Applications > Protein Production > General Protein Production

MaxCyte flow electroporation reproducibly transfects adherent and suspension cell types enabling production of proteins in the quantities needed for most pre-clinical product development. While stable cell lines have been the standard for protein production for over two decades, MaxCyte electroporation has become a practical solution to the time, labor and cost challenges faced when relying exclusively on stable cell lines. The MaxCyte® STX™ provides a rapid, scalable method for transient transfection of primary cells, cell lines and importantly, historically difficult to transfect cell lines such as CHO cells and can produce proteins faster than stable cell lines.

MaxCyte Electroporation for Protein Production:

Decrease reliance on stable cell lines
Fully scalable, able to transfect up to 1x10¹⁰ cells in < 30 minutes
Specialized protocols optimized for protein production
Compatible with primary cells, cell lines and other difficult-to-transfect cells
Express secreted, membrane-associated or cytoplasmic proteins

Optimized Protocols
Secreted Proteins
Membrane Proteins
Enzyme Production
Recommendations

Increased Protein Expression with Optimized Electroporation Parameters

The MaxCyte STX instrument is preloaded with specialized electroporation (EP) protocols for individual cell types. Standard MaxCyte protocols provide an optimal blend of loading efficiency and cell viability, which are ideally suited for generating cells for use in cell-based assays. MaxCyte has developed additional EP protocols for CHO and HEK cells that are designed specifically for high level protein expression. These protocols are used for transfecting cells both in small scale (5x10⁵ to 4x10⁷ cells in seconds) and large scale formats (up to 1x10¹⁰ cells in less than thirty minutes). After identifying a DNA concentration that yields optimal assay results at small scale, the EP process can be scaled up without impacting transfection efficiency or cell viability.

Figure 1 illustrates the relative effects of electroporation energy and DNA concentration on cell viability and protein expression in CHO cells. The increased electroporation energy of the CHO Protein Expression protocol resulted in the ability to load cells with greater quantities of pGFP DNA and in turn, lead to an increase in average GFP expression per cell when compared with the standard CHO protocol. Note that in the absence of DNA, electroporation had little impact on viability, even when using the higher energy protocol. The average cell viability was reduced due to DNA toxicity, and correlated directly with the amount of DNA loaded into the cell. Additionally, there was a further reduction in viability at higher DNA concentrations when using higher electroporation energy. The MaxCyte CHO and HEK protein expression protocols are optimized for higher level protein production rather than cell viability which is in contrast to the general electroporation protocols which balance transgene expression and cell viability levels.

Figure 1: Increased Cell Loading with Protein Expression Protocols. CHO cells were electroporated (EP) with 0, 200 or 400 μg/mL pGFP in small scale format (OC-100 processing assemblies) using either MaxCyte's standard CHO protocol or a CHO-specific protocol optimized for protein expression. Cells were seeded in shake flasks at approximately 1x10⁶ cells/mL and assayed by FACS ~20 hrs post electroporation. Data are expressed for % cell viability, % GFP+ cells and the mean fluorescence intensity (MFI).

Sustained Production of Secreted Proteins

The data in these figures demonstrate the ability to produce secreted proteins from CHO cells for an extended period of time and the influence of post electroporation culture conditions on protein production levels. In Figure 2, CHO cells were transfected with varying concentrations of an expression plasmid encoding soluble TNFα receptor using the Standard CHO electroporation protocol or the CHO Protein Expression protocol and cultured at either 30°C or 37°C post electroporation. After culturing TNFαR transfected cell populations for 3 days at 37°C, the effects of DNA toxicity at 400 mg/mL were manifested as lower relative TNFαR titers for cells transfected using either the standard of expression-optimized protocol. TNFαR titers in all three cell populations were higher after three days in culture when incubated at 30°C than in the parallel 37°C cultures. The amount of DNA needed to reach peak TNFaR production at 30°C could be cut in half by using the Protein Expression electroporation protocol.

Figure 2: Post Electroporation Culture Temperature Optimization. Cells were EP'd with psTNFαR expression plasmid in small-scale processing assemblies using either the standard or protein expression CHO protocol. Cells were seeded into duplicate shake flasks at 1x10⁶ cells/mL, and cultured at 37°C or 30°C. Conditioned media samples were collected without replacement at days 1 and 3 post EP and assayed for TNFαR production by ELISA.

To examine the duration of protein expression, cells transfected with the TNFαR plasmid were cultured at 30°C for over two weeks. Although the MaxCyte STX system transiently transfects cells, TNFαR protein titers increased for up to 2 weeks post EP. The duration of protein production will depend on factors such as protein half life, media composition, cell type and vector design, but these data strongly suggest that transient transfection using the MaxCyte STX is a viable alternative to stable cell lines for pre-clinical protein production.

Figure 3: Sustained sTNFαR Production at 30°C. Cells were EP'd with 200 μg/mL psTNFαR plasmid using the CHO protein expression protocol, seeded into shake flasks at 5x10⁵ cells/mL and cultured at 30°C. Conditioned media samples were collected daily for 15 days post EP and TNFαR quantified.

Expression of Membrane Proteins in Suspension and Adherent Cells

Many assays require the over expression of membrane receptors. MaxCyte electroporation can be used to express a variety of surface receptors including receptor tyrosine kinases (RTKs), GPCRs, and Ion Channels in primary cells or cell lines. The data in Figure 4 summarize studies examining the expression of CD40L in suspension and adherent cells following transfection with the MaxCyte STX. Approximately 95% of both suspension-adapted HEK 293F and adherent CHO K1 transfected cells exhibited CD40L on their surfaces, demonstrating utility of the MaxCyte STX system for efficient expression of cell surface proteins.

Figure 4: Expression of CD40 ligand in transiently transfected HEK 293F and CHO K1 cells. Suspension-adapted HEK 293F cells and adherent CHO K1 cells were transfected with 200 μg/mL of a plasmid encoding CD40 ligand (CD40L/CD154) via small-scale electroporation. Cells were analyzed by FACS using a fluorescently labeled anti-CD40L antibody at 24 & 48 hours post electroporation. A). Structure of CD40L protein. B). Model of homotrimeric CD40L protein interacting with CD40. C). FACS analyses of transfected 293F and CHO K1 cells.

Expression of Functional Enzymes

The MaxCyte STX can be used to express a variety of enzymes. The studies below describe the expression of two different enzymes: an aldo-keto-reductase & a beta-secretase (BACE). Figure 5 summarizes data from a functional enzyme activity assay. Not only does the level of enzyme activity increase with increasing DNA concentrations, but addition of an enzyme inhibitor blocks the activity of the expressed Aldo-Keto-Reductase. Cell viability was reduced with increasing concentrations of DNA (data not shown). Cell viability was approximately 90% for cells transfected with 300μg/mL DNA.

Figure 5: Transient Expression of an Aldo-Keto-Reductase. 3x10⁷ HEK 293 cells were transfected with varying concentrations of an aldo-keto-reductase expression plasmid using small-scale MaxCyte electroporation. Data courtesy of Cell Culture Services (CCS).

The levels of Beta-secretase expression were assessed for varying concentration of DNA using the MaxCyte STX and compared to Lipofectamine. Western blot analysis showed that BACE levels were DNA concentration dependent and significantly higher than cells transfected with a lipid-based transfection reagent even at the lowest DNA concentration.

Figure 6: BACE Protein Expression via Western Blot Analysis. 4x10⁷ HEK293 cells were transfected with a BACE expression plasmid using small-scale MaxCyte electroporation or standard lipid-based reagent. Cells were cultivated for 24 hours post electroporation in S500 plates. Cells were harvested, pelleted and lysed in SDS buffer. Western blot analysis conducted using monoclonal anti-BACE antibody (Santa Cruz). Data courtesy of Cell Culture Services (CCS).

Protein Production Method Recommendations

To optimize conditions for high titer production of a particular protein in CHO and HEK cells, MaxCyte STX users should conduct several small scale electroporations with varying concentrations of their expression plasmid using both the standard and protein expression protocols. DNA concentrations in the range of 200-400 µg/mL are a good starting point. In some cases, lower or higher concentrations may yield better titers, depending on the relative toxicity of the expressed protein, strength of the promoter and physiology of the cell line.

In addition to DNA concentration and electroporation protocol type, cell culture conditions after electroporation have a significant impact on protein titers. Factors such as media composition, cell seeding density, culture temperature and media collection schedules will influence the amount of protein that can be generated by transfected cells.

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