Tackling Advanced Challenges in Development, CMC and Manufacturing of pDNA, mRNA and LNP

12:00 Registration, Welcome Lunch and Refreshments

13:00 Bring & Test your Samples

• Hand-on demonstration in the lab and testing your own samples*
• Receive the results on Tuesday.

08:30 Registration, Coffee and Snacks

09:10 Welcome & Intro

09:30 Seminar by Rok Sekirnik | Head of Process Development for mRNA and pDNA

• Optimizing mRNA Manufacturing: Achieving High Purity and Yield through Advanced Purification Techniques

10:30 Break for PATfix Demo and Coffee

• Demonstration of the all-in-one chromatographic solution for LNP-specific analytics
• Enjoy Coffee & refreshments

11:30 Seminar by Dan Peer | Professor and Director of the Laboratory | Precision NanoMedicine

• RNA Delivery: Challenges and Innovations in Gene Expression Modulation

12:15 Seminar by Heinrich Haas  | Chief Technology Officer | NeoVac

• Relevance of Supramolecular Organization for Quality and Activity of mRNA LNPs

12:45 Lunch

13:45 Seminar by Tristan Kovačič | Project Manager – Process Analytics Development  | Sartorius BIA Separations

• Tackling CMC and Manufacturing Challenges of Complex LNP-based Biopharmaceuticals for Oncological, Gene editing and Vaccine Applications

14:15 Seminar by Mojca Tajnik Sbaizero | Process Development Manager | Sartorius BIA Separations

• Enhanced Purification Methods for Manufacturing of Complex Lipid Nanoparticle Formulations with Multiple Payloads

14:45 Break for PATfix demo and coffee

• Demonstration of the all-in-one chromatographic solution for LNP-specific analytics
• Enjoy Coffee & refreshments

15:45 Seminar by Jacek Lubelski | Chief Technology Officer | Nanocell Therapeutics

Novel Non-Viral DNA-based Gene Therapy Vector for CAR T Engineering In Vivo

16:15 Wrap up

16:25 Drinks and snacks

8:30 Welcome Coffee

9:00 Bring & Test your Samples

• Hand-on demonstration in the lab and testing your own samples*
• Receive the results by email.

12:00 Free Afternoon & Travel Home

• Suggestions for activities and culinary experiences in the Vipava Valley

RNA Delivery: Challenges and Innovations in Gene Expression Modulation

Abstract:

Modulating gene expression for tumor cell death, protein replacement therapy, or gene editing is a major medical challenge. Strategies have been developed to target cell surface receptors and create amino ionizable lipids for repeated dosing. Novel mRNA sequences with enhanced immunogenicity are designed for bacterial infections. Dan Peer will discuss the journey from Lipid Nanoparticles (LNPs) conceptualization to clinical application, including cancer targets, the first bacterial mRNA vaccine, and gene editing strategies. He will also cover targeting LNPs to the gut in inflammatory bowel diseases and the lung epithelium without targeting agents. Delivering RNA payloads to specific cells is a milestone in personalized medicine.

Relevance of supramolecular organization for quality and activity of mRNA LNPs

Abstract:

Afte their application in the vaccines against Covid-19, lipid nanoparticles (LNPs) comprising messenger RNA (mRNA) have been gaining increasing attention in the community. They are not only being successfully been applied as vaccines against infectious diseases, but also for delivery of short interfering RNA (siRNA) for treatment of rare diseases, and they are promising for a scope of therapeutic purposes.

Despite of this broad interest, many critical quality attributes of LNPs still are only poorly understood. This refers particularly to aspects which are related to the supramolecular organization of the different molecular moieties from which the nanoparticles ae formed. Here we present recent results on approaches for advanced characterization of size and structure of LNPs as a function of their local environment. These insights may lead to refined paradigms for quality evaluation of LNP formulations and to a better understanding of biological processes, such as endosomal release. Criteria for tailoring future mRNA delivery systems may be derived.

Optimizing mRNA Manufacturing: Achieving High Purity and Yield through Advanced Purification Techniques

Abstract:

This presentation will focus on a streamlined approach to mRNA manufacturing, including the preparation of high-quality DNA templates needed to achieve high RNA purity. We introduce a scalable purification workflow featuring continuous in-line lysis and selective hydrophobic interaction chromatography (SHIC) for purification of supercoiled (sc) DNA. The process includes efficient sc pDNA linearization monitored by the PATfix analytical system. In vitro transcription (IVT) reaction, used to produce mRNA from DNA template, is monitored with rapid PATfix at-line monitoring which also tracks nucleoside triphosphate (NTP) consumption. Using a data-driven design-of-experiment (DOE) approach, we achieve a reaction yield of 25 g/L, and can transform the reaction from batch to fed-batch mode. mRNA is purified with Oligo dT affinity chromatography which is selective for polyadenylated mRNA. Residual dsRNA is removed either with reverse-phase chromatography or with aqueous denaturing chromatography also based on Oligo dT. The process is scalable from ug to multi-gram scale and suitable for clinical manufacturing.

Novel Non-Viral DNA-based Gene Therapy vector for CAR T engineering in vivo

Abstract:

The development of immune cells engineered to express Chimeric Antigen Receptors (CARs) has transformed cancer therapy. Ex vivo CAR-T cell treatments have achieved notable clinical and commercial success. However, they are also associated with significant limitations, including high costs, complex and time-consuming manufacturing, potential toxicities, and logistical challenges tied to patient-specific cell production. These barriers underscore the urgent need for next-generation approaches that can enhance accessibility, reduce complexity, and improve overall treatment efficiency.

We present an innovative cell-targeted lipid nanoparticle (tLNP) platform designed to deliver both DNA and RNA directly to T cells, enabling in situ CAR-T cell generation. This tLNP system consists of lipid nanoparticles carrying transposon DNA (encoding CAR), mRNA (encoding transposase), and targeting/activating protein components. We demonstrate that this approach can activate resting primary T cells and facilitate stable CAR expression without requiring traditional ex vivo expansion or external activation steps.

Beyond in vitro modification, we show that this non-viral vector drives functional CAR-T cell generation in vivo. We would like to present and discuss our latest data demonstrating successful generation of CAR T in an NSG mouse model injected with a human B-cell leukemia cell line. We will discuss that a single dose of tLNP (carrying DNA/RNA) results in durable CAR-T cell generation, tumor control, and prolonged survival. Furthermore, we will present a biodistribution analysis based on whole-organ imaging, using fluorescence imaging of the Cy5.5 label alongside bioluminescence imaging (BLI) of luciferase mRNA and CAR–luciferase minicircle DNA (mcDNA).

By transitioning CAR-T engineering from a labour-intensive, facility-based process to a simplified in vivo approach, this platform offers the potential to address key challenges in cell therapy—enhancing scalability, reducing costs, and increasing accessibility of CAR-T treatments.

Tackling CMC and Manufacturing Challenges of Complex LNP-based Biopharmaceuticals for Oncological, Gene editing and Vaccine Applications

Abstract:

Lipid nanoparticles (LNPs) are the most established platform for delivery of nucleic acid payloads. Their tunability and streamlined manufacturing facilitated an unprecedentedly rapid scale-up during the COVID-19 pandemic. However, being multicomponent, complex systems also poses a challenge of controlling their quality and safety. Emerging novel modalities like targeted LNPs are powerful for active targeting, but additionally increase the complexity of CMC and manufacturing.

Monolithic columns offer a unique solution for intact chromatography of LNPs. Advanced chromatographic platforms can address some of these CMC challenges, using multiple analytical columns and multiple detectors, operating without sample pre-treatment to simplify workflows. Multiple cargo formulations, like those in use for CAR-T therapies, CRISPR gene editing and combination vaccines need to be handled analytically properly by separating the cargos, which is achieved with such chromatography. Critical quality attributes, such as nucleic acid content, including free NA, fragmented NA, and NA-lipid adducts, can be determined, while also determining parameters like particle heterogeneity, lipid composition, and purity. This approach enhances the correlation between in vitro characteristics and therapeutic efficacy, accelerating LNP-based therapeutics and vaccines.

Enhanced Purification Methods for Manufacturing of Complex Lipid Nanoparticle Formulations with Multiple Payloads

Abstract:

Lipid nanoparticles (LNPs) are pivotal in nucleic acid delivery technologies, advancing therapeutic applications. However, challenges in manufacturing and characterization must be addressed, including ensuring drug product integrity, achieving sufficient recovery during purification while maintaining functionality, and producing a well-purified product. To achieve precise separation and a uniform, functional product, purification using CIM monolithic columns have been developed.

Purification and characterization become increasingly critical as product complexity grows, such as encapsulating multiple payloads or adding targeting moieties to the surface. Innovative analytics, like the PATfix LNP Switcher, play a crucial role in developing efficient processes. Payloads of varying sizes and types are characterized and encapsulated with high efficiency into LNPs with diverse compositions.

On larger scale, monolithic columns can be used for efficient purification of LNPs, as an alternative to standard processes (TFF and dialysis). Mobile phases for purification are optimized for high recovery, stability, and functionality. Compared to standard processes, the novel chromatographic method using CIM monoliths demonstrates superior activity and uniformity due to reduced size distribution and enhanced activity. Integrating monolithic technology at each stage, from RNA production to LNP integrity assessment, ensures consistent product quality and process efficiency, supporting the development of safe and effective LNP-based therapies.