC and report.	- Selected and optimized stable pool. - Purified protein sample. - Data and QC report.	Inquiry​
P1526 – Stable cell line Mammalian stable cell line generation service 23 – 30 Weeks	All of the content from P1521 Stable pool generation service, together with the following: 1. Monoclonalization and best clones selection. 2. Fed-batch 1 (small culture volume) primary study and characterization. At least 6 characteristics will be tested for a duration of 8 – 15 days. 3. Fed-batch 2 (midi culture volume) enhanced study and characterization, optional. 4. Cell banking. 5. Whole genome sequencing of the selected clone, and backup plasmid establishment.	- Selected stable cell line. - Purified protein sample. - Data and QC report.	Inquiry​
More test options, including: 1.      Bioactivity test: binding/blocking assay, ADCC assay, etc. 2.      Affinity test by biacore or ForteBio method. 3.      Cell culture monitoring: Ammonia test, lactic acid test, pCO2 analysis, etc. 4.      Charge heterogeneity test. 5.      MS test: molecular weight, aggregation, etc. Would like to lean more? Seeking a specific testing method? Click on Inquiry​ and we will get back to you soon.

 

Bio Bench has successfully established the following host into stable cell line
CHO	CHO-K1	expiCHO	CHO-S
293F	CHO-DG44	HEK293	293T
expi293F	Vero (insect cell)	Your cell line here! Get a quote for free

 

Several typical applications including:

1. Hit screening / identification in therapeutic drug discovery.
2. Cell signaling.
3. Protein production, including antibody drug production.
4. Toxicity study.
5. Gene/mRNA function study.​

General process of stable cell line development

​Transient expression and its pros and cons

The GOI (Gene of Interest) is commonly expressed by transient infection of mammalian cell line, and lead to successful expression of X milligram of protein. In small scale expression, transient transfection is 1) easy to operate – maximum 10 days training of new staff, 2) fast – 5 days from transfection to protein, and 3) reliable – relatively reproducible result >80%. However, once your project requires XX milligram, XXX milligram or even gram level of protein,  transient expression can be costly, laborious, time-consuming and risky. You may need to prepare large amount of plasmid and redundant cell culture priorly of transfection; all of the cells have to be in healthy condition at the time of transfection; if any problem happens – misoperation, contamination, low transfection efficiency and/or low expression level,  all of the procedures will have to be re-done.

​Stable pool/Stable cell line and its pros and cons

While talking about "stable", 2 concepts will be often discussed: stable pool and single clonal stable cell line. After transfection, the transfectants will be cultured in a medium containing growth inhibitors to remove the untransfected cells and low expression transfectants. The remaining group of cells is considered a stable pool. The stable pool may be seeded in 96-well (or 384-well, 1536-well, etc.) plate and subcloned to find out the stable single clone. This clone is defined as a stable single clonal cell line, or stable cell line. The stable cell line shows many advantages: constant GOI expression, easy cell culture and stable cell banking. It is thus ideal for midi-to-large scale protein expression or for long duration project.

Comparison among transient expression, stable pool and stable cell line

Service type Specification	Transient transfection	Stable pool	Stable cell line
Initial cost	+	++	+++
Midi scale cell culture or production cost	+++	++	++
Large scale cell culture or production cost	+++++	++	+
Operation complexity at small scale	++	+	+
Operation complexity at midi scale	+++	+	+
Operation complexity at large scale	+++++	+	+
Operator-to-operator variation	++++	+	+
Batch-to-batch variation (by the same operator)	++	+	+
Protein expression level	+	+++	+++++
Short term expression stability (1 - 3 days)	++++	+++++	+++++
Long term expression stability (10 – 20 days)	\	++	+++++
Regeneration stability	\	++	+++++
Conclusion	1.       Cell culture volume <100mL, or occasionally used cell culture. 2.       Protein amount <10mg, or occasionally used protein. 3.       Transfection is request by experiment protocol. 4.       Co-transfection of more than 1 plasmid. 5.       Small scale and proof-of-concept experiment.	1.       100mL < Cell culture volume < 20L, or regularly used cell culture (without specific requirement). 2.       10mg < Protein amount < 5g, or one time production but enough for a long period of usage. 3.       Non-commercial purpose, or less sensitive to cost. 4.       Low/no requirement for cell homogeneity. 5.       Low requirement for stability.	1.       Small to high volume cell culture, with or without high requirement. 2.       Small to high amount of protein production. 3.       Commercial purpose, cost effective. 4.       Experiment requires high level of cell homogeneity. 5.       High requirement for cell stability.
	Inquiry	Inquiry	Inquiry

 

Case study: Mammalian-ST™ Stable pool/Stable cell line

A drug potential antibody was discovered from human naive antibody library. After in-house test, one of the selected clones was considered “great performance”, and further experiment will require large amount of purified antibody. Client requests Bio Bench to establish a stable cell line and purify optimum quantity of antibody.
CHO-K1 was selected as expression host to establish the stable cell line. G418 was chosen as selection marker in order to simplify the process. A vector containing G418 resistance was use to carry the antibody gene.
After the operation, 1.2g/L titer was achieved by Bio Bench. Would like to learn more? Request full case study!

Fig. Transient pool antibody expression analysis by SDS-PAGE

Fig. Single clone tracking for 8 days (Celigo)

Fig. Fed-batch cell proliferation and viability test