European Pharmaceutical Review explores how plants can be used for large-scale, glycosylated protein bioproduction for the pharma industry.
Plants can be used to produce large quantities of complex proteins, particularly glycosylated proteins, which are becoming more widely used in a range of therapies. Monoclonal antibodies (mAbs) are among the types of glycosylated proteins that plants can produce, but while there are multiple benefits to their use as ‘bioreactors’, there are also some key considerations.
This article explores why and how plants can be used to produce proteins for use in therapies, but also the factors that show this method may not be applicable to all protein products.
How can plants be used to produce proteins?
For plants to produce synthetic proteins, they must first be expressed somewhere within their genome. This requires some form of recombinant protein expression or genetic engineering, and to achieve optimum yield just implanting the gene is insufficient. To achieve a high level of transcription, which allows for downstream translation and protein modification for stability, the regulatory gene elements – including the promoter and polyadenylation site – must also be expressed.1
This report addresses the key factors shaping pharmaceutical formulation, including regulation, QC and analysis.
Access the full report now to discover the techniques, tools and innovations that are transforming pharmaceutical formulation, and learn how to position your organisation for long-term success.
What you’ll discover:
Key trends shaping the pharmaceutical formulation sector
Innovations leading progress in pharmaceutical formulation and how senior professionals can harness their benefits
Considerations and best practices when utilising QbD during formulation of oral solid dosage forms
There are three commonly used types of expression mechanisms for plant bioproduction: nuclear, chloroplast and transient expression.
Nuclear expression involves genetically modifying the genome in the nuclei of plants cells to express a protein. This is the simplest and most widely used approach in the pharmaceutical industry, as it can be achieved with viral vectors, but a more modern technique is CRISPR-Cas9 technologies.1 A 2018 study showed that in cotton, CRISPR showed no off‐target editing and an editing efficiency of 66.7 to 100 percent at each of multiple sites.2 The nuclear expression techniques, although reliable, are becoming less popular as they typically require more time to develop.
The second method involves expression of a recombinant protein in the chloroplasts requires a particle gun to insert the transgene. There are several benefits to this technique, including the ease of manipulating the chloroplast genome compared with the nucleus and the number of chloroplasts per cell, which increases yield. Using a transgene cassette to precisely target and insert the foreign gene avoids placing it into a poorly transcribed part of the genome, ensuring a high level of expression and little chance of silencing. Transgenes are commonly integrated between the trnl‐trnA genes in the rrn operon, as this is a transcriptionally active region offering high levels of gene expression.1
The third mechanism, transient expression, is becoming more common as it allows the rapid insertion of proteins, with little time required for the production, modification and optimisation of the expression system. Some companies have begun marketing this kind of expression for the rapid, large-scale production of proteins for therapeutics. The Agrobacterium‐mediated transient expression technique is purported to have better efficiency than the integrated gene systems and the ability to reach a high percentage of cells in a treated tissue, resulting in higher yields.1
Why use plants as bioreactors?
Molecule size
In prokaryotic cells, like Escherichia coli (E. coli), protein size is limited to less than 30 kilodaltons, mainly due to reliability of production and yield. However, in eukaryotic cells, eg, Chinese hamster ovary (CHO) and plant cells, it is easier to produce larger proteins with high yields.1
Scalability
According to experts, when using cell line or bacterial production methods – such as CHO cells and E. coli – to produce proteins, once the initial cell line is created it is often difficult to scale up, as glycosylation profiles become variable.3 The inconsistencies in protein product both cost money and result in waste.
On the other hand, dependent on expression mechanisms, plants can reliably maintain the glycosylation profile required even as bioreactor volume increases.
Speed
As a result of consistent production capabilities, plants do not require scale-up protocols. This saves both time and money when setting up a bioreactor.
A further advantage is that, if the plant is made to generate the protein through a transient expression system, there is very little time required to set up a production system. One company claims their tobacco plant-based system can be tailored for large-scale fabrication of a protein product in under 12 months, compared to 20-22 months with CHO or E. coli, 3 and one study suggests this could be done in a matter of weeks.1
Adaptability
There are multiple options for plant expression systems, particularly with regards to species, and each is best suited to produce different proteins. Genetic engineering can also be employed to allow customised N-glycosylation to generate different target products.
Cost
The plant industry is well established, with conditions for growth often being less complex than that of cell lines or bacteria and, dependent on choice of plant species, cultivation costs can be further reduced.
A techno-economic analysis of the theoretical set-up of a new large-scale biomanufacturing facility, producing mAbs using tobacco plants, found that compared to CHO production platforms, the plant system resulted in significantly reduced capital investment. Moreover, the model calculated that there would be more than a 50 percent reduction in the cost of goods, compared with published values for similar products at this production scale.4
Biobetters
One company has paved the way for the creation of ‘biobetters’, using their FastGlycaneering Development Service™. iBio has shown that certain methods of plant bioproduction can improve the potency and homogeneity of biological medicines and ensure fully humanised glycosylation patterns.
Compatible with AI and blockchain
iBio have also stated that their system, due to its consistencies in upstream processing, is compatible with artificial intelligence (AI). The company aim to implement a new end-to-end manufacturing process using AI and blockchain to reduce costs through optimising both the process and workflows.3
Overcoming concerns
Some of the major challenges include regulatory approval, environmental contamination, protein stability and the immunogenicity of non-human post-translational modifications.1
Environmental concerns are predominantly from the possibility of spreading genetic modifications to food crops through pollination. This is more of a concern with the nuclear expression systems than transient or chloroplast expression. However, this can be overcome with geographical or physical containment, using a less transferable genetic modification method or through using a self-pollenating species.1
A review suggested that companies are unlikely to go through the cost of a shift from an already approved production system to seek regulatory approval for a new one.1 While altering an approved process is often unfeasible, setting up systems for the production of new products in the pipeline could prove to be more cost effective in the long run. Another consideration is the rising need for quick, large-scale vaccine production in response to pandemics and epidemics – such as the Covid-19 coronavirus and Ebola – which, due to the speed at which a transient expression production system can be constructed, could encourage companies to branch into this type of production.
Protein stability is a concern, as plants have endogenous enzymes that can break down the protein products. Some methods to overcome this include changing plant species and co-expressing peptides to fuse and stabilise the produced proteins together.
Post-translational modifications such as Asparagine-linked glycosylation (N-glycosylation) are one of the key worries, as they can be immunogenic. Particularly likely to cause unfavourable side effects are N-glycan modifications, because they differ in plants and humans.
What is N-glycosylation?
N-glycosylation is a post-translational modification conducted on many secreted or membrane proteins in plants and mammals. Endogenously, it enables protein folding, stabilisation and protein-protein interactions. It is similarly used in pharmaceutical bioproduction to stabilise products and provide antibodies and other proteins the correct pharmacokinetic properties and immunogenicity.5,6
The plant industry is well established, with conditions for growth often being less complex than that of cell lines or bacteria”
While early N-glycosylation and N-glycan modifications are highly conserved between yeast, mammals and plants, later N-glycan modifications differ; they are more simplified in plants than mammals.5,6 So, to use plants as producers of fully humanised proteins, the plant glycosylation machinery is often removed and replaced with human machinery when the plant is modified to express the protein. Of note, chloroplasts have no glycosylation machinery, so cannot perform these modifications without the insertion of foreign DNA; although this can reduce immunogenicity of the products, it can limit which proteins can be produced by chloroplast expression.
Production species:
Tobacco
Tobacco is the most widely used plant for production of recombinant proteins in the lab. High yield and rapid scale-up, due to large numbers of seeds produced, are the primary benefits. However, proteins stored in the leaves are vulnerable to degradation and must be stored or extracted appropriately, in a timely manner. Tobacco tissues can also contain phenols and toxic alkaloids that must be removed in downstream processing to make products safe.1
Cereals
Cereals are primarily used due to their seed protein storage capabilities; cereal seeds have protein storage vesicles and a dry intracellular environment. Once dried, the seeds can be stored at room temperature with limited degradation to protein products or loss of activity. Use of food crops is particularly attractive as they offer the opportunity to administer oral vaccines produced in the crop by feeding them to patients with minimal processing. Some edible vaccines have reached Phase I trials.1
Legumes
Peas are a particularly attractive option, as they have high protein content in their seeds – similar to cereals – and have lower nitrogen requirements, reducing cultivation costs. However, legumes usually have less leaf biomass than tobacco, meaning they require a larger area to produce the same quantity.1
Conclusion
Plants can be modified through several methods to express proteins and the requisite promoters and transcription controllers, for the production of therapeutic proteins. There are several important considerations, including protein expression methods and plant species; however, the many benefits, including reduced costs, adaptability and speed associated with plant bioproduction systems make them an attractive option.
A particular driver of this bioproduction process is the possibility of using transient expression to produce vast quantities of highly potent, fully humanised vaccines in response to pandemics and epidemics.
References:
Burnett, M. and Burnett, A. Therapeutic recombinant protein production in plants: Challenges and opportunities [Internet]. Plants, People, Planet. 28 November 2019. [Cited: 10 February 2020]. Available at: https://nph.onlinelibrary.wiley.com/doi/full/10.1002/ppp3.10073
Miki, D., Zhang, W., Zeng, W. et al. CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation [Internet]. Nature Communications. 17 May 2018. [Cited: 12 February 2020]. Available at: https://www.nature.com/articles/s41467-018-04416-0
Dr Morrow Jr., K. J. Building Therapeutic Proteins through Plant Glycosylation: A Sweet Solution [Internet]. Genetic Engineering and Biotechnology News. 4 February 2020. [Cited: 12 February 2020]. Available at: https://www.genengnews.com/topics/bioprocessing…
Nandi, S., Kwong, A., Holtz, R. et al. Techno-economic analysis of a transient plant-based platform for monoclonal antibody production [Internet]. August 2016. [Cited: 12 February 2020]. Available at: https://www.researchgate.net/publication/306928430…
This website uses cookies to enable, optimise and analyse site operations, as well as to provide personalised content and allow you to connect to social media. By clicking "I agree" you consent to the use of cookies for non-essential functions and the related processing of personal data. You can adjust your cookie and associated data processing preferences at any time via our "Cookie Settings". Please view our Cookie Policy to learn more about the use of cookies on our website.
This website uses cookies to improve your experience while you navigate through the website. Out of these cookies, the cookies that are categorised as ”Necessary” are stored on your browser as they are as essential for the working of basic functionalities of the website. For our other types of cookies “Advertising & Targeting”, “Analytics” and “Performance”, these help us analyse and understand how you use this website. These cookies will be stored in your browser only with your consent. You also have the option to opt-out of these different types of cookies. But opting out of some of these cookies may have an effect on your browsing experience. You can adjust the available sliders to ‘Enabled’ or ‘Disabled’, then click ‘Save and Accept’. View our Cookie Policy page.
Necessary cookies are absolutely essential for the website to function properly. This category only includes cookies that ensures basic functionalities and security features of the website. These cookies do not store any personal information.
Cookie
Description
cookielawinfo-checkbox-advertising-targeting
The cookie is set by GDPR cookie consent to record the user consent for the cookies in the category "Advertising & Targeting".
cookielawinfo-checkbox-analytics
This cookie is set by GDPR Cookie Consent WordPress Plugin. The cookie is used to remember the user consent for the cookies under the category "Analytics".
cookielawinfo-checkbox-necessary
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Necessary".
cookielawinfo-checkbox-performance
This cookie is set by GDPR Cookie Consent WordPress Plugin. The cookie is used to remember the user consent for the cookies under the category "Performance".
PHPSESSID
This cookie is native to PHP applications. The cookie is used to store and identify a users' unique session ID for the purpose of managing user session on the website. The cookie is a session cookies and is deleted when all the browser windows are closed.
viewed_cookie_policy
The cookie is set by the GDPR Cookie Consent plugin and is used to store whether or not user has consented to the use of cookies. It does not store any personal data.
zmember_logged
This session cookie is served by our membership/subscription system and controls whether you are able to see content which is only available to logged in users.
Performance cookies are includes cookies that deliver enhanced functionalities of the website, such as caching. These cookies do not store any personal information.
Cookie
Description
cf_ob_info
This cookie is set by Cloudflare content delivery network and, in conjunction with the cookie 'cf_use_ob', is used to determine whether it should continue serving “Always Online” until the cookie expires.
cf_use_ob
This cookie is set by Cloudflare content delivery network and is used to determine whether it should continue serving “Always Online” until the cookie expires.
free_subscription_only
This session cookie is served by our membership/subscription system and controls which types of content you are able to access.
ls_smartpush
This cookie is set by Litespeed Server and allows the server to store settings to help improve performance of the site.
one_signal_sdk_db
This cookie is set by OneSignal push notifications and is used for storing user preferences in connection with their notification permission status.
YSC
This cookie is set by Youtube and is used to track the views of embedded videos.
Analytics cookies collect information about your use of the content, and in combination with previously collected information, are used to measure, understand, and report on your usage of this website.
Cookie
Description
bcookie
This cookie is set by LinkedIn. The purpose of the cookie is to enable LinkedIn functionalities on the page.
GPS
This cookie is set by YouTube and registers a unique ID for tracking users based on their geographical location
lang
This cookie is set by LinkedIn and is used to store the language preferences of a user to serve up content in that stored language the next time user visit the website.
lidc
This cookie is set by LinkedIn and used for routing.
lissc
This cookie is set by LinkedIn share Buttons and ad tags.
vuid
We embed videos from our official Vimeo channel. When you press play, Vimeo will drop third party cookies to enable the video to play and to see how long a viewer has watched the video. This cookie does not track individuals.
wow.anonymousId
This cookie is set by Spotler and tracks an anonymous visitor ID.
wow.schedule
This cookie is set by Spotler and enables it to track the Load Balance Session Queue.
wow.session
This cookie is set by Spotler to track the Internet Information Services (IIS) session state.
wow.utmvalues
This cookie is set by Spotler and stores the UTM values for the session. UTM values are specific text strings that are appended to URLs that allow Communigator to track the URLs and the UTM values when they get clicked on.
_ga
This cookie is set by Google Analytics and is used to calculate visitor, session, campaign data and keep track of site usage for the site's analytics report. It stores information anonymously and assign a randomly generated number to identify unique visitors.
_gat
This cookies is set by Google Universal Analytics to throttle the request rate to limit the collection of data on high traffic sites.
_gid
This cookie is set by Google Analytics and is used to store information of how visitors use a website and helps in creating an analytics report of how the website is doing. The data collected including the number visitors, the source where they have come from, and the pages visited in an anonymous form.
Advertising and targeting cookies help us provide our visitors with relevant ads and marketing campaigns.
Cookie
Description
advanced_ads_browser_width
This cookie is set by Advanced Ads and measures the browser width.
advanced_ads_page_impressions
This cookie is set by Advanced Ads and measures the number of previous page impressions.
advanced_ads_pro_server_info
This cookie is set by Advanced Ads and sets geo-location, user role and user capabilities. It is used by cache busting in Advanced Ads Pro when the appropriate visitor conditions are used.
advanced_ads_pro_visitor_referrer
This cookie is set by Advanced Ads and sets the referrer URL.
bscookie
This cookie is a browser ID cookie set by LinkedIn share Buttons and ad tags.
IDE
This cookie is set by Google DoubleClick and stores information about how the user uses the website and any other advertisement before visiting the website. This is used to present users with ads that are relevant to them according to the user profile.
li_sugr
This cookie is set by LinkedIn and is used for tracking.
UserMatchHistory
This cookie is set by Linkedin and is used to track visitors on multiple websites, in order to present relevant advertisement based on the visitor's preferences.
VISITOR_INFO1_LIVE
This cookie is set by YouTube. Used to track the information of the embedded YouTube videos on a website.