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 Culture Media: Balancing Resources with Results

Why does medium matter?

It all starts with media. Without an appropriate growth medium, no biopharmaceutical process would exist. The proper medium allows for cells to grow and generate product. Going beyond the fact that cell culture processes would simply not exist without cell culture media, the medium (and associated feeds for fed-batch processes) represents a large portion of the cost of a biopharmaceutical process. Cell culture media are complex entities that encompass large quantities of raw materials as well as a high level of technical expertise, driving up the cost per litre.

What are the advantages of an optimized medium formulation compared to an off the shelf product? 

Many people will choose to start with standard off the shelf products for their cell culture processes. While there are a wide variety of products available on the market, there are several important reasons to consider using an optimized medium formulation instead. The first is to tailor a medium formulation specifically for your own cell line. Every cell line will respond differently to standard products, and optimizing a medium formulation is the best way to ensure that you use a medium that works best for your application and requirements. Additionally, as you generate different cell lines with varying genetic constructs for new products, your cells may begin to respond differently. Having an optimized medium formulation allows you to adapt to these changes while maintaining growth and productivity. Using a standard product also means you are bound to a single vendor with no access to proprietary formulations. With your own optimized medium, you have full access to the formulation, with the ability to manufacture it with a vendor of your choice.

What factors should I consider when developing an optimized medium?

As discussed previously, optimizing a medium formulation to get you the best growth and productivity is the main driving factor, and allows you to minimize the amount of medium used and therefore your overall COGS. It is important to also consider what the criteria are for your optimized medium. For many industries, having a fully chemically defined, non-animal origin medium is critical. This minimizes lot to lot variability and is highly desirable from a regulatory standpoint. Ease of use is also important to consider. This encompasses storage aspects, such as shelf life, and shipping and handling conditions. Ease of use also involves simplifying the process for the operator. As media and feeds become increasingly complex, how difficult does it become to formulate? Will operators have to perform risky pH adjustments with large volumes of corrosive reagents? Will high temperatures be needed to dissolve certain components, and can these high temperatures be achieved as the industry moves towards more single-use technologies? These and other factors must be considered when developing an optimized medium formulation.

What resources are required to develop an optimized medium?

The actual raw materials that form the bulk of a cell culture medium are generally inexpensive. The resources required for an optimized medium formulation that drive up the ultimate cost per liter are time, personnel, equipment, and expertise. With the increasing use of multivariate approaches (Design of Experiments, Principal Component Analysis), the use of high throughput systems become highly advantageous, reducing personnel requirements. Finally, expertise in media development is something that comes only with years of experience in the industry and cannot be bought with any amount of money.

So how do I develop an optimized medium formulation?

There are several approaches to media optimization. Some methods include titration, reverse engineering of other media, and metabolomics. While some of these methods are effective, when implemented on their own, they are time consuming, labor intensive, and heavily resource-dependent. A modern, DoE based approach to media development for CHO based processes is the CHOptimizer® Media Builder. CHOptimizer® consists of three distinct phases. The package is designed to be integrated into our ambr™15 system for automation and ease of use. In the first phase, four chemically defined, non-animal origin based media are blended in different ratios, according to a mixtures design DoE approach. Subsequent phases incorporate spent media analysis and fractional factorial DoE approaches to develop an optimized medium formulation, feed formulation, and corresponding feeding strategy. CHOptimizer® base media are developed using the expertise of Lonza Biologics, and combines a modern, high throughput approach with traditional ideas to deliver an optimized medium in a short timeline, with field based support. It also offers full access to the formulation for ultimate flexibility in the future.

To learn more about CHO media optimization join Sartorius Stedim Biotech’s workshop, chaired by Dr. Michael Gillmeister, Lonza at the IBC Cell Line Development & Engineering Conference, San Francisco, 13th June at 11.45am.

About the author:

Dr. Michael Gillmeister received his Ph.D. in Chemical and Biomolecular Engineering from the Johns Hopkins University in collaboration with the University of Maryland School of Medicine specializing in glycosylation, transient protein production, and neurobiology. In 2009, Mike joined the Gibco® research and development group and was responsible for next-generation media and sera projects. He then led media and process development projects to modulate product quality and maximize titer for PD-Direct® Custom Media Services using high-throughput and bioreactor technologies. Currently, Mike leads Protein Expression Media R&D and the CHOptimizer™ media optimization service for Lonza Walkersville.

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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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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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2013 BPI Conference Pre-Event Podcast Series: Upstream Processing

We’d like to share with you 2013 BPI Conference Pre-Event Podcast Series! This edition is Upstream Processing and previews what you will experience while attending the 2013 BioProcess International Conference & Exhibition this coming September.

Each of these podcast speakers will be joining us this September 16-19, 2013 in Boston for the BioProcess International Event. Featured Podcasts in this month’s upstream series are:

• The BioProcess International Conference & Exhibition: 10 Years of Industry Excellence
• By Barry Walsh and Michael Keenan, Conference Directors, IBC Life Sciences
• Apoptosis of CHO Cells Monitored by a Novel Dielectrophoretic (DEP) Cytometer
• By Michael Butler, PhD, Professor of Microbiology, University of Manitoba
• Challenges to the Entry of New Biologics into the Market
• By Joanne T. Beck, Ph.D., Vice President, Process Development, Shire Human Genetic Therapies
• Investigation and Reduction of Performance Variability in Single-Use Cell Culture Bioreactors
• By Rajesh Krishnan, PhD, Associate Director, Cell Line & Upstream Process Development, Gilead Sciences
• Fast Identification of Reliable Hosts for Targeted Cell Line Development
• By Yongping Crawford, PhD, Scientist, Early Stage Cell Culture, Genentech

To listen to each of these podcasts, visit the official webpage of the podcast series. 

Want to join these speakers and more at BioProcess International?  As a reader of this blog, we’re offering you a save 20% savings off the standard conference rates when you register to join us and mention priority code BLOG13JP when you register!*

Have any questions about the event or the program or want to get involved? Contact me jpereira@iirusa.com.

*This discount only applies to new registrations.

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