Gene Editing in CHO Cells

The team behind Cell Line Development & Engineering event have recently produced an exclusive whitepaper titled "Gene Editing in CHO Cells". Below you will find a brief summary of the whitepaper and download the complete whitepaper now.

Whitepaper Summary:

Recently, several exciting advances in CHO cell line engineering have received significant media and research attention due to efforts in genome sequencing, systems biology, and bioinformatics combined with the relatively new field of targeted gene editing platforms. Three key gene editing technologies have been at the forefront of the recent developments in CHO cell line engineering. Early efforts to introduce targeted site specific edits to the CHO genome focused on implementing the zinc finger nucleases (ZFNs) and the transcription activator-like effector nucleases (TALENs). The ZFN platform has been successfully deployed in a variety of post translational modification applications aimed at increasing specificity of recombinant protein production, but the efficiency of this platform can be limited in mammalian cell lines.

The clustered regularly interspaced short palindromic repeats associated 9 (CRISPR/Cas9) targeted gene editing system has recently exploded onto the research scene in almost every organism. This targeted gene editing platform allows for the creation of multiplexed edits in a single cost-effective step with a specificity previously unachievable in the genome editing arena. This complex is composed of short guide RNAs (sgRNAs) and a CRISPR-RNA that form a site specific construct that is complimentary to the target DNA, which introduces a double stranded DNA break upon binding. Repair of the break site by endogenous enzymes then creates a highly specific change to the DNA which can be customized for a variety of applications.

These advances in genome editing have helped enable high-throughput development of CHO cell lines that can be utilized as economically viable commercial expression vectors. The CRISPR/cas9 gene editing system has shown to be an extremely useful tool for customizing the metabolic pathways of CHO cell lines for use in biopharmaceutical production. One of the most useful applications of this exciting technology has been the creation of multiplexed targeted knock out screening systems. Previously, knock out experiments had to rely on mutagenesis, drug knock out, or media screening to identify the effect of a mutation on a desired cell type. These methods are inefficient and sometimes lead to less desirable off-target effects. It is now possible to develop very large gene knockout libraries to be targeted by CRISPR/cas9 using bioinformatics software specific to this platform.

Customizing metabolic pathways in CHO cell lines is of paramount importance for developing “cell factories” capable of biopharmaceutical production. Reducing the energy expenditure associated with mitochondria production and oxidative metabolism is one approach that has been shown to increase the efficiency of the cell by directing metabolism towards production of the target product. Modulating these pathways has traditionally been accomplished using interfering RNAs, but the specificity of the CRISPR/cas9 platform offers another tool with which researcher can customize the energy profile of CHO cell lines destined for biopharmaceutical production.

Future efforts in the field will be focused on increasing the efficiency of the CRISPR/cas9 system, as this platform is poised to become the model for the majority of biopharmaceutical development. Currently, 60 to 70% of all biopharmaceutical production is accomplished with recombinant mammalian cell lines, and this market share is expected to grow. Multiplexed editing efforts will also continue to increase the rate at which specific customizable CHO cell lines can be produced, as this process is highly critical to increasing the rate at which CHO cell line engineering moves forward.

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Cell Line Development: MicroRNAs in a Commercial Process

A few weeks ago, we wrote about an interview with Dr. Niall Barron, who’ll be speaking at this year’s Cell Line Development & Engineering conference, in which he explained why researchers should be paying attention to MicroRNAs.  Given that an estimated 50% to 70% of all protein encoding genes are under the control of MircoRNAs themselves, it’s clear that these molecules should be peaking the interest of researchers.  But how can they be leveraged in the commercial sense?  We go back to Dr. Barron for the answer.

How might they be implemented by industry in a commercial process?

Dr. Barron: So, as I mentioned previously, there are two main approaches. Ideally, an industry would use this technology before going through regulatory submission. So, with a new cell line making a particular product of interest, a cell line would be manipulated stably by using something like the technology I just described. For instance, over expression sponge that would target the microRNA of interest, or indeed, over expressing a short hairpin that will mimic the microRNA of interest if over expression is desired. So, that forms a stable cell line that can act as the parent, into which the product gene or the transgene is placed and that would go through the normal regulatory process.

The alternative in existing process where full regulatory submission is not desirable would be to add either mimics or inhibitors, which are short molecules that are typically complexed with some kind of transfection reagent and that can be fed directly into the culture medium where it is transported across the outer cell membrane to mediate its effect directly in the process.

You can hear the complete interview with Dr. Barron here, or you can download the transcript included in our brochure here.

Get the latest from Dr. Barron and other industry experts at this year’s Cell Line Development and Engineering conference, September 8-10, Berkeley, CA.  Now, SAVE 20% off the standard rate*.  Register here and use code XB14189BLOG.

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MaxCyte's New Technology Could Help Vaccine Shortages

The outbreak of the H1N1 virus in 2009 and the resulting vaccine shortage demonstrated the costly and often lengthy process associated with vaccine production.  While stable cell lines have been the standard in biotherapeutic protein production for the last 20 years, researchers have been anxious for more efficient technology.  Transient transfection technology, including MaxCyte’s Flow Electroporation, largely meet some of these efficiency demands.  MaxCyte recently published a whitepaper looking at how this meets some of the demands in vaccine production.  You can download the article here, but you may want to see what Dr. Weili Wang, one of the lead scientists on this study, had to say on the implications. 

The report says that no other single expression system has the capability to produce the series of next-gen vaccines. What are the implications of this on the field?

MaxCyte electroporation is a clinically proven transient transfection method that enables early risk reduction and accelerated development of antibodies, recombinant antigens, VLPs, VRPs, viral vectors, and cell immunotherapies. In addition, it enables the progression from gene to gram-scale quantities of proteins within days rather than weeks. This means, in the field vaccines for pandemics, seasonal outbreaks, or biodefense needs could be generated much quicker than it currently is.

What can this contribute to the field of cell line engineering?

With more than a dozen adherent and suspension cells lines currently in use by vaccine manufacturers, a transfection platform that provides cell type flexibility is clearly needed. MaxCyte electroporation consistently results in high levels of transfection efficiency and cell viability for a wide range of cells, including CHO, MDCK, BHK-21, Vero, NS0, insect cells, and other cell types commonly used for protein expression. Furthermore, cell immunotherapy using patient-isolated primary cells, most frequently hematopoietic cells, requires high transfection efficiency and low cell toxicity while meeting stringent sterility and safety needs. MaxCyte electroporation was originally developed for this application, which is why it has extremely high levels of cell viability and transfection efficiencies for a range of primary cells. Together the ability to transfect a wide range of cells, including difficult-to-transfect cells, and the high transfection efficiencies and cell viabilities could greatly expand the field of cellular immunotherapy.

See also: Timeline Reduction in Cell Line Development

Looking at this data, what jumps out at you the most as something you didn’t expect?

The data reinforce our previous experiences with the wide range of cell types it can transfect, including insect cells. Insect cells are commonly used in vaccine production since they post translationally modify proteins in a manner similar to that of mammalian cells and are easy to culture with simplified cell growth that is readily adapted to high-density suspension. While both transient transfection and recombinant baculovirus platforms are commonly used for insect cell protein expression, MaxCyte electroporation offers a more rapid means of production since there is no need to make baculovirus and it provides a higher yield because there is no need to remove the baculovirus, which the literature has cited a reason for yield loss.

Let your imagination run wild for a second, what’s the next innovation we could expect from this type of technology?

The use of therapeutic antibodies and vaccines is a large and growing area of interest. Over the next several years we can expect to see more therapeutic products come to market, particularly concerning rheumatoid arthritis, multiple sclerosis, Alzheimer's disease, and different types of cancers. This next generation of therapeutic products will be better at targeting cancerous cells as exemplified by the exponential growth in Antibody-Drug Conjugates (ADCs) and they will have dual targeting properties, as exemplified by the growing body of research in bi- and multi-specific antibodies, immunotherapies, and VLP vaccines. Flow electroporation will be an integral part of the growing reliance therapeutic antibodies, since it provides the best transfection efficiency and cell viability and accelerates the process of biotherapeutic development by providing higher titers of protein and significant savings in time, cost, and resources.

If you had one sentence to express the implications of this paper, how would you explain it?

It is possible that we will never face a vaccine shortage like we did in 2009-2010 with the H1N1 flu outbreak.

To explain, large-scale cell culture-based vaccine has many advantages over egg-based production. It provides a reduction in lead time, greater reliability, and greater flexibility. The egg-based method requires 5-6 months for the production of enough eggs and then another 9-10 months for the production of the final vaccine. With the cell-based method, the 5-6 months needed to establish the egg supply is eliminated as well as a portion of the 9-10 months since there would be no need to adapt the virus to grow in eggs. Using the cell-based method also provides a more reliable source for vaccine production. While eggs are perishable, cell lines can be established and cryopreserved. Furthermore, there is growing concern over the increase in avian influenza (H5N1). This is real threat to the supply of chickens and embryonic egg used in vaccine production. A cell-based method for vaccine production truly means we may never face a shortage again.

What’s one implication from the findings that might be obvious to most people?

No one wants to be faced with a vaccine shortage. We need to be able to quickly produce vaccines at the quantity needed. MaxCyte electroporation is an important step in that direction. With its ability to quickly scale-up to the necessary quantities much faster than using stable cell lines. 

Access exclusive speaker interviews from leaders in cell line development. Download the Cell Line Engineering & Development conference brochure here to check them out.  

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IBC’s Cell Line Development & Engineering Conference: Leverage Cutting-edge Technical Innovation in Bioproduct Development

Join us for IBC’s 2014 Cell Line Engineering & Development conference—Now celebrating its 10^th anniversary.  This year’s program is aimed to help you leverage cutting-edge technical and process innovation in order to improve bioproduct development. 

What can you expect from this year’s edition?  How about 40 case studies and presentations of new data to help you:

• - Implement new flexible, automated platforms with integrated data management to improve predictability
• - Utilize synthetic biology principles to create new and better bioproduction lines
• - Integrate cell line and process development to dramatically increase production for a difficult to express protein
• - Develop biosimilars with novel techniques to control cell line and product quality
• - Reduce risk and build success for process scale-up of PER.C6 processes
• - Apply In silico models of metabolism and protein secretion that provide insights to guide cell engineering efforts
• - Isolate clones with desired properties to assure stability and clonality through regulated-expression using flow cytometry
• - Use directed evolution to optimize the cell line development process and identify cell lines with desired characteristics

Download the brochure to check out the complete agenda

Keynote Presentations

Accelerated Path to Probe the Biology through Deferred Cloning and Applying Platform Manufacturing Processes
Rohini Deshpande, Ph.D., Executive Director, Drug Substance Development, Amgen, Inc

The Genome as an Enabler of Product Attribute Control
Kelvin Lee, Ph.D., Gore Professor of Chemical Engineering, Delaware Biotechnology Institute Faculty Fellow, University of Delaware

Design of Next Generation CHO Cell Factories
David James, Ph.D., Professor of Bioprocess Engineering, Department of Chemical and Process Engineering, University of Sheffield, United Kingdom

Access our exclusive speaker interviews when you download the brochure today.

Book your trip now to join us in Berkeley, California from September 8-10. Save 20% off the standard rate when you register here and use code XB14189BLOG.

Questions? Comments? Want to get involved? Email me at mmadarasz@iirusa.com.

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Removing Bottlenecks in Cell Line Engineering

There are several known bottlenecks in generating mammalian cell clones to produce therapeutic proteins.  Dr. Nic Mermod, Director of the Institute of Biotechnology at the University of Lausanne, cites biological issues, such as transcription levels, as well as process issues, including the cloning and maintenance of many cells, as causes of these bottlenecks.  Dr. Mermod, who will be speaking at this year’s Cell Line Development & Engineering conference, has several ideas on removing said bottlenecks.  Some thoughts from this leader in the field:

What are the current approaches when removing such bottlenecks?

Dr. Mermod: The first bottleneck is transcription of the transgene. We have been working on the element called MAR, which can remove some of the negative effects of integration of the transgene into a non-favorable genetic environment. And other people are also working on other elements. That – for the MAR elements –allows to, pretty much, dampen some silencing effects and also to prevent oscillation of transcription, whereby you get the transgene active at all times within all cells.

So, that’s one way to alleviate low levels of transcription. Another way, of course, is to basically increase transgene copy number. That is to work on the recombination mechanism to allow your CHO cells to more efficiently integrate transgene, if you want to further boost the level up.

And then for basic protein metabolism and secretion, there are efforts by a number of laboratories to perform genomic engineering of the CHO cell. For instance, we have reengineered the human secretion machinery in CHO-M cells which allows to increase protein secretion of easy or difficult to express proteins; both of them. So, that’s basically the current approach that we and others are taking.

To check out our full interview with Dr. Nic Mermod, download the Cell Line Development and Engineering brochure.

We’ll have more from Nic at this year’s Cell Line Development & Engineering conference.  Join us September 8-10 in Berkeley, CA.  Save 20%* off the standard rate as a reader of this blog when you register here and use discount code XB14189BLOG.   

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Timeline Reduction in Cell Line Development

What are the most important attributes in developing recombinant cell lines?  For Alison Porter, Head of Mammalian Cell Culture R&D for FUJIFILM Diosynth Biotechnology, the list includes high titer, growth properties that are suitable for a manufacturing process and production of product with the correct characteristics.  The importance of these qualities can’t be overstated, however, achieving them in an efficient way is a priority as well.   We went ahead and got Alison’s take on how to improve some of these characteristics with a reduced timeline in mind.  Check out what she had to say:

What can you target if you want to reduce timelines whilst improving some of these desirable attributes?

Alison: To answer this, I’m going to concentrate on the idea of improving titer whilst reducing timelines. Probably, increasing titer is the most common goal when looking to improve desirable attributes further. When people are thinking about increasing titer over the past 20 to 25 years, the obvious and main contributor that comes to mind is, of course, design of media and feeds in particular.

But there are other contributors from the cell line development stage that can play a role in improving titer whilst reducing timelines. The host cell line, vector and the cell line development process itself. If, for example, we think about the cell line development process, introduction of improved screens can allow earlier selection of the best cell lines. By improved screens, I’m thinking about things like multi-well plate systems, which are operated in suspension mode and have feeds applied, and micro bioreactor systems. So, you hopefully end up using these as being both better at identifying cell lines capable of high expression and identifying them earlier on.

September 8-10, Berkeley, CA

To think about host cell lines, work can be done to develop new host cell lines with increased expression capability and you can also improve them with regards to growth characteristics - and that will also potentially help reduce timelines.

At FUJIFILM, we’ve actually been looking at all three of these areas recently – host cell lines, vector and cell line development process. Looking to further improve our cell line development capabilities.

To check out the full interview with Alison Porter, download the Cell Line Development and Engineering brochure.

We’ll have more from Alison at this year’s Cell Line Development & Engineering conference.  Join us September 8-10 in Berkeley, CA.  Save 20%* off the standard rate when you register here and use discount code XB14189BLOG.   

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MicroRNAs: Should Researchers Be Paying Attention?

According to Niall Barron, it’s estimated that somewhere between 50 and 70% of all protein encoding genes are under the control of MicroRNAs themselves.  That said, MicroRNAs are an important part of cell line development going forward.  Dr. Barron, who will be presenting on the topic at this year’s Cell Line Development and Engineering conference, believes these molecules should have researcher’s attention.  We were recently able to pick his brain and get some of his thoughts as to exactly why that is:  

Should biopharma companies and researchers be interested in MicroRNAs?

Dr. Barron: As I say, harkening back to this characteristic – which is their ability to influence the expression of lots of different proteins – one of the challenges in the past for engineering CHO cells is that we have to do it gene by gene. This is not proven to be particularly useful, although there has been some success in that area. So, with the ability to over-express or down-regulate individual microRNA, that opens up the possibility of changing the expression of multiple downstream genes or proteins. This really allows us to start thinking of about ways of engineering entire biological pathways within cells.

One of the other interesting aspects is that these molecules are not translated into proteins themselves. Therefore, as we know CHO cells, what we’re using them for is to over-express our product. Therefore, engineering something in there that will compete without over-expression is likely to be detrimental to efficiency. So, by using a microRNA as a tool, you reduce the translational burdens that you place upon the cell. We would hope that by doing that you are less likely to negatively impact on the expression of your product.

Finally, genetic engineering of CHO cells typically would involve a full submission to the regulatory authorities. Stably expressing CHO cells to up or down-regulate a particular microRNA would require that also, but there is the possibility of transiently transfecting them into an existing CHO cell line within the bioreactor, which would just be a process-related change in the same way that adding a feed of nutrients or something to a bioreactor is not considered a new cell line.

Download the brochure to check out Dr. Barron’s full interview.

Get the latest from Dr. Barron and other industry experts at this year’s Cell Line Development and Engineering conference, September 8-10, Berkeley, CA.  Now, SAVE 20% off the standard rate.  Register here and use code XB14189BLOG.

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