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  • OVERVIEW
New Lipid Nanoparticle Formulation Development
  • OVERVIEW

LipoXTM New Lipid Nanoparticle Formulation Development

Seattle Genova has launched our LipoX platform to assist our customers with new lipid nanoparticle formulation development. Our LipoX platform uses lipid libraries and proprietary formulations to facilitate targeted delivery and tailored biodistribution solutions. Combined with the expertise of our specialist team, our new lipid nanoparticle formulation development service has been devised to allow customers to maximize the delivery of their RNA-products. Our LipoX LNP formulation platforms reportedly employ a range of mRNA and lipid mixing technologies and will provide the market with differentiated alternatives to the LNP formulations in use currently.

The formulations we create are designed to match our clients’ requirements and resources precisely. Significantly, they are provided royalty-free, so there is no commitment or limiting factor on any future development. Our work is carried out on a purely fee-for-service basis, with our aim to provide the best formulation development and the best chance of project success. An extensive suite of new lipid nanoparticle formulation development services is offered at Seattle Genova.

One of the key aspects of developing LNP-based formulations with the desired target properties is the composition. Typically, LNPs contain the following lipid components:

• Ionisable lipid, positively charged at low pH (enabling RNA complexation)
• Helper lipid that contributes to delivery efficiency and stability
• Structural lipid, such as cholesterol
• PEGylated lipid to minimise reticuloendothelial clearance and increase circulation time

Our solutions for new LNP formulation development


High-throughput synthesis and screening of next-generation lipid nanoparticles
We utilize high-throughput synthesis techniques that are based on solid-phase synthesis, Michael addition, click chemistry and so on, to facilitate the development of a large variety of LNP structures. Our LNP diversity is built-in by varying molecular factors such as lipid type, tail length, charge and structure, as well as overall LNP size.
Molecular barcoding allows for HTS of the respective LNPs upon binding to their designated targets at the cellular and tissue levels. Quantification of LNP biodistribution is subsequently based on target DNA sequence amplification via PCR followed by next-generation sequencing (NGS).

DNA Barcoded Lipid Nanoparticles
To facilitate testing of many nanoparticles in a single trial, we designed and optimized a high-throughput DNA barcoding system to simultaneously measure nucleic acid delivery mediated by dozens of distinct nanoparticles in a single trial. This nano-barcoding system can be used to study hundreds, or even thousands, of nanoparticles directly in vivo and could dramatically accelerate the discovery and understanding of nanoparticle drug delivery systems.

Improved Long-Term Stability of LNPs
The long-term storage and stability of mRNA-LNPs is a major consideration in the design of the formulations.
1.      Freezing LNPs, cryoprotectants such as sucrose or trehalose should be added after mRNA-LNP formulation. At Seattle Genova we can develop new cryoprotectants to offer better protection during the process of freezing.
2.      Lyophilization of mRNA LNPs, this can be accomplished by the addition of lyoprotectants.
3.      mRNA degradation within LNPs occurs at a faster rate and dictates the storage time and temperature. In Seattle Genova, we design and use optimized mRNA sequences and mRNA modifications for long-term storage.

Formulation of LNPs With Target Destination in Mind
Organ specific modifications of LNPs include manipulation of LNP charge that promotes lung-, spleen- or liver- specific delivery of therapeutic RNA. Along with standard LNP components including an ionizable cationic lipid, phospholipids, cholesterol and PEG, the authors proposed adding SORT molecules which allow lung-, spleen- or liver-specific gene delivery.
In addition to organ-specific delivery, we have pursued targeting specific cell subsets in the liver by engineering ionizable lipid nanoparticles for selective RNA delivery into hepatocytes and liver sinusoidal endothelial cells (LSEC).
Tumor targeting can be engineered by adding tumor-specific antibodies to the surface of LNPs.

Process Optimization & Large-Scale Production
With our technology, we can accomplish direct scaling from screening through process optimization and GMP production saving process validation steps.


Characterization of LNPs
1.      Particle Size, ZP, and Surface Morphology
PDI indicates the extent of particle size distribution with a range of 01, and a PDI of less than 0.2 is often considered as narrow size distribution whereas most of studies set PDI value less than 0.3 as the upper limit. Normally, particle size and PDI can be determined by dynamic light scattering (DLS) which measures the intensity differences of fluctuated light due to the motion of particles.
ZP refers to the surface charge of particles measured by a ZP analyzer. It can be used to indicate the stability of formulated colloidal dispersions by determining the degree of repulsion force.
Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) are often be employed to determine particle size, as well as to observe particle surface morphology.


2.      Drug EE(mRNA encapsulation efficiency)
To determine the amount of drug encapsulated in nanoparticles, UV–vis spectrometry or high-performance liquid chromatography (HPLC) is normally used.


3.      Drug Release Studies
In vitro studies are very useful to predict the in vivo behaviors of drug loaded LNPs. In vitro drug release studies simulating in vivo drug release are often conducted in phosphate-buffered saline(PBS) or simulated body fluids using side-by-side diffusion cells with biological or artificial membrane-like reverse dialysis sacs, ultracentrifugation, dialysis bags, centrifugal ultrafiltration, and ultrafiltration. Then UV spectrophotometer or HPLC is used to analyze the drug release profile.


4.      Crystallinity
It is very important to determine the crystallinity of LNP components because the lipids and the drugs encapsulated may undergo polymorphic transformation during storage, leading to drug expulsion and instability. Lipid crystallinity also strongly affects drug incorporation and drug release. DSC and X-ray diffractometry (XRD) are often used for the investigation of structure, content, and size of lipid crystalline.


5.      LNPs Stability
Experience from formulating therapeutic proteins, including the monoclonal antibodies, aids in the prediction of the issues that may arise in LNP preparations, including chemical stability of the LNP components, physical stability of the LNP(disintegration, aggregation, and adsorption on surfaces), and stability of the RNA within the LNP.

Through our long-established focus on RNA manufacture and delivery, and associated proprietary technologies, we have engineered libraries of custom lipids, which enables our LNP formulation team to design the most appropriate LNP for your mRNA application.

LipoXTM New Lipid Nanoparticle Formulation Development

Seattle Genova has launched our LipoX platform to assist our customers with new lipid nanoparticle formulation development. Our LipoX platform uses lipid libraries and proprietary formulations to facilitate targeted delivery and tailored biodistribution solutions. Combined with the expertise of our specialist team, our new lipid nanoparticle formulation development service has been devised to allow customers to maximize the delivery of their RNA-products. Our LipoX LNP formulation platforms reportedly employ a range of mRNA and lipid mixing technologies and will provide the market with differentiated alternatives to the LNP formulations in use currently.

The formulations we create are designed to match our clients’ requirements and resources precisely. Significantly, they are provided royalty-free, so there is no commitment or limiting factor on any future development. Our work is carried out on a purely fee-for-service basis, with our aim to provide the best formulation development and the best chance of project success. An extensive suite of new lipid nanoparticle formulation development services is offered at Seattle Genova.

One of the key aspects of developing LNP-based formulations with the desired target properties is the composition. Typically, LNPs contain the following lipid components:

• Ionisable lipid, positively charged at low pH (enabling RNA complexation)
• Helper lipid that contributes to delivery efficiency and stability
• Structural lipid, such as cholesterol
• PEGylated lipid to minimise reticuloendothelial clearance and increase circulation time

Our solutions for new LNP formulation development


High-throughput synthesis and screening of next-generation lipid nanoparticles
We utilize high-throughput synthesis techniques that are based on solid-phase synthesis, Michael addition, click chemistry and so on, to facilitate the development of a large variety of LNP structures. Our LNP diversity is built-in by varying molecular factors such as lipid type, tail length, charge and structure, as well as overall LNP size.
Molecular barcoding allows for HTS of the respective LNPs upon binding to their designated targets at the cellular and tissue levels. Quantification of LNP biodistribution is subsequently based on target DNA sequence amplification via PCR followed by next-generation sequencing (NGS).

DNA Barcoded Lipid Nanoparticles
To facilitate testing of many nanoparticles in a single trial, we designed and optimized a high-throughput DNA barcoding system to simultaneously measure nucleic acid delivery mediated by dozens of distinct nanoparticles in a single trial. This nano-barcoding system can be used to study hundreds, or even thousands, of nanoparticles directly in vivo and could dramatically accelerate the discovery and understanding of nanoparticle drug delivery systems.

Improved Long-Term Stability of LNPs
The long-term storage and stability of mRNA-LNPs is a major consideration in the design of the formulations.
1.      Freezing LNPs, cryoprotectants such as sucrose or trehalose should be added after mRNA-LNP formulation. At Seattle Genova we can develop new cryoprotectants to offer better protection during the process of freezing.
2.      Lyophilization of mRNA LNPs, this can be accomplished by the addition of lyoprotectants.
3.      mRNA degradation within LNPs occurs at a faster rate and dictates the storage time and temperature. In Seattle Genova, we design and use optimized mRNA sequences and mRNA modifications for long-term storage.

Formulation of LNPs With Target Destination in Mind
Organ specific modifications of LNPs include manipulation of LNP charge that promotes lung-, spleen- or liver- specific delivery of therapeutic RNA. Along with standard LNP components including an ionizable cationic lipid, phospholipids, cholesterol and PEG, the authors proposed adding SORT molecules which allow lung-, spleen- or liver-specific gene delivery.
In addition to organ-specific delivery, we have pursued targeting specific cell subsets in the liver by engineering ionizable lipid nanoparticles for selective RNA delivery into hepatocytes and liver sinusoidal endothelial cells (LSEC).
Tumor targeting can be engineered by adding tumor-specific antibodies to the surface of LNPs.

Process Optimization & Large-Scale Production
With our technology, we can accomplish direct scaling from screening through process optimization and GMP production saving process validation steps.


Characterization of LNPs
1.      Particle Size, ZP, and Surface Morphology
PDI indicates the extent of particle size distribution with a range of 01, and a PDI of less than 0.2 is often considered as narrow size distribution whereas most of studies set PDI value less than 0.3 as the upper limit. Normally, particle size and PDI can be determined by dynamic light scattering (DLS) which measures the intensity differences of fluctuated light due to the motion of particles.
ZP refers to the surface charge of particles measured by a ZP analyzer. It can be used to indicate the stability of formulated colloidal dispersions by determining the degree of repulsion force.
Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) are often be employed to determine particle size, as well as to observe particle surface morphology.


2.      Drug EE(mRNA encapsulation efficiency)
To determine the amount of drug encapsulated in nanoparticles, UV–vis spectrometry or high-performance liquid chromatography (HPLC) is normally used.


3.      Drug Release Studies
In vitro studies are very useful to predict the in vivo behaviors of drug loaded LNPs. In vitro drug release studies simulating in vivo drug release are often conducted in phosphate-buffered saline(PBS) or simulated body fluids using side-by-side diffusion cells with biological or artificial membrane-like reverse dialysis sacs, ultracentrifugation, dialysis bags, centrifugal ultrafiltration, and ultrafiltration. Then UV spectrophotometer or HPLC is used to analyze the drug release profile.


4.      Crystallinity
It is very important to determine the crystallinity of LNP components because the lipids and the drugs encapsulated may undergo polymorphic transformation during storage, leading to drug expulsion and instability. Lipid crystallinity also strongly affects drug incorporation and drug release. DSC and X-ray diffractometry (XRD) are often used for the investigation of structure, content, and size of lipid crystalline.


5.      LNPs Stability
Experience from formulating therapeutic proteins, including the monoclonal antibodies, aids in the prediction of the issues that may arise in LNP preparations, including chemical stability of the LNP components, physical stability of the LNP(disintegration, aggregation, and adsorption on surfaces), and stability of the RNA within the LNP.

Through our long-established focus on RNA manufacture and delivery, and associated proprietary technologies, we have engineered libraries of custom lipids, which enables our LNP formulation team to design the most appropriate LNP for your mRNA application.

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