mRNA related process development

     In the process of IVT (in vitro transcription) process development, achieving high yield and integrity are the primary fundamental quality indicators. High yield is closely linked to production costs, while high integrity forms the cornerstone of product quality.

         T7 RNA Polymerase, a pivotal ingredient in IVT, plays a decisive role in mRNA quality. Hzymes, based on five core technology platforms involving enzyme gene resource exploration, directed evolution of enzyme, enzyme artificial intelligence design, large-scale enzyme manufacturing, and enzyme catalytic system application, offers a complete set of ingredients for mRNA synthesis.

· case1: Leveraging the T7 RNA Polymerase mutant (HBP000330) and the buffer library to assist clients in optimizing the IVT reaction, thereby enhancing yield and integrity. ·
/Improve yield and purity/
· Case 2: Utilizing the T7 RNA Polymerase low dsRNA mutant (HBP000340) and high-temperature-resistant mutant (HBP000350) to aid clients in IVT reaction procedure optimization. This approach aims to reduce side reactions and minimize the byproduct dsRNA content in the final product. ·
/Reducing dsRNA content in the final product/
· Case 3: Employing the T7 RNA Polymerase mutant variant (HBP000330) and the buffer library to facilitate the development of an IVT reaction capable of producing ultra-short fragments below 300 nt ·
/Produces ultra-short fragments and improves purity/
· Case 4: Utilizing the T7 RNA Polymerase mutant (HBP000330) and the buffer library to optimize an IVT reaction procedure for producing ultra-long fragments, while also enhancing integrity. ·
/Produces ultra-long fragments and improves purity/
At present, the main process steps of Hzymes biotech mRNA preparation include mRNA encapsulatio, dilution, complex purification, sterilization and filtration

RUN

TestLoadUFDF pool filter

1

entrapment efficiency(%)97.6694.84
size(nm)67.3771.51
PDI0.090.12

2

entrapment efficiency(%)
95.6995.00
size(nm)
66.9073.94
PDI0.130.16
LNP process development services
Service and data display
1.mRNA encapsulatio
2. Dilution
3.Complex purification
4.Sterilization and filtration
LNP Encapsulation
Dilution
TFF
0.22um Filtration

The foundation of the LNP encapsulation process is the design and development of the delivery system. A well-designed delivery system is necessary to prevent mRNA molecules from being degraded by RNase after entering the human body, effectively delivered to the target site, passed through the cell membrane, and released within the cell. Currently, Hzymes adopts the mainstream delivery system for lipid nanoparticles.

The key to the preparation of mRNA-LNP encapsulation is not only the formulation of lipid components, but also the control of the process, that is, how to control the contact and interaction between mRNA and lipid components to form stable, uniform, and high yield mRNA-LNP complexes. At present, Hzymes also adopts the current mainstream Microfluidics mixing technology. Due to the solubility of mRNA in the slightly acidic aqueous phase and the solubility of liposomes in ethanol, the mRNA solution and liposome solution are mixed by two opposing jets under high pressure. The strong turbulence causes each component to fully mix, while the ethanol phase is diluted and the pH of the solution changes. Liposomes precipitate to form lipid nanoparticles and form encapsulation complexes with mRNA.

After mRNA encapsulation, it is necessary to purify and remove unencapsulated/unloaded mRNA, free polymers or lipid materials, and adjust the final complex concentration, exchange the solvent buffer system, adjust pH value, etc. This step is usually achieved through tangential flow filtration, where the mRNA-LNP complex is intercepted, impurities and solvents are washed and filtered. Taking 4000nt mRNA as an example, it was diluted 20 times after encapsulation and perform tangential flow filtration using a hollow fiber column.

Delivering firefly luciferase mRNA-LNP into mice, the data showed that mRNA-LNP was successfully expressed in situ, targeting the liver and spleen.

In general, the purification design of mRNA downstream process includes the following four Unit operation, which can be used to remove enzyme reactants, residual DNA and unwanted high molecular weight (HMW) substances.

For 4000nt mRNA, the purification process related data of Case  is as follows:

Step

Cycle No.
Load
Elution

CE 

(%)


SEC_HPLC

(H/M/L)

(%)

Res. Protein

(ug/mg)

Yield

(%)

CE 

(%)

SEC_HPLC

(H/M/L)

(%)

Res. Protein (ug/mg)

dsRNA

(ug/mg)


HCD

(ug/mg) 

Res.DNA template

(ug/mg) 

Endotoxin

(EU/mg)

TFF 1

Cycle 1

76

-
-
100
78.8

ND/97.3/2.8

8.5
0.69
ND

2.07E-15

<9.4

AC

Cycle 1

71.5

ND/97.9/2.1

6.3
92.1
82.5

3.7/95.1/1.2

0.2
0.60
ND

1.04E-15

<17.5

Cycle 2
76.7

ND/96.9/3.1

6.3
94.0
83.0
0.8/98.0/1.2
0.2
0.94
ND

9.36E-16

<17.2

TFF 2

Cycle 1

82.8
3.6/95.4/0.9
0.2
99.6
82.5

ND/99.6/0.4

/
0.68
/
/

<6.8

Purification process development
Purification Process and Function of Hzymes mRNA Downstream Process
1.UF/DF filtration
2. use oligo dT resin to capture mRNA
3.UF/DF filtration for concentration and buffer exchange
4.sterilization and filtration

Purification Process and Function of Hzymes mRNA Downstream Process

In general, the purification design of mRNA downstream process includes the following four Unit operation, which can be used to remove enzyme reactants, residual DNA and unwanted high molecular weight (HMW) substances.

· The first step is UF/DF filtration. Start from receiving IVT reaction products, then install UF/DF filters, flush with Nuclease free water, and balance buffer solution. Next, the product is concentrated to the target volume and exchanged into a buffer solution. Finally, the product is recovered by cleaning the UF/DF components. The purpose of this operation is to reduce impurities such as RNA polymerase, DNA template, NTPs, capping enzymes and reagents, inhibitors, and to exchange the buffer system.

· The second step is to use oligo dT resin to capture mRNA or use multimodal chromatography resin. The former uses oligo dT (polythymidine) ligand (18-25 bp Thymine deoxynucleotide) and polyadenylate (PolyA tail) at the 'end of mRNA 3' to form stable hybridization through hydrogen bond to capture mRNA. The purpose of this operation is to remove impurities lacking PloyA tail, such as free nucleotides, short chain transcripts, enzymes, etc. The latter is the mRNA flow through resin, which collects the flow through and rinse solution into a single fraction and proceeds to the next step. The purpose of this step is to remove the IVT reaction components contaminants.

· The third step is UF/DF filtration for concentration and buffer exchange. Replace the target product into the final formulation buffer and top wash the UF/DF components to recover the product.

· The final step is 0.2 µm sterilization and filtration. Then fill the closed drug substrate solution into a sterile container. It should be noted that any pipeline, chromatography or filter components that cannot be detected as free of nuclease should be kept with 0.5M NaOH for at least 1 hour under environmental conditions, and then washed with nuclease free water and appropriate buffer solution for the next Unit operation.

Finally, these purified mRNA drug substrates can be further encapsulated into LNP.

Purification process development and data display

In the field of mRNA, in addition to designing optimized mRNA sequences and developing efficient and safe delivery vectors, the development of simple, fast, large-scale, and cost-effective mRNA preparation processes is also one of the most important innovations in mRNA technology. In the production process of mRNA, purification after IVT is crucial for the safety and effectiveness of the final mRNA product, as the content of impurities can affect the translation efficiency of mRNA and alter immunogenicity. Therefore, it is necessary to remove impurities, including residual substrates, dsRNA, abnormal mRNA, DNA templates, protease residues, etc. In general, downstream purification methods include ultrafiltration, chromatography, precipitation, etc.

At present, the downstream purification scheme of mRNA that Hzymes biotech can provide is as follows:
Linearized pDNA
IVT
Dnase I Digestion
TFF 1
AC/MMC
Polishing Step
TFF 2
Final Filtration And Dispense
Drug Substance
Proteinase K Digestion
Two-stage TFF
MRNA vaccines and drug-related services
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Expression CRDMO Services
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