THE RNA PLATFORM A REVOLUTION IN THERAPEUTICS

Pharmaceutical

Paul-Philippe Champagne, PhD, PEng Senior Process Engineer Divyang Patel CQV and Compliance Specialist The RNA platform is a revolution in therapeutics

The recent coronavirus pandemic has revolutionized medical innovation. + The rapid growth in requirement for therapeutics, including vaccines, 30 has brought the RNA platform to the forefront. However, the relevant literature is focused either exclusively on the molecular biology of RNA or on the development of RNA products. As such, we provide you with YEARS an accessible introduction on this revolutionary technology that bridges OF EXPERIENCE IN THE the gap between the science and its application. We begin by giving you PHARMA an overview of RNA and how biopharmaceutical companies are using BIOTECH & this novel technology in a wealth of diverse pharmaceutical products. medical device We also discuss key processes for the development and manufacture of RNA therapies, with mRNA vaccines as a case study. Lastly, we MARKET highlight the potential of this technology and discuss facility delivery.

Messenger ribonucleic acid (mRNA) is a naturally occurring A plethora of RNA products molecule in the cell. It is primarily used as an intermediate in the transfer of genetic information encoded in deoxyribonucleic acid Owing to the versatile of the RNA platform, it is being used (DNA) to produce proteins. DNA is frst transcribed into mRNA in to develop vaccines for infuenza, coronavirus, and other infectious the nucleus of the eukaryotic cell, and then the mRNA is translated diseases (Jackson et al., 2020; Pardi et al., 2018). mRNA vaccines into proteins in the cell’s cytoplasm. are also in progress for human (Pardi et al., 2018). There are numerous other therapeutics in the pipeline that use some mRNA technology allows for the safe delivery of an intact sequence facet of the RNA platform, and it is likely more therapies will be of interest to the host cell (Hoecke & Roose, 2019; Jackson et al., approved. For example, mRNA is currently being explored to 2020; Pardi et al., 2018). The sequence can code for an antigen, a express mAbs (Hoecke & Roose, 2019). These molecules would monoclonal (mAb), or any other therapeutic target. The then trigger the immune response in the presence of their platform is revolutionary because modifed mRNA is more stable respective antigens. Likewise, the mRNA platform is being used for than native mRNA and is not immunogenic, thus reducing the the development of single-stranded antisense oligonucleotides immune response and improving the effciency. In (ASOs) (Deweerdt, 2019). ASOs bind to their complementary target addition, the formulation and encapsulation of the mRNA is critical mRNA sequence and can obstruct the splicing process, hinder 5’ to the technology since it allows the molecule to be delivered intact cap formation, mark the mRNA for destruction, and block to target cells. The mRNA is typically encapsulated in a lipid translation by the ribosome with steric obstruction. Similarly, nanoparticle, which further limits the immune response that would mRNA technology can be utilized to block translation of the mRNA trigger its destruction by the immune system. Crucially, the mRNA of interest through RNA interference (RNAi) (Deweerdt, 2019). sequence does not penetrate the cell nucleus and does not insert RNAi can occur through and small interfering itself into the . Therefore, there is no genetic modifcation. (siRNAs), which can tag the mRNA for degradation and inhibit These features have allowed the rapid development of current protein translation in a manner akin to ASOs. In addition, mRNA can mRNA vaccines for the SARS-Cov-2 coronavirus pandemic. Indeed, also be employed to modulate the function of a target protein via responding to the threat of emerging coronavirus variants, mRNA the expression of RNA (Deweerdt, 2019). Table 1 vaccine manufacturers have been able to adjust their existing illustrates a sample of RNA therapies. mRNA sequences to develop new vaccine candidates.

PHARMACEUTICAL / / THE RNA PLATFORM / / 2 Therapy Disease Generic/ Target Organization(s) Status Approval Type Type Brand

Breast BioNTech Ongoing N/A N/A

Cancer Melanoma Radboud University Ongoing N/A N/A

Prostate cancer Oslo University Hospital Ongoing N/A N/A

HIV Erasmus Medical Centre Ongoing N/A N/A

Infuenza Ongoing N/A N/A Vaccine mRNA-1273/ EMA/FDA/ COVID-19 Infectious Moderna Completed HPFB/MHRA Vaccine SARS-Cov-2 Moderna

EMA/FDA/ Tozinameran/ Pfzer-BioNTech Completed HPFB/MHRA Comirnaty

Akcea Therapeutics-Ionis EMA/FDA/ Inotersen/ Autoimmune Amyloidosis Completed Pharmaceuticals MHRA Tegsedi

Duchenne muscular Eteplirsen/ ASO Sarepta Therapeutics Completed FDA dystrophy Exondys 51 Genetic Ionis EMA/FDA/ Nusinersen/ Spinal muscular atrophy Completed Pharmaceuticals-Biogen HPFB/MHRA Spinraza

BioNTech-Johannes Cancer Lymphoblastic leukemia Ongoing N/A N/A Gutenberg University

Georgia Institute of mAb Infectious RSV Technology-Emory Ongoing N/A N/A University

CureVac-Acuitas Toxin Botulism Therapeutics-Tufts Ongoing N/A N/A University

RNA Neuro- / AMD Astellas Pharma Completed EMA/FDA/HPFB degenerative Macugen

Alnylam Patisiran/ siRNA Autoimmune Amyloidosis Completed EMA/FDA Pharmaceuticals Onpattro

Table 1: Overview of the array of RNA therapeutics.

Table Legend: AMD, age-related ; ASO, antisense oligonucleotides; EMA, European Medicines Agency; FDA, Food and Drug Administration of the United States; HIV, human immunodefciency virus; HPFB, Health Products and Food Branch of Health Canada; mAB, monoclonal antibody; MHRA, Medicines and Healthcare products Regulatory Agency of the United Kingdom; mRNA, messenger ribonucleic acid; RSV, respiratory syncytial virus; SARS-Cov-2, severe acute respiratory syndrome coronavirus 2 (COVID-19); siRNA, small interfering ribonucleic acid. Source: Deweerdt, 2019; Hoecke & Roose, 2019; Jackson et al., 2020; Pardi et al., 2018.

PHARMACEUTICAL / / THE RNA PLATFORM / / 3 A PRIMER ON THE mRNA MANUFACTURING PROCESS

Generation of the DNA template (or transcription template) The mRNA transcript manufacturing process begins with the synthesis of the template DNA (pDNA). Molecular biologists design the DNA template which includes the genetic sequence of interest that encodes for either a candidate antigen or any therapeutic protein. Once designed and produced in small quantities, the pDNA is thereafter introduced into a bacterial host (usually ) for its amplifcation through cell expansion and plasmid replication. pDNA amplifcation begins in the recombinant bacterial shake fask culture and is then scaled up to a microbial fermenter. At the end of the cultivation period, the pDNA is extracted from the bacterial culture and is purifed. Typically, the harvested cells are lysed using a cell homogenizer and the pDNA is subsequently recovered from the clarifed lysate. A continuous centrifuge or a microfltration system is normally used for the clarifcation step. The pDNA is further purifed by chromatography to remove host cells and endotoxins, and flter sterilized. A diafltration step may be added to exchange the buffer for in vitro mRNA transcription. The production of pDNA can be outsourced or implemented in-house by any biopharmaceutical organization depending on the DNA requirements and scale of production.

In vitro transcription of mRNA Once generated, the purifed pDNA is enzymatically linearized and added to a mixture of enzymes and nucleotides (the RNA building blocks) for the synthesis of mRNA by the enzyme RNA polymerase. This enzyme transcribes the target genetic sequence of the linearized pDNA into mRNA. After mRNA generation is complete, the template DNA is enzymatically digested with a deoxyribonuclease (DNase), and the bulk mRNA is purifed via a series of chromatography steps. The mRNA transcripts are subsequently capped on their 5’ end and then further purifed. Active research and development efforts are underway to combine the mRNA transcription and 5’-capping into one step. The capped mRNA is further purifed by chromatography, diafltered for buffer exchange, and sterile fltered for the subsequent formulation. The purity of the mRNA transcript is the key to minimizing risks of side effects and/or lack of effcacy after vaccine administration.

Formulation of the mRNA transcript (drug substance) Various technologies for the formulation of mRNA vaccines exist, with microfuidic encapsulation with the customary choice. We recommend this technology, compared to others such as sonication and extrusion, since it has the advantage of consistently achieving greater control over the size and favoring higher encapsulation effciencies of the drug substance. The mRNA transcript in an aqueous buffer and the lipids dissolved typically in ethanol are homogenized by microjet impingement mixing. During the mixing process, the lipids and mRNA molecules spontaneously self- assemble into loaded liposomes. The lipid types are carefully selected and buffer conditions optimized for the effcient encapsulation of the mRNA and for the correct liposome self-assembly (Buschmann et al., 2021; Kis et al., 2020). We believe, and experts agree, that this step may be one of the major bottlenecks of the manufacturing process as technologies allowing the scale up of this formulation are scarce relative to the current unprecedented demand (Lowe, 2021). The fnal drug product, the encapsulated and capped mRNA transcript, is diafltered and sterile fltered for vial flling.

Lipid Cholesterol PEG-lipid mRNA

PHARMACEUTICAL / / THE RNA PLATFORM / / 4 Final Packaging and Quality Considerations Fill, fnish, and packing are the last steps of the drug manufacturing process. Vials are automatically flled with the mRNA drug product solution under strictly controlled sterile and aseptic environments. The execution of the mRNA manufacturing process must be stringently controlled to ensure quality assurance and fulfllment of product specifcations. Quality control testing must be performed at appropriate steps depending on pre-determined critical drug intermediate specifcations. Several quality control tests may be employed, and their use depends on the target mRNA treatment. Our compliance, quality, and regulatory teams can provide additional guidance as per client specifcations. We suggest in-process control testing to include capping effciency, residual plasmid, ribonuclease (RNase) and DNase contamination, and concentration of the product. We further propose quality control testing parameters for batch release to include identity, pH, purity (via UV A260/A280 ratio), and endotoxin levels.

The current mRNA vaccine supply chain and material fow. Controlled manufacturing areas in a vaccine production facility must provide environmental conditions to The unprecedented demand for COVID-19 vaccines combined with ensure process and product integrity against contamination and compressed delivery times have put tremendous pressure on cross-contamination when multiple production lines are in biomanufacturing capacity and on the vaccine supply chain. operation. Segregation and containment of manufacturing pro- Although critical steps such as mRNA formulation are implemented cesses will be key drivers of equipment and facility design to in-house by vaccine manufacturers, specialty chemicals like lipids support production fexibility. and 5’-capping agents are produced for the most part by third party manufacturers across several countries. mRNA vaccine producers needed to assemble a manufacturing supply chain in a highly As such, we foresee that biopharmaceutical compressed schedule to allow rapid deployment of vaccine manufacturers will increasingly use modular production capacities to fulfll the urgent deliveries to save lives. systems to add and replace production lines For example, current manufacturers were creative in setting up their vaccine manufacturing supply chains at their existing as required to adjust to demands during production facilities as there was insuffcient time to build new the next decade. Facilities will need dedicated manufacturing facilities. As such, the current mRNA to be designed in such a way that future manufacturing supply chain is spread over multiple sites across different geographies. production capacity expansions will not interfere with existing and ongoing Evolution of the biopharmaceutical manufacturing operations. facility design

We believe that reducing the situational complexity of the current Plant digitalization will also become essential to ensure fexible mRNA manufacturing supply chain by regrouping the manufacturing and agile manufacturing in a multi-purpose RNA biomanufacturing of the mRNA and the specialty chemicals ‘under one roof’ will facility. We recommend to our clients that the implementation of improve productivity and will become a growing trend. As plant digitalization be supported by a network of high-resolution personalized medicines accounted for 33% of all new drugs process analytical technologies. These will be crucial to ensure approved from 2016 to 2020 (Brooks, 2021), RNA technology has accurate and real time visibility of building and manufacturing the potential to be a strong enabler of innovative and personalized processes. The potential quality improvement of real time process therapies. The growing needs for these types of therapeutic information will allow better and timely process troubleshooting treatments, as refected in Table 1, will drive the growth of drug and interventions as required. In addition, a growing number of candidates and licensed product portfolios of biopharmaceutical biopharmaceutical manufacturers are recognizing the value of organizations globally for the next few years. plant digitalization for reducing the incidence of quality deviations and for securing their supply chains. Models derived from collected Therefore, we anticipate that manufacturing facilities will need to process data (e.g. Digital Twins) can be used to identify potential be fexible to accommodate a diversity of RNA-based drugs (or issues or improvement opportunities for supply chain debottle- drug candidates) produced for clinical trials or commercialization. necking (Demesmaeker et al., 2021). Facility and layout designs are centered around process, personnel,

PHARMACEUTICAL / / THE RNA PLATFORM / / 5 FACILITY DESIGN, COMPLIANCE, QUALITY, AND REGULATORY STANDARDS

PROGRAM 5 MANAGEMENT

6 DESIGN AND ENGINEERING BUILD AND 7 QUALIFICATION

8 FACILITY DELIVERY 4 CALCULATING RETURN ON INVESTMENT 11 DISTRIBUTION

APPROPRIATE FACILITY 3 LOCATION AND TYPE 1 GOVERNMENT/REGULATORY COMPLIANCE

OPERATIONS & 2 IMPROVED VISUALIZATION 10 AND DATA INTEGRATION MAINTENANCE

9 PRODUCTION

SNC-Lavalin has designed and built many biopharmaceutical RNA technology is a gamechanger for the biopharmaceutical facilities and houses a large multidisciplinary team with all the industry. The ability for rapid formulation and modifcation of expertise needed to deliver, manage, and execute a bio- mRNA relative to traditional therapeutic development platforms manufacturing facility that will support its clients in bringing life - will enable its adoption and further refnement by the feld. With savings drugs and therapies to patients. Our team of process and a wide range of technical and project management services, we validation experts deliver projects with an end-to-end mindset from provide support for all phases of the pharmaceutical product conceptualization, through feasibility studies, preliminary and lifecycle. SNC-Lavalin is thus unique in offering full spectrum detailed engineering designs, execution, commissioning, technology services and is well positioned to serve clients in the bio- transfer, and validation to commercial production. Our biomanu- pharmaceutical sector having RNA as its technology platform for facturing expertise includes in-process design and improvement, innovative and personalized therapies. with specialty areas in upstream and downstream processes, formulation, and adjuvant development. We provide preliminary process modelling and simulation services to support process scale-up, layout, and cleanroom design. As part of the full range of our service portfolio, our process engineering, automation, and validation teams work together early in any project to provide validation forecasting to ensure that our biomanufacturing facility and its processes that we design can be validated. Our team provides the entire spectrum of commissioning, qualifcation, and validation (CQV) services for all types of process equipment, single use technologies, laboratory instruments, and mechanical systems.

Our comprehensive quality and compliance services, and SNC - Lavalin’s global presence, ensures that your facility and products meet all applicable international and regional regulatory standards. This mitigates risk for our clients and leads to smooth processes of technology transfer, regulatory approvals, and production ramp up of the facility. Pharmaceutical

PHARMACEUTICAL / / THE RNA PLATFORM / / 6 References

Brooks K. Pharmaceutical Manufacturing Equipment Trends. Contract Pharma (2021). https://www.contractpharma.com/issues/2021-03-01/view_features/pharmaceutical-manufacturing-equipment-trends/

Buschmann M.D., Carrasco M.J., Alishetty S., et al. Nanomaterial Delivery Systems for mRNA vaccines. Vaccines 9, 65-95 (2021). https://doi.org/10.3390/vaccines9010065

Demesmaeker M., Kopec D., Arsenio A.M. Bioprocessing 4.0 – Where Are we with Smart Manufacturing in 2020? Pharmaceutical Outsourcing (2020). https://www.pharmoutsourcing.com/Featured-Articles/568001-Bioprocessing-4-0-Where-Are-We-with-Smart-Manufacturing-in-2020/

DeWeerdt S. RNA therapies explained. Nat 574, S2-S3 (2019). https://doi.org/10.1038/d41586-019-03068-4

Jackson N.A.C., Kester K.E., Casimiro D. et al. The promise of mRNA vaccines: a biotech and industrial perspective. npj Vaccines 5, 11 (2020). https://doi.org/10.1038/s41541-020-0159-8

Kis Z., Kontoravdi C., Dey A.K., et al. Rapid development and deployment of high-volume vaccines for pandemic response. J Adv Manuf Process 2, e10060 (2020). https://doi.org/10.1002/amp2.10060

Lowe D. Myths of Vaccine Manufacturing. Sci Transl Med 13, 579 (2021). https://blogs.sciencemag.org/pipeline/archives/2021/02/02/myths-of-vaccine-manufacturing

Pardi, N., Hogan, M., Porter, F. et al. mRNA vaccines — a new era in vaccinology. Nat Rev Drug Discov 17, 261–279 (2018). https://doi.org/10.1038/nrd.2017.243

Van Hoecke, L., Roose, K. How mRNA therapeutics are entering the monoclonal antibody feld. J Transl Med 17, 54 (2019). https://doi.org/10.1186/s12967-019-1804-8

Authors

Paul-Philippe Champagne Divyang Patel PhD, PEng Senior Process Engineer CQV and Compliance Specialist HEAD OFFICE Pharmaceutical 455 René-Lévesque Blvd. West Montreal, QC, H2Z 1Z3, Canada Telephone: +1 514 393-1000

#BeyondEngineering www.snclavalin.com