General Design Methods for mRNA Drugs

 Image: By Thomas Shafee CC BY 4.0, https://commons.wikimedia.org/w/index.php?curid=39630174

With the rapid development of biotechnology and molecular medicine, the introduction of mRNA as a vaccine or therapeutic agent enables the production of almost any desired functional protein/peptide within the human body. This represents an increasingly promising field of precision medicine, holding significant prospects for preventing and treating numerous challenging or genetic diseases. Scientists have been striving to optimize mRNA stability, immunogenicity, translation efficiency, and delivery systems to achieve efficient and safe mRNA delivery.

By Carrier Taylor

Messenger RNA (mRNA) is a single-stranded ribonucleic acid transcribed from the DNA chain, carrying the encoding information for protein synthesis. In vitro transcribed (IVT) mRNA has been successfully transcribed and expressed in mouse skeletal muscle cells, establishing the feasibility of mRNA therapy. When mRNA can be successfully transfected and elicit an immune response by direct injection into the mouse body to express therapeutic proteins in a dose-dependent manner, mRNA-based therapeutic approaches have been proposed. In contrast to DNA-based drugs, mRNA transcripts have relatively high transfection efficiency and low toxicity, as they do not need to enter the cell nucleus to function. Importantly, mRNA carries no potential risks of unintended infection or opportunistic insertion mutations. Compared to traditional transient protein/peptide drugs, mRNA has the ability to continuously translate proteins/peptides, thus holding broad potential in the treatment of various diseases.

Key discoveries and advances in mRNA-based therapeutics (Qin, Shugang, et al. 2022)

General Design Methods for mRNA Drugs

The design and synthesis of mRNA are critical steps in the development of mRNA drugs. mRNA consists of five functional regions, including the 5′ cap structure, 5′ untranslated region (UTR), open reading frame (ORF), and 3′ untranslated region and 3′ poly(A) tail structure, which mediate mRNA translation efficiency and decay rate.

 

Structure of in vitro transcribed (IVT) mRNA and commonly used modification strategies (Wadhwa, Abishek, et al. 2020)

5′ Cap: The 5′ cap is located at the 5′ end of mRNA, and its methylation degree varies. In eukaryotes, the 5′ cap (m7G ppp) contains 7-methylguanosine (m7G), linked to the next nucleotide through a 5′-5′ triphosphate bridge (ppp). During the translation initiation stage, the cap binds to eIF4E through hydrophobic cation-π interactions of m7G and the negatively charged triphosphate bridge. Strategies optimizing m7G or the triphosphate bridge have been employed to achieve cap analogs with high affinity for eIF4E and low sensitivity to decapping enzymes.

Poly(A) Tail: The poly(A) tail typically consists of 10-250 adenine ribonucleotides. Dynamically added to mRNA, the poly(A) tail plays a crucial role in regulating mRNA translation efficiency and protein expression. Mechanistically, the 3′ poly(A) tail binds to poly(A)-binding proteins (PABPs), subsequently interacting with the 5′ cap through translation initiation factors eIF4G and eIF4E, promoting a “closed-loop structure” that regulates mRNA translation efficiency.

In vitro transcribed (IVT) mRNA and translation initiation (Qin, Shugang, et al. 2022)

 

5′-UTR and 3′-UTR: The 3′ and 5′ UTRs of mRNA do not directly encode proteins but play essential roles in regulating mRNA translation and protein expression. UTRs are involved in mRNA subcellular localization, regulating translation efficiency, and mRNA stability. While 5′-UTR and 3′-UTR both regulate protein expression levels, the former primarily participates in translation initiation, while the latter mainly influences mRNA stability and half-life.

 

ORF: ORF design focuses on codon optimization, introducing functional peptides, and the replication process. Codon optimization, a widely used but controversial improvement method, involves replacing rare codons with synonymous codons decoded by abundant tRNAs, enhancing mRNA translation efficiency. However, it may alter protein conformation, generating unknown peptides with in vivo biological activity. Increasing the GC content by substituting rare codons in the ORF protects mRNA from ribonuclease degradation and enhances in vivo mRNA protein expression. Additionally, functional peptides are crucial for mRNA drugs, and signal peptides encoded by mRNA are necessary for proteins to function extracellularly. Optimizing mRNA by introducing signal peptides into the ORF region improves the functionality of therapeutic mRNA.

In conclusion, each step in the quality control of mRNA directly impacts its efficacy. Therefore, the production and preparation of mRNA are crucial for mRNA therapy.

 

mRNA Manufacturing

Synthesis and Optimization of mRNA

In vitro transcribed (IVT) mRNA is synthesized using a linearized plasmid DNA template or a PCR template, requiring at least one promoter and the corresponding mRNA coding sequence. IVT mRNA is generated by adding a polymerase (T7, T3, or SP6), but additional capping is necessary. Uncapped mRNA is rapidly degraded by RNase and contains a 5′-ppp group, causing greater immune stimulation. This can be treated with phosphatase to reduce adverse effects. There are two methods for capping IVT mRNA: co-transcriptional capping and post-transcriptional capping. The Poly(A) tail of IVT mRNA is typically encoded in the DNA template or attached post-synthesis through polyadenylation, with the former providing more precise control over Poly(A) tail length. It’s worth noting that II-type restriction endonucleases used for plasmid template linearization can promote 3′ overhangs, hindering IVT mRNA translation efficiency. This can be avoided by substituting II-type restriction endonucleases with IIS-type endonucleases.

mRNA Purification

Since IVT mRNA is mixed with RNA polymerase and DNA template after synthesis, purification is crucial. This involves removing immune-stimulating contaminants, free ribonucleotides, short mRNA, and DNA templates. DNase is commonly used to degrade excess DNA templates. Commercial purification kits are often employed for purification and separation of synthesized mRNA. The purified mRNA is then precipitated with ethanol or isopropanol to eliminate most contaminants and obtain high-purity mRNA. Further purification methods, such as precipitation with high concentrations of LiCl or alcohol, chromatography, or elution from silica membrane columns, are used to remove proteins, free nucleotides, or other components. However, these methods may not effectively eliminate double-stranded RNA impurities.

RNase III is a novel purification method that has been proposed to eliminate double-stranded RNA (dsRNA) contaminants. It has been shown to significantly reduce the immunogenicity of mRNA and enhance the cytotoxic killing effect of CAR-T cells by reducing the immunogenicity of mRNA through electroporation with RNase III. Its potential drawback is that RNase III may cut double-stranded secondary structures formed by single-stranded RNA.

Various purification methods can be chosen based on research or application purposes, addressing different purity requirements and scales of mRNA. Regardless of the purification method used, stringent mRNA quality control standards are essential to ensure the maximization of therapeutic benefits in mRNA therapy.

References

1.      Boczkowski D, Nair SK, Snyder D, Gilboa E. Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo. J Exp Med. 1996 Aug 1;184(2):465-72.

2.      Wadhwa A, Aljabbari A, Lokras A, Foged C, Thakur A. Opportunities and Challenges in the Delivery of mRNA-based Vaccines. Pharmaceutics. 2020 Jan 28;12(2):102.

3.      Qin S, Tang X, Chen Y, Chen K, Fan N, Xiao W, Zheng Q, Li G, Teng Y, Wu M, Song X. mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct Target Ther. 2022 May 21;7(1):166.

About the author:

Carrier Taylor

R & D Director and Business Devlopment Director of BOCSCI 

2014 - Present, working in BOCSCI

2012-2014 Study in Rice University, MBA

2004-2008 Study in Rice University,Pharmacy 

Linkedin profile: https://www.linkedin.com/in/carrier-taylor/ 

Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Incite a Greener Future With Sustainable Pharma Logistics

Image by Combustion2016 - Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=52032751

Many leaders are prioritizing better sustainability in the pharmaceutical industry. They know customers and other stakeholders increasingly prefer engaging with companies that make progressively greener choices to protect the planet’s future and eliminate unnecessary resource usage. Making progress takes time and dedication, but modern executives are finding the most appropriate options to explore. Pinpointing logistics-related activities is a particularly popular option.

 

By Emily Newton

 

Reducing the Environmental Impacts

In a 2023 study that was the first of its kind, researchers found pharmaceutical businesses could reduce their environmental impacts by 45% by changing in a few targeted ways. More specifically, they could get those results by:

 

●     Optimizing supply chain networks

●     Improving production processes

●     Switching to renewable energy

 

The study examined the entire life cycle carbon impact of the Tenofovir Disoproxil Fumarate, an HIV antiretroviral. While looking at the supply chain activities, the team found factors such as the long distances between raw material sources, manufacturing plants and patients were some of the biggest aspects to focus on for meaningful improvements. Doing so could cause a carbon footprint reduction of up to 9.3% throughout the product’s life cycle.

 

However, the researchers confirmed the energy sources used in manufacturing are the most significant carbon contributors. Besides finding clean alternatives, drugmakers can reduce emissions by altering internal processes or sourcing strategies, such as reducing how much packaging a drug requires or enhancing processes to recycle chemical solvents.

 

The results of this study should be a strong reminder to industry professionals that sustainability in the pharmaceutical industry happens through numerous actions. Even if people primarily emphasize logistics networks, they should also recognize the industry’s interconnectedness and how making changes in one non-logistics-centered area could ultimately improve supply chain performance, too.

Exploring Transport Methods to Curb Temperature-Related Losses and Excessive Emissions

Thinking about distances and energy sources is a great start for working on sustainability in the pharmaceutical industry. However, leaders must also remember loss prevention is another area of improvement to explore. After all, even a method that seems eco-friendly on paper would fall short if it results in products arriving in unsellable conditions.

 

Many pharmaceutical products must stay in temperature-controlled environments during transit and distribution to prevent spoilage. While referencing internal transport data about loss prevention, one pharmaceutical executive clarified only 10% of known temperature excursion events occurred during sea transport. In contrast, 50% happened during flights and enterprise representatives linked 40% to road journeys.

 

Technology solutions such as Internet of Things (IoT) sensors can give pharmaceutical logistics managers better oversight of products in motion, no matter where they are. Besides providing location-based information, many IoT options gather and transmit temperature-related information. Then, whether a refrigerated truck’s storage area fails or supply chain workers place a box of chilled medication in a warm place for too long, the relevant alerts can help managers take corrective action before the goods spoil.

 

Another option is to set and track metrics with all suppliers. Pharmaceutical executives should stipulate that all supply chain partners must meet certain minimum standards for achieving on-time deliveries, eliminating temperature-related accidents and tackling all other factors that could affect the bottom line.

 

Leaders should consider assisting suppliers and service providers in gradually reducing emissions through better supply chain management as the next step for improving sustainability in the pharmaceutical industry. That might mean logistics partners invest in electric delivery vehicles, using intelligent routing software or supporting efforts to develop greener aviation fuels.

Setting Goals and Examining the Options

As revealed in a small 2023 survey of biopharmaceutical supply leaders, many are getting serious about specific things they could do within and outside logistics to make sustainability progress. For example, 64% said they are actively pursuing or scaling up efforts to minimize adverse impacts on water quality. Additionally, 59% are working on sourcing sustainable materials and 57% are creating sustainable value chains.

 

Smart factories were another area of interest for many respondents, with 47% either starting to build them or scaling up existing ones. Such facilities will help enhance eco-friendliness by allowing companies to reduce emissions and resource usage during production, providing more opportunities to continue that momentum through logistics opportunities.

 

Elsewhere, research published in 2023 about the green efforts of more than 200 firms in 15 European countries found 90% planned to increase their corporate sustainability efforts. Seventy-six percent of respondents categorized in the study as sustainability governance experts felt optimistic about their performance. Conversely, significantly fewer — 45% — expressed that opinion at the beginning of their corporate environmental efforts.

 

These takeaways highlight how enhancing sustainability in the pharmaceutical industry takes time, but committing to efforts that will cause steady progress should pay off. Once logistics leaders set measurable goals and get everyone involved, they have set the foundation for greener pharmaceutical operations and supply chains.

Sustainability in the Pharmaceutical Industry Is Worthwhile

There is no single or best way to strive for enhanced sustainability in the pharmaceutical industry. Instead, logistics leaders will get the best results by studying what is working well in their organizations, and which operations or practices need the most improvement. Examining factors like budgets, workforce sizes and how many locations the pharmaceutical company operates worldwide will help them make targeted decisions, too.

 

Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

New books for your pharmaceutical microbiology library

Two key microbiology texts from Euromed and PDA, published in 2024:
 

Industrial Pharmaceutical Microbiology: Standards and Controls 6th Edition

 

Industrial Pharmaceutical Microbiology: Standards and Control covers the entire spectrum of industrial pharmaceutical microbiology, as applicable to pharmaceuticals and healthcare. 

To meet the latest regulatory expectations, the role of the microbiologist is essential. In addition there is important input requirement from quality assurance personnel, engineers, and process specialists. Whilst there is a continuing need for monitoring of the environment and conducting standardised laboratory tests, industrial pharmaceutical microbiology has moved on a great deal in the past decade. 

 

It now has to embrace microbiological audits; rapid microbiological methods; conducting risk assessments, both proactive in terms of minimising contamination, and reactive in terms of addressing microbial data deviations; and also ensuring that processes meet ‘quality by design’ principles. In this new 700 page book a team of 20 international authorities will assist you in all your questions.

 

For details see Euromed

 

Industrial Pharmaceutical Microbiology: Quality Control

The reference text brings together the hitherto unavailable background, fundamental science, principles, development, intended purpose, and specific answers to questions of execution and qualification of compendial and related microbiological test methods. Key topics include the types of microbiological tests, reference strains and culture collections, and equivalence of reference strain.  

 

Industrial Pharmaceutical Microbiology: Quality Control with its 20 chapters, 19 international authors and over 500 pages, enables the practitioner to have a complete understanding of these microbiological methods, and to ensure better developed, compliant, appropriate procedures and accurate meaningful data.

 

For details see: Euromed

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

The Top 5 Benefits of Working With a Pharmaceutical Facility Cleaning Service

 

As a pharmaceutical company, maintaining a clean and sterile environment is crucial to ensuring the safety and effectiveness of your products. One way to achieve this is by working with a pharmaceutical facility cleaning service. These professionals are trained to monitor your clinical environment and keep it clean to the highest standards. Here's a quick look at the five benefits of partnering with a pharmaceutical facility cleaning service.

 

By Lizzie Weakley

 

Expertise in Pharmaceutical Cleaning Protocols

Pharmaceutical facility cleaning services are well-versed in the specific cleaning protocols and regulations that are required in the pharmaceutical industry. They understand the importance of maintaining a sterile environment to prevent contamination and ensure product quality. By working with a professional cleaning service, you can relax, knowing that your facility is being cleaned according to industry standards.

State-Of-The-Art Cleaning Equipment and Techniques

Pharmaceutical facility cleaning services have access to the latest cleaning equipment and techniques to clean and disinfect your facility effectively. From specialized cleaning solutions to high-tech equipment, these professionals have the necessary tools to ensure a thorough and comprehensive cleaning process. By utilizing their expertise and resources, you can maintain a clean and safe environment for your employees and products.

Compliance With Regulatory Requirements

The pharmaceutical industry is highly regulated, with strict guidelines and requirements in place to ensure product safety and efficacy. A pharmaceutical facility cleaning service understands these regulations and works diligently to ensure compliance with all applicable standards. By partnering with a professional cleaning service, you can rest assured that your facility meets all regulatory requirements and avoids potential fines or penalties.

Enhanced Cleanliness and Hygiene

Maintaining a clean and hygienic environment is essential for the health and well-being of your employees and patients. A pharmaceutical facility cleaning service can help you achieve this by thoroughly cleaning and disinfecting all areas of your facility. From common areas to production zones, these professionals can ensure that every surface is clean and free of contaminants. By investing in professional cleaning services, you can create a safer and healthier environment for everyone.

Improved Efficiency and Productivity

A clean and organized work environment can have a positive impact on employee morale, productivity, and overall efficiency. By working with a pharmaceutical facility cleaning service, you can create a clean and welcoming space that promotes employee satisfaction and engagement. When employees feel safe and comfortable in their work environment, they are more likely to perform at their best and contribute to the success of your organization.

 

Partnering with a pharmaceutical facility cleaning service offers numerous benefits for pharmaceutical companies. From expertise in pharmaceutical cleaning protocols to compliance with regulatory requirements, these professionals can help you maintain a clean and sterile environment for your products and employees. By investing in professional cleaning services, you can enhance cleanliness, improve efficiency, and promote a healthier work environment. Consider working with a pharmaceutical facility cleaning service to monitor your clinical environment and keep it clean to the highest standards.

 

Tim Sandle's new book is now available.

 

 

Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)