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  • OVERVIEW
LNP Formulated mRNA Production Service
  • OVERVIEW

LNP Formulated mRNA Production Service


mRNA has shown therapeutic potential in a range of applications, including viral vaccines, protein replacement therapies, cancer immunotherapies, cellular reprogramming and genome editing. To achieve therapeutic effects, mRNA molecules have to reach specific target cells and produce sufficient proteins of interest.  Lipid nanoparticles (LNPs) are the leading nonviral technologies for the delivery of exogenous RNA to target cells in vivo. A major challenge to advance lipid nanoparticles (LNPs) for RNA therapeutics is the development of formulations that can be produced reliably across the various scales of drug development.


Seattle Genova is highly specialized in synthesizing top-quality mRNA and lipid nanoparticles for research and development applications, focusing on tailored production to meet your specific needs. We also offer a standard portfolio with optimized stability, solubility, and handling characteristics.

LNPs are typically composed of glycerophospholipids, canonic lipids, sterol lipids, and PEGylated lipids that encapsulate oligonucleotides, which are contained within an aqueous phase.

Table 1: Commonly used mRNA delivery systems

Lipid Nanoparticle

Liposome

Solid Lipid Nanoparticle

Nanostructured Lipid Carrier

Lipid Shell

Monolayer

Bilayer

Surfactant

Surfactant

Internal Core

Reverse micelles

Aqueous

Solid lipids

Solid and liquid lipids

Cargo

Oligonucleotides

Hydrophobic and/or hydrophilic small molecules

Hydrophobic and/or hydrophilic small molecules

Hydrophobic and/or hydrophilic small molecules

Size

~20-100 nm

~50-1,000 nm

~40-1,000 nm

~40-1,000 nm


Generally, mRNA-LNP design is comprised of several elements: (1) mRNA sequence design and nucleotide modification choice, (2) Optimization of a LNP formulation to encapsulate and deliver mRNA and (3) long-term storage. Each one of these phases entails a plethora of choices and considerations to make.


Workflow of mRNA Preparation:

1.      1. Antigen design

2.      2. Plasmid production

3.      3. In vitro transcription

4.      4. Characterization

A, primary structure of mRNA

-Sequencing

-Mapping

-mRNA modification analysis

B, mRNA secondary structure

-CD

-Cryo-EM

-Secondary structure prediction

C, mRNA product analysis

-mRNA integrity and purity analysis

-Analysis of mRNA capping efficiency

-Poly A tail length analysis

-Double-Stranded RNA Analysis


Procedures for the preparation of LNPs:

1.      1. Design

-Lipid molar ratio

-Lipid:oligonucleotide weight ratio

-Ionizable lipid nitrogen:oligonucleotide phosphate (N:P) molar ratio

-Lipid acid dissociation constant (lipid pK)

-Buffer composition, ionic strength, and pH

-Particle size and pharmacokinetics

-Routes of administration for LNPs

2.      2. Preparation and mixing

-Mixing

-Extrusion

-Dialysis

-Filter-sterilize


3.      3. Stability

-Physical stability

-Chemical stability

-Biological stability


4.      4. Storage

-Short-term storage at 4°C

-Long-term storage at -80°C

-Lyophilization


5.      5. Characterization

-LNP size: Dynamic light scatering (DLS)

-PDI: DLS

-Ζeta potential: Laser Doppler electrophoresis

-Encapsulation efficiency: Fluorescent dyes; UV spectroscopy with Triton-X

-Particle concentration: DLS; fluorescence correlation spectroscopy; UV-vis spectroscopy; UPLC

-Lipid and cargo integrity: RP-HPLC; SE-HPLC; IP-HPLC; LC-MS/MS


6.      6. mRNA-LNP Efficacy Analysis

-In vitro efficacy analysis: qPCR, Western blot, cell-based reporter assays, ELISA, multiplexed assays

-In vivo efficacy analysis: tissue distribution, lipid tissue concentrations

LNP Formulated mRNA Production Service


mRNA has shown therapeutic potential in a range of applications, including viral vaccines, protein replacement therapies, cancer immunotherapies, cellular reprogramming and genome editing. To achieve therapeutic effects, mRNA molecules have to reach specific target cells and produce sufficient proteins of interest.  Lipid nanoparticles (LNPs) are the leading nonviral technologies for the delivery of exogenous RNA to target cells in vivo. A major challenge to advance lipid nanoparticles (LNPs) for RNA therapeutics is the development of formulations that can be produced reliably across the various scales of drug development.


Seattle Genova is highly specialized in synthesizing top-quality mRNA and lipid nanoparticles for research and development applications, focusing on tailored production to meet your specific needs. We also offer a standard portfolio with optimized stability, solubility, and handling characteristics.

LNPs are typically composed of glycerophospholipids, canonic lipids, sterol lipids, and PEGylated lipids that encapsulate oligonucleotides, which are contained within an aqueous phase.

Table 1: Commonly used mRNA delivery systems

Lipid Nanoparticle

Liposome

Solid Lipid Nanoparticle

Nanostructured Lipid Carrier

Lipid Shell

Monolayer

Bilayer

Surfactant

Surfactant

Internal Core

Reverse micelles

Aqueous

Solid lipids

Solid and liquid lipids

Cargo

Oligonucleotides

Hydrophobic and/or hydrophilic small molecules

Hydrophobic and/or hydrophilic small molecules

Hydrophobic and/or hydrophilic small molecules

Size

~20-100 nm

~50-1,000 nm

~40-1,000 nm

~40-1,000 nm


Generally, mRNA-LNP design is comprised of several elements: (1) mRNA sequence design and nucleotide modification choice, (2) Optimization of a LNP formulation to encapsulate and deliver mRNA and (3) long-term storage. Each one of these phases entails a plethora of choices and considerations to make.


Workflow of mRNA Preparation:

1.      1. Antigen design

2.      2. Plasmid production

3.      3. In vitro transcription

4.      4. Characterization

A, primary structure of mRNA

-Sequencing

-Mapping

-mRNA modification analysis

B, mRNA secondary structure

-CD

-Cryo-EM

-Secondary structure prediction

C, mRNA product analysis

-mRNA integrity and purity analysis

-Analysis of mRNA capping efficiency

-Poly A tail length analysis

-Double-Stranded RNA Analysis


Procedures for the preparation of LNPs:

1.      1. Design

-Lipid molar ratio

-Lipid:oligonucleotide weight ratio

-Ionizable lipid nitrogen:oligonucleotide phosphate (N:P) molar ratio

-Lipid acid dissociation constant (lipid pK)

-Buffer composition, ionic strength, and pH

-Particle size and pharmacokinetics

-Routes of administration for LNPs

2.      2. Preparation and mixing

-Mixing

-Extrusion

-Dialysis

-Filter-sterilize


3.      3. Stability

-Physical stability

-Chemical stability

-Biological stability


4.      4. Storage

-Short-term storage at 4°C

-Long-term storage at -80°C

-Lyophilization


5.      5. Characterization

-LNP size: Dynamic light scatering (DLS)

-PDI: DLS

-Ζeta potential: Laser Doppler electrophoresis

-Encapsulation efficiency: Fluorescent dyes; UV spectroscopy with Triton-X

-Particle concentration: DLS; fluorescence correlation spectroscopy; UV-vis spectroscopy; UPLC

-Lipid and cargo integrity: RP-HPLC; SE-HPLC; IP-HPLC; LC-MS/MS


6.      6. mRNA-LNP Efficacy Analysis

-In vitro efficacy analysis: qPCR, Western blot, cell-based reporter assays, ELISA, multiplexed assays

-In vivo efficacy analysis: tissue distribution, lipid tissue concentrations

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