mRNA vaccines and drugs
The mRNA vaccine broke the traditional immune activation mode of inactivated and attenuated vaccines, and innovatively used human cells to produce antigens to activate specific immunity, which was brilliant during the COVID-19 epidemic. The application prospects of mRNA vaccines and drugs are very broad. In addition to being used for preventing infectious diseases, treating tumors and immune diseases, they can also be applied in various biopharmaceutical platform technologies, such as protein supplementation therapy, cell therapy, antibody drugs, gene editing, and so on. In the post COVID-19 era, mRNA vaccines still have the potential for substantial growth.

Preparation of mRNA immune antigen antibody

mRNA has the potential to encode almost all proteins. MRNA vaccine technology can be applied to antigen preparation to solve the difficulties in protein preparation, such as long preparation cycles, complex production processes, and poor flexibility in customized production of traditional protein antigens. MRNA, as a platform technology, has the advantages of simple production process, short production cycle, and high flexibility. It can provide customers with antigen preparation faster and more cost effectively, and conduct rapid target feasibility verification.

Hzymes provides one-stop mRNA vaccine & drug development concept validation services to help customers quickly verify the feasibility of drug targets

– Experienced team has successfully delivered multiple concept validation projects

– Diversified tools are used to predict the secondary structure of proteins using alphafold, providing guidance for customers’ antigen design and mRNA sequence design.

– Our independently established mRNA technology and application platform, comprehensive quality system, and full process services from sequence design, mRNA LNP preparation to in vivo biological evaluation.

Delivery Standard

Cases
mRNA vaccine design
Sequence design

step1： Confirm amino acid sequence
Based on the AI intelligent tool – Alphafold protein structure prediction and analysis tool, and in cooperation with the State Key Laboratory of Microbiology of Shanghai Jiaotong University, a protein structure prediction and optimization system were built.

step2： Decoding DNA nucleotide sequences
Hzymes utilizes various sequence optimization tools to output nucleotide sequence files, providing support for the next optimization step

1.Target determination: Identify the targets for drug development.
2.Optimization design: Based on MFE and CAI multi parameter combination, output multiple candidate sequences and sort them.
3.Screening scheme: Based on the MFE and CAI of the selected sequence, with the standard of covering all sequence sets as comprehensively as possible, select candidate sequences and use them for wet experiments
4.Experimental verification: Through wet experiments, select the best sequence.
5.Delivery of optimal sequence: The sequence delivered to the customer will be rigorously screened and most likely to become the optimal candidate for drug phenology.

Step 3: mRNA sequence optimization
1.Kozak sequence significantly increases protein expression

— Kozak


+ Kozak

UTR adaptability affects the expression effect of CDS sequences


Transcription initiation sequence


Verification of A-tail sequence of mRNA.

step4: Software predicts the secondary structure of candidate sequences

Predict the secondary structure of candidate sequences to avoid sequences with stable or hyperstable complementary structures at the 5 ‘level.

EGFP mRNA, MFE：-314.62


saRNA-EGFP, MFE -2718.90

Plasmid prep

1. Strain screening – controlling yield and super helix ratio


2. Fermentation process – control OD600 and plasmid yield

Optimize various parameters for different receptive states and plasmids:


· Cultivation medium formula
· Fermentation tank parameters
· Feeding formula and feeding strategy
· Fermentation tank control parameters


3. Important indicator for plasmid optimization – PolyA tail stability

Different receptive state transformation plasmids, selecting monoclonal antibodies with stable A tail


After fermentation amplification of different plasmid skeletons, the heterozygosity of PolyA tail in sequencing quality inspection varies


By screening the receptive/monoclonal/plasmid backbone and optimizing the fermentation process, we ultimately obtained plasmids with high helix ratios and stable PolyA tails

4. Purification process

1.Optimization of Cracking Process


2. Molecular sieve chromatography to remove RNA


3. Affinity chromatography to remove open-loop plasmids

4. Ion exchange chromatography to remove endotoxins


5. Product release testing


3. In vitro transcription synthesis of mRNA

Product release testing
https://www.hzymescro.com/en/mRNA-Vaccine-and-Drug-Proof-of-Concept-Services.html

4. Translation proficiency testing
https://www.hzymescro.com/en/mRNA-Vaccine-and-Drug-Proof-of-Concept-Services.html

5. LNP encapsulation
LNP microfluidic control preparation process


Hzymes has explored and optimized the downstream purification process for LNP, mainly focusing on the development of inlet flow rate, Trans Membrane Pressure Drop (TMP), membrane pore size, membrane area, membrane material, hollow fibers, and flat plate membrane package. The process time can be controlled at around 3.5 hours, and the stability of mRNA LNP preparation at -80 ℃ can reach more than 18 months, and it can be frozen and thawed 10 times without affecting physical and chemical indicators

Product release testing 
https://www.hzymescro.com/en/mRNA-Vaccine-and-Drug-Proof-of-Concept-Services.html