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Determination of DNA and RNA molecular weight
Sequencing of the DNA or RNA molecules is the most reliable approach to ensure that the target molecule has been produced; nevertheless, sequencing alone cannot easily identify partially synthesized molecules. Our Pace Life Sciences Boston, MA development site develops custom methods for the determination of molecular weight and polydispersity of nucleic acid molecules ranging between a few nucleotides and large polynucleotides thousands of units long. (CE, SEC-MALS, LC-MS)

Conformation of nucleic acids
RNA can self-assemble through intramolecular base pairing to form complex secondary and tertiary structures. Using orthogonal methods, we determine size and volume of RNA molecules, gaining insights on how changing process parameters and formulation conditions affect their overall molecular organization. (DLS, SEC-MALS)

Thermal and chemical stability
Several chemical-physical factors can jeopardize the stability of gene therapy vectors and derail a gene therapy development program. At Pace, we assess physicochemical stability applying a range of orthogonal techniques. (DLS, SEC-UV, CD, CE)

Determination of molar extinction coefficient
Accurate determination of nucleic acid concentration at several development stages is measured through the UV absorbance at 260 nm of a standard nucleic acid solution. The molar extinction coefficient for each molecule is determined through orthogonal methods based on UV absorbance and refractive index measurements. (UV spectroscopy, SEC-UV-MALS-dRI).

Capsid protein composition
A common vector for the delivery of DNA-based gene therapeutics is virus like particles (VLPs), e.g., adeno associated virus. To ensure proper assembly o VLPs it is essential to determine the composition in capsid proteins.

Pace Life Sciences determines the composition in virus proteins with a combination of techniques, looking at the ratio among the individual particle components and their integrity and post translational modifications. (RP-HPLC, CE, LC-MS)

Capsid integrity
The production and formulation process can lead to incomplete synthesis or fragmentation of viral particle, which can decrease the potency of DP and compromise preclinical studies where titer is measured as protein concentration. Capsid integrity (and encapsulation efficiency, see below) in a critical quality attribute that needs to be monitored at all steps of development.(SEC-MALS, FFF-MALS)

Lipid Nanoparticle (LNP) size distribution

LNPs are a common vehicle for the administration of nucleic acids of any size and chemical identity; nanoparticles exceeding the recommended dimension can lead to toxicity and modify the encapsulation efficiency and needs to be monitored. Our Pace Life Sciences Boston, MA development site can determine hydrodynamic radius, polydispersity and zeta potential to ensure proper packaging and limiting risks related to toxicity.(DLS, z-potential)

Encapsulation efficiency (VLP)
Dosing of DNA using VLPs as a vector is often based on infective particles per body weight; the manufacturing process for VLP vectors relays on cell culture expression and packaging of virus like particles, a system with a potential for high variability which may lead to the production of DS lots with highly variable amounts of empty capsids and the administration of a poorly defined dose of capsid proteins, which may result in toxicity, undesired immune response against the vector, or ineffective dosage. (SEC-MALS, IEX-MALS, FFF-MALS)

LNP composition
The formulation of nucleic acids with a mixture of lipids to obtain the final formulation and the formation of lipid nanoparticles is a stochastic process which requires monitoring of the final product to ensure that the required composition in lipids is achieved. Pace develops custom methods able to achieve the highest resolution and sensitivity for each lipid mixture used during the formulation process.

Encapsulation efficiency (LNP)
Dosing of mRNA delivered using LNPs as a vector is based on mRNA concentration; if encapsulation efficiency is not well controlled, a fixed dose of nucleic acid may correspond to highly variable dose of lipids, which may lead to modified effectiveness of the treatment or to toxicity due to high doses of lipids. Pace Life Sciences utilizes a panel of orthogonal methods for the determination of encapsulation efficiency, ensuring a robust and reliable control of this critical quality attribute.