New hope in the post-new crown epidemic era: HEK293 cells and key upstream technologies

The new crown virus is raging around the world with unprecedented destructive power.
Various mutant viruses are emerging one after another.
Epidemics are one after another, and there are no specific therapeutic drugs
.

Therefore, how to quickly develop the new crown and the therapeutic drugs for all future epidemics is the focus of common concern all over the world
.

The biopharmaceutical production process based on large-scale mammalian cell culture technology can quickly produce therapeutic drugs such as virus vaccines and neutralizing antibodies to efficiently prevent and treat epidemic diseases such as new coronary pneumonia
.

Application of HEK293 cells The human embryonic kidney cell line HEK293 cultured in a large-scale bioreactor is one of the main cells for the production of biological drugs
.

Because of its easy transfection characteristics, it is widely used in the production of various therapeutic proteins, virus-like particles (VLP) used in the production of vaccines, virus vectors used in cell and gene therapy and other products or to carry out related scientific research (table 1)
.

Table 1 Product types and examples based on HEK293 cell culture technology However, for a long time, since HEK293 cells can only be cultured adherently, it is difficult to scale up, which limits their application scope to a large extent
.

Nevertheless, compared with the CHO cell line commonly used in industrial production, the post-translational modification (PTM) capability of HEK293 cells is more powerful, and it can carry out complex human glycosylation modifications to products.
The HEK293 cell line has appeared one after another, and its productivity and scalability have been effectively improved
.

Therefore, the future application prospects of HEK293 cells may be broader, which is expected to fill the technical gap based on CHO cell technology
.

The origin of HEK293 cells and their types HEK293 cells can be traced back to 1973.
Since then, many different HEK293 cell lines have been derived and used in different directions
.

HEK293 cells are immortalized human embryonic kidney cells developed by transfection of sheared human adenovirus 5 (Ad5).
Its genome integrates the genes of adenovirus proteins E1A and E1B.
The expression of these two proteins can be regulated by Cell cycle and apoptosis promote the growth of HEK293 cells, and can be used as basic cofactors to increase the production of recombinant adeno-associated virus (AAV) particles, thereby turning it into an efficient "cell factory"
.

Since the establishment of the HEK293 cell line, it has been fully characterized, and many unique derived cell lines have been developed, including: HEK293T, HEK293E, HEK293-6E, HEK293F and its derivative cell lines HEK293FT, HEK293FTM, HEK293S And HEK293H and so on
.

Figure 1 HEK293 cell line HEK293 culture technology based on ADCF platform.
In HEK293 cell culture, materials and equipment such as medium, digestion agent, microcarrier, culture flask, bioreactor, etc.
are designed.
These materials or materials are in direct contact with cells and may be introduced.
The pollution will ultimately affect the safety of the product
.

Moreover, the regulations and systems of various governments or related global organizations have higher and higher requirements for products, so more manufacturers will choose to use animal-free ingredients (ADCF) platforms for production
.

There are two main categories of ADCF products: animal-free (ACF) or animal-derived: products or materials that do not contain animal tissues or body fluids or are separated or purified from animal tissues or body fluids
.

XF-free (XF): refers to products containing recombinant materials derived only from human, plant, bacterial or yeast sequences, and products or materials that do not contain non-human animal components
.

1.
HEK293's ADCF adherent culture technology The HEK293T adherent culture technology based on ADCF platform is mainly shown in Figure 2.
The general process mainly includes: cell cryopreservation and recovery, cell culture, cell digestion, microcarrier culture, WAVE bioreactor For seed amplification, large-scale bioreactor production, etc.
, Cytiva can provide a complete set of integrated solutions
.

Including: Figure 2 ADCF adherent production platform of HEK293 (1) Non-liquid nitrogen program cooling instrument for cell bank cryopreservation VIA Freeze traditional liquid nitrogen program cooling instrument will volatilize nitrogen after the sample is cooled, which may contain bacteria and fungi Contamination sources such as spores, viruses, and prions have always been controversial in the production and application of new therapies
.

Moreover, the stability of temperature control directly affects the state of cell seeds, so the instability of the traditional liquid nitrogen program cooling apparatus is also a major challenge in the freezing of cell seeds
.

The non-liquid nitrogen programmed cooling instrument VIA Freeze can be used for the freezing of HEK293T cell banks and can accurately control the temperature
.

Cytiva VIA Freeze is a non-liquid nitrogen program cooling instrument, which is a better choice in terms of cost and safety
.

Cytiva tested various cell lines sensitive to freezing procedures, and the results proved that Cytiva VIA Freeze can guarantee the consistency and stability of the recovered cells to the greatest extent
.

Figure 3 Cost analysis and cell cryopreservation effect of non-liquid nitrogen program cooling instrument VIA Freeze (2) Adherent medium In traditional adherent cell culture, cell factories, roller bottles, microcarriers, etc.
are usually used to provide adherent medium for cells
.

However, the cell factory tends to cause uneven growth and waste the effective adhesion area.
It is difficult to scale up, and it requires a higher area occupied by the delivery room.
In addition to the same shortcomings as the cell factory, the roller bottle is also relatively easy Pollution
.

Therefore, microcarriers (such as the Cytodex series) are often used in large-scale industrial production to provide cells with adherent media
.

The microcarrier culture technology allows cells to evenly attach to tiny spheres, effectively expanding the cell attachment area per unit volume, and in this way achieves high-density suspension culture of adherent cells
.

Figure 4 HEK293 attachment solution in large-scale production (3) Trypsin digestion solution is in the process of seed expansion and scale up of adherent cells.
When the cells are laid on the adherent medium, the cells need to be removed from the adherent medium.
After digestion, a larger area of ​​adherent medium is used for re-adherent culture
.

Generally speaking, trypsin digestion solution is mainly used for digestion
.

HyrTryp ADCF Recombinant Trypsin Digestion Solution is a new product launched by HyClone, a subsidiary of Cytiva, and can be used as a perfect alternative to traditional pancreatin
.

HyrTryp ADCF Recombinant Trypsin Digestion Solution has the following advantages: all links in the production process have been fully verified to ensure compliance with cGMP, ISO 9001:2015, ISO13485:2016 and other standards; it is ADCF raw material, regardless of regulatory safety or downstream purification It is a better choice from the perspective of difficulty; it is suitable for various adherent cell types, which can effectively separate cells from the adherent medium without affecting cell growth and viability; HyrTryp reaction environment is mild, not only suitable for Vero, MRC5 and other cells The digestion is also suitable for HEK293T, primary cells, stem cells and other cells that are sensitive to digestive enzymes
.

Figure 5 HyrTryp ADCF recombinant trypsin digestion solution Figure 6 Comparison of HyrTryp with porcine trypsin and other commercially available recombinant trypsin 2.
HEK293 ADCF suspension culture technology domesticated HEK293 cells from adherent growth to single cell suspension growth.
The process flow can be effectively simplified, and it is easier to scale up
.

Using Cytiva's HyClone series ADCF medium and suspension cell acclimation technology, HEK293 cells can be acclimated into a full suspension cell line that can grow in a single cell suspension like CHO cells, and applied to industrial large-scale production
.

Figure 7 CCRM uses Cytiva to construct the cell therapy and gene therapy plant layout with the overall solution of CCRM.
HEK293 cell culture process optimization 1.
Improve the growth performance of HEK293 and increase the yield through genetic engineering technology.
Cell line is the most important production material in industrial production, cell Inherent biological characteristics are the biggest limiting factor for process production efficiency
.

By introducing chromosomal mutation can effectively regulate cell growth and yield of the desired product, for example: nucleotide substitutions, deletions or insertions gene, overexpression of homologous or heterologous genes and the like
.

Through the use of RNA interference, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clusters of regularly spaced short palindromic repeats (CRISPR) related proteins (CRISPR-Cas9), it is possible to target Temporarily or permanently modify specific genes involved in the expression of the target product
.

In recent years, with the rapid development of global genetic engineering, cell engineering and whole-genome analysis technologies, random mutations, RNA interference screening, whole-genome flow cytometry, metabolomics, proteomics, and transcriptomics analysis can now be used Methods such as identifying new target molecules related to product expression, optimization directions include: protein synthesis, folding (including PTM) secretion, degradation and other related gene modification
.

Engineered gene targets involved in the following mainly focused on the intracellular pathways: cell proliferation, apoptosis, the central carbon metabolism, glycosylation, protein folding, and the like secretory pathway
.

Figure 8 Improve HEK293 process performance and increase yield through genetic engineering technology 2.
Improve HEK293 cell growth performance and increase yield through optimization of culture process and culture environment.
In addition to cell lines, cell culture environmental conditions are also important factors that determine process efficiency.
, Usually single factor or DOE method can be used to optimize the culture environment conditions to improve process performance
.

For example: optimizing cell seeding density, medium, culture mode, nutrient replenishment strategy, bioreactor control parameters (pH, DO, temperature, etc.
) and other factors to establish platform biotechnology; evaluate transgene dosage, packaging and auxiliary plasmid ratio, and total DNA concentration The influence of parameters such as cell density on AAV
.

At present, most of the HEK293 processes are still fed-batch culture processes, but the feasibility of using other culture modes is also being tried, such as packed bed bioreactors, ATF and other perfusion culture systems
.

Figure 9 HEK293 perfusion culture system based on ATF With the increasing demand for new coronavirus vaccines and other therapeutic biological drugs, there is an urgent need to develop more efficient cell culture processes
.

HEK293 cells have many advantages, and the performance of PTM is better than CHO cells and other mouse-derived cells
.

Therefore, after CHO, HEK293 is expected to become the new favorite of the future biological product production process
.

For more related content, you can scan the code to watch the course topic of the Cytiva Academy course HEK293 suspension/adherent ADCF scalable process optimization and case sharing course introduction HEK293 cell line has been widely used in new vaccines, gene and cell therapy, recombinant protein domain proteins and For the production of viral vectors, this lecture will focus on the optimization of HEK293 industrialization and culture, and share the transformation direction of 293 cell lines at home and abroad, culture environment and culture methods
.

The transformation direction of HEK293 cell line includes: suspension domestication, Knock out cell line establishment, optimization purpose and sharing of cryopreservation methods suitable for new therapies
.

Production auxiliary materials may directly contact cells and products, affecting the safety of the final product
.

With the rise of new vaccines and emerging therapies and increasingly stringent regulatory requirements, most industries in the industry are more inclined to use ADCF auxiliary materials for production
.

Thus, the contents of the optimization of the culture material comprising: defining ADCF materials and supplier management, can be amplified ADCF culture media and culture environment digestive medium, optimization of transfection conditions
.

At present, the culture of HEK293 cells is mainly based on fed-batch culture, and the continuous culture mode is one of the development directions of the future bioindustry
.

Therefore, this lecture will also focus on the knowledge of the production process based on the continuous culture of HEK293 cells, the scale-up strategy, and the establishment of the TFF platform
.

Course outline 1) The purpose of constructing HEK293 ADCF production platform and supplier management 2) HEK293 cell line transformation direction and case sharing 3) HEK293 cell culture related ADCF production material optimization and case sharing 4) HEK293 fed and perfusion culture process Case Sharing Reference Materials 1.
Abaandou, Laura, David Quan and Joseph Shiloach.
"Affecting Hek293 Cell Growth and Production Performance by Modifying the Expression of Specific Genes.
" Cells 10, no.
7 (2021): 1667.
2.
Bloom, Fredric R, Paul Price, Guifang Lao, Jiu Lin Xia, John H Crowe, John R Battista, Richard F Helm, Steve Slaughter and Malcolm Potts.
"Engineering Mammalian Cells for Solid-State Sensor Applications.
" Biosensors and Bioelectronics 16, no.
7-8 (2001): 603-608.
3.
Casademunt, Elisabeth, Kristina Martinelle, Mats Jernberg, Stefan Winge, Maya Tiemeyer, Lothar Biesert, Sigurd Knaub, Olaf Walter and Carola Schröder.
"The First Recombinant Human Coagulation Factor Viii of Human Origin: Human Cell Line and Manufacturing Characteristics.
"European journal of haematology 89, no.
2 (2012): 165-176.
4.
Geisse, Sabine and Cornelia Fux.
"Recombinant Protein Production by Transient Gene Transfer into Mammalian Cells.
" Methods in enzymology 463, (2009): 223-238.
5 .
Genzel, Yvonne, Thomas Vogel, Johannes Buck, Ilona Behrendt, Daniel Vazquez Ramirez, Gudrun Schiedner, Ingo Jordan and Udo Reichl.
"High Cell Density Cultivations by Alternating Tangential Flow (Atf) Perfusion for Influenza a Virus Production Using Suspension Cells.
" Vaccine 32, no.
24 (2014): 2770-2781.
6.
Jäger, Volker, Konrad Büssow, Andreas Wagner, Susanne Weber, Michael Hust, André Frenzel and Thomas Schirrmann.
"High Level Transient Production of Recombinant Antibodies and Antibody Fusion Proteins in Hek293 Cells.
" BMC biotechnology 13, no.
1 (2013): 1-20.
7.
Kim, Keun-Soo, Min Soo Kim,Ji Hyun Moon, Moon Sik Jeong, Jinhong Kim, Gyun Min Lee, Pyung-Keun Myung and Hyo Jeong Hong.
"Enhancement of Recombinant Antibody Production in Hek 293e Cells by Wpre.
" Biotechnology and bioprocess engineering 14, no.
5 (2009) : 633.
8.
Lin, Yao-Cheng, Morgane Boone, Leander Meuris, Irma Lemmens, Nadine Van Roy, Arne Soete, Joke Reumers, Matthieu Moisse, Stéphane Plaisance and Radoje Drmanac.
"Genome Dynamics of the Human Embryonic Kidney 293 Lineage in Response to Cell Biology Manipulations.
" Nature communications 5, no.
1 (2014): 1-12.
9.
Malm, Magdalena, Rasool Saghaleyni, Magnus Lundqvist, Marco Giudici, Veronique Chotteau, Ray Field, Paul G Varley, Diane Hatton, Luigi Grassi and Thomas Svensson.
"Evolution from Adherent to Suspension: Systems Biology of Hek293 Cell Line Development.
" Scientific reports 10, no.
1 (2020): 1-15.
10.
Pulix, Michela, Vera Lukashchuk, Daniel C Smith and Alan J Dickson.
"Molecular Characterization of Hek293 Cells as Emerging Versatile Cell Factories.
" Current Opinion in Biotechnology 71, (2021): 18-24.
11 .
Stepanenko, AA and VV Dmitrenko.
"Hek293 in Cell Biology and Cancer Research: Phenotype, Karyotype, Tumorigenicity, and Stress-Induced Genome-Phenotype Evolution.
" Gene 569, no.
2 (2015): 182-190.
Gene 569, no.
2 (2015): 182-190.
Gene 569, no.
2 (2015): 182-190.