It’s been a HEK of a journey

Celebrating 10 glorious years of working with HEK293-6E cells to produce high quality recombinant proteins

In 2025 we celebrated 10 years of licencing the HEK293-6E mammalian suspension expression system from the National Research Council (NRC) in Canada. Since then, it has become a reliable expression workhorse for us with the result that the HEK293-6E system has played a key role in delivering mammalian cell grows for the company. This anniversary seems like an ideal moment to look back at what we have achieved with them over the past decade.

HEK293-6E were developed at NRC and are engineered to express a truncated form of Epstein-Barr virus nuclear antigen 1 (EBNA-1), which allows for episomal replication of plasmids with oriP. Transfection of the cells with the pTT5 vector, which has both oriP and a strong hCMV promoter, enables enhanced expression of recombinant proteins, making them ideal for quickly generating useful quantities of transiently expressed protein.

Back in 2015 the HEK293-6E were brought in to complement the E. coli and insect cell lines which had already been established at Peak Proteins (now The Protein Science & Structural Biology department at Sygnature Discovery Ltd). The HEK293-6E were selected based on previous experience of working with this cell line and the enhanced expression characteristics mentioned above. What made it a “no brainer”, in the words of our founder Mark Abbott, was that the commercial terms on the HEK-6E cells were much better than alternatives on the market and the media was a lot cheaper. Being a small start-up company, these were key factors for us at the time.

So, what types/classes of proteins have we grown?

We tend to choose mammalian host cell lines for extracellular targets, especially when glycosylation is a consideration. With the HEK293-6E system, well, we have produced everything from membrane proteins to proteases, extracellular domains of receptors and some really challenging molecules including the production of some intrinsically disordered proteins and even a protein complex that involved co-transfection of 12 separate plasmids. They have proved excellent for the production of antibodies and antibody derived proteins (e.g. Fabs) which alongside our other mammalian cell lines have become a key component in the support of our ADC workflows.

A few selected examples are presented below:-

Production of the extracellular domain of G6b-B

One of our early successes was the production of the extracellular domain (ECD) of G6b-B protein, which was used in an X-ray crystallography project. The team successfully determined the structure of the G6b-B ectodomain bound to a recombinant Fab fragment and a crystal structure of the ECD in complex with a 12 saccharide-unit heparin fragment (DP12) that was predicted to be an analogue of the physiological ligand. To date this is still the only structure of G6b-B in the PDB (reference 6R0X).

Production of single chain Fvs

Bivalent single chain Fvs (scFv) expressed in our HEK293-6E cells featured in a publication on a new family of anti-thrombotic biotherapeutics from the lab of Dr. Yotis Senis, Research Director, INSERM, Strasbourg, France. Christened CAPRIs (cis-acting platelet receptor inhibitors), the ScFv act by clustering the platelet inhibitory receptor G6b-B (G6B, MPIG6B) with the platelet stimulatory receptors GPVI-FcR γ-chain or FcγRIIA, thus blocking platelet activation and laying the foundation for a novel class of anti-thrombotics with potential for greater efficacy.

Production of the protein API for a biotherapeutic

HEK293-6E have also been used for the development of a production process that was suitable for full scale manufacture of the protein component of a biopharmaceutical. The small, secreted <10kDa glycosylated protein featured all of the desired traits for excellent SISPQ and a robust process for manufacture: fully active, amenable to subsequent conjugation, high yielding (~80-100 mg per L culture), low endotoxin (0.1 EU/mL), a single species observed by MS, reproducible, scalable and with a single step purification, relatively inexpensive with respect to Cost of Goods.

in vivo biotinylation of target proteins in HEK293-6E

Working with HEK293-6E for a long period of time has allowed us to develop additional tools such as an in vivo biotinylation method (well of course it is not strictly in vivo, more during translation by co-expression of BirA) , which provides further options for our clients’ projects.

Glycosylation

We are also able to modulate the post-translational glycosylation of proteins which can be especially helpful for X-ray crystallography projects. Oligosaccharides attached to asparagine or N-linked structures are very common and we can remove the N-linked glycans by either mutating the asparagine or more commonly culturing the cells in kifunensine and then treating the protein with endoglycosidase H during purification.

Much less common are oligosaccharides linked to the -OH group of either threonine or serine. Protein characterisation using mass spectrometry has revealed O-glycosylation on proteins from time to time. Since O-glycosylation can have a significant impact on proteins produced for biotherapeutic purposes this highlights the importance of identifying any post translational modifications proteins may possess.

CURRENT STATUS OF CELL CULTURE AT SYGNATURE

As described in our recent blog ‘More Protein Targets More Cell Cultures’ there has been a dramatic rise in the number of cell expression grows, culture volumes and expressed protein targets during the last five years. We have kept up with this demand by expanding the range of volumes the cells (including the HEK293-6Es) are grown at and can now cover everything from small scale feasibility studies in as little as 4mL in a plate, up to 20L wavebag grows. In addition to this, we have developed an in house ‘toolbox’ of tricks and tweaks we use to coax more difficult proteins to express.

What about the future?

And what might the next decade bring for HEK293-6E? We’re currently developing a method for expressing labelled proteins for NMR so watch this space!