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10 Pharmaceutical Technology ® Innovations in mRNA eBook April 2024 PharmTech.com Manufacturing well-known firms with candidates in the clinic. But it was the small biopharma company Arcturus Ther- apeutics, in collaboration with CSL, that received the first marketing approval of a lyophilized mRNA-LNP product. Its self-amplifying mRNA COVID-19 vaccine ARCT-154 was approved by Japan's Ministry of Health, Labor, and Welfare (MHLW) in November 2023 (1). The main challenge: maintaining integrity While lyophilization is an important means of ad- dressing stability issues associated with mRNA ther- apeutics, allowing the transport and storage of these products without the need for ultracold chain infra- structure, there have been challenges to effectively harnessing the benefits of lyophilization for these products, according to Vincenza Pironti, strategic marketing director at Recipharm. The sensitiv it y of large, high ly charged m RNA molecules, even when encapsulated in LNPs, to oxy- gen, light, heat, and temperature must be taken into consideration, contends Joseph E. Payne, president and CEO of Arct ur us Therapeutics. The tempera- ture transfer during lyophilization and the use of a vacuum places a strain on the LNPs. "It is necessary to develop a subtle and sophisticated method that does not perturb or discombobulate the nanoparti- cles during both freeze-drying and reconstitution," he says. Doi ng so, Pay ne adds, requi res a considerable amount of resources and years of innovation, and typically involves the use of the right combination of buffers and excipients to stabilize the mRNA-LNPs during dehydration and rehydration. These challenges, Pironti says, include ensuring the lyophilization process is effective at optimizing product stability and protecting the integrity of the LNPs. "In practice this means there needs to be a good final cake within the vial that can be readily reconstituted when it is time to administer without detrimental effects to the formulation," she explains. That can be par ticularly dif f icult to achieve for low-dos a ge f or mu l at ion s s uc h a s m R NA-ba se d t herapies due to t he rat io of i ng red ient s to dose volume, observes Pironti. "What is needed in these cases is the use of appropriate cr yoprotectants or bulking agents within the formulation, which must be compat ible w it h t he for mu lat ion a nd t he v ia l and ensure the stability of final drug product," she obser ves. She also notes that comprehensive ana- lytical testing of both the formulation and the LNPs well ahead of commercialization can help identif y the right lyophilization methods to suppor t prod- uct stabi l it y a nd shel f-l i fe once t he t herapy is i n commercial use. E q u a l l y i m p o r t a n t , a c c o r d i n g t o C h r i s t i a n D oh men , e xe c ut ive d i re c tor, te c h nolog y de ve l- opment & CMC w it h Et h r is, is to ensure t hat t he LNPs are fully intact after reconstitution. There are many factors that contribute to product deteriora- tion of lyophilized mRNA/LNPs, including particle size, mRNA integrity, and encapsulation efficiency. "If lef t unaddressed, t he combined ef fect of t hese fac tors leads to deter iorat ion of t he produc t a nd decreased biological activit y as well as undesired immune recognition (e.g., due to formation of ag- gregates), ultimately leading to reduced therapeu- Learning curve at Arcturus Therapeutics Arcturus Therapeutics began working on a lyophilization process for messenger RNA–lipid nanoparticles (mRNA- LNP) vaccines prior to emergence of the COVID-19 pandemic. The company, says President and CEO Joseph E. Payne, was looking at large-patient-population disease, for which the supply chain would be an issue. "We fortunately had strategic partners that enabled us to spend the necessary time, effort, and resources to develop a robust, repeatable lyophilization process to ensure we could provide a stable product for our late- stage trials taking place across the world," he notes. As a self-amplifying mRNA drug substance about three times larger than regular mRNA molecules, the Arcturus candidate had an extended learning curve, according to Payne. "Generally, the larger the mRNA, the more challenging is the development of manu f ac turing , puri f i c ati on , f ormulati on , and lyophilization processes," he observes. The Arcturus process was developed after extensive empirical screening conducted in combination with the application of internal knowhow and innovative approaches. The product formulation includes a unique combination of excipients and buffers and a carefully established, sophisticated lyophilization process involving specifically designed cycles with optimum time, temperature, and vacuum settings. The process is being implemented for the production of its Japanese-approved COVID-19 vaccine by its manufacturing partner in Japan (ARCALIS, joint venture between Axcelead and Arcturus), and Arcturus anticipates it will also transfer the technology to CSL, its partner for commercialization and distribution of the COVID-19 vaccine in Europe. In addition, Payne comments that the company is working to expand the lyophilization process to different types of vial presentation for different markets.