Seminars & Webinars

Event: GEN
Date: November 16, 2023
Presenter: Aleš Štrancar, Sartorius BIA Separations

Adeno-associated virus (AAV) capsids are an effective mechanism for delivering gene therapies intended for therapeutic use. A critical production step is the separation of full capsids from empty ones, which are considered a product-related impurity in downstream processing. Partial and heavy AAV capsids in gene therapy products are problematic due to the potential carry-over of host cell DNA and plasmid DNA fragments. In large-scale vector manufacturing, separating full and empty AAV capsids requires high-resolution chromatography media and scale-to-scale reproducibility. To qualify chromatographic columns for separating capsids, it is important to use column release criteria that are based on the AAV elution profile. 

In this GEN webinar, Dr. Aleš Štrancar will discuss various chromatographic methods to consistently separate empty, partial, and full capsids in large-scale AAV manufacturing. He will introduce the CIMmultus High Reproducibility columns by Sartorius BIA Separations, which have better resolution and use narrow acceptance criteria to ensure consistent results. These columns allow for step gradient elution using the same buffer strength at any scale. Two order of magnitude removal of empty capsid should be possible at any scale.

Event: Cell and Gene Therapy Insights Webinar
Date: November 8, 2023
Presenter: Aleš Štrancar, Sartorius BIA Separations

Separation of empty and full AAV capsids with very similar chromatographic characteristics requires very high-resolution chromatography media and scale-to scale reproducibility within just few percent. Qualification of chromatographic columns using small molecules might be misleading when separation of big molecules or virus particles is in question. Column release criteria based on the AAV elution profile must therefore be adopted and used for all column sizes. Recently, the presence of partial and heavy AAV capsids in gene therapy products has raised concerns due to potential carryover of host cell DNA and plasmid DNA fragments. Their removal from the product is therefore crucial. To address these needs, Sartorius BIA Separations developed columns with better resolution and narrower acceptance criteria. These columns allow for batch-to-batch and scale-to-scale reproducibility within just 3% of the AAV8 empty capsid isoconductivity elution, and step-gradient elution using the same buffer strength at any scale. Two orders of magnitude removal of empty capsid should be possible at any scale.

Learn how to achieve:

• Five times better resolution of empty/full capsids
• Large scale removal of empty, partial and heavy AAV capsids
• Batch-to-batch and scale-to-scale reproducibility within +/- 3%

To address the recent needs Sartorius BIA Separations developed new CIMmultus HR line (HR stands for High Reproducibility) columns with better resolution and narrower acceptance criteria. These columns allow for step gradient elution using the same buffer strength at any scale. Two order of magnitude removal of empty capsid should be possible at any scale.

Event: Cell and Gene Therapy Insights Webinar
Date: October 12, 2023
Presenter: Aleš Štrancar, Sartorius BIA Separations

Separation of empty and full AAV capsids with almost same chromatographic characteristic request very high-resolution chromatography media and scale-to scale reproducibility within just few percent. Qualification of chromatographic columns using small molecules might be misleading when separation of big molecules or virus particles is in question. Column release criteria based on the AAV elution profile therefore needs to be adopted and used for all column sizes. Recently the presence of partial and heavy AAV capsids in gene therapy products gathered more attention due to potential carry-over of the hcDNA and plasmid DNA fragments. Their removal from the product is therefore becoming crucial. To address the recent needs Sartorius BIA Separations developed new CIMmultus HR line (HR stands for High Reproducibility) columns with better resolution and narrower acceptance criteria. These columns allow for batch-to-batch and scale-to-scale reproducibility within just 3% of the AAV8 empty capsid isoconductivity elution. Such columns allow for step gradient elution using the same buffer strength at any scale. Two order of magnitude removal of empty capsid should be possible at any scale.

Event: Cell and Gene Therapy Insights Webinar
Date: September 26, 2023
Presenter: Nejc Pavlin, Sartorius BIA Separations

Lipid nanoparticles (LNPs) have emerged as versatile drug delivery systems capable of encapsulating various payloads, including different nucleic acids. LNPs offer numerous advantages, such as protection of the payload from degradation, enhanced cellular uptake, and controlled release, making them promising candidates for therapeutic applications. However, the complex composition and structure of LNPs pose significant challenges for their characterization and analysis. In addition to physical properties such as size, polydispersity, surface charge and physical stability, parameters such as encapsulation efficiency, lipid identification and quantification, and the integrity of encapsulated nucleic acids are of great importance. Accurate and reliable analytical methods are essential to ensure the quality, efficacy, and safety of LNPs in various applications. By utilizing liquid chromatography as an analytical tool, researchers and formulation scientists can gain valuable insights into the encapsulation efficiency, lipid composition and quantification, load integrity and size of LNPs loaded with nucleic acids. These analytical methods contribute to the development of robust and efficient lipid-based drug delivery systems, ultimately enhancing the therapeutic outcomes of nucleic acid and protein-based therapies. In this work, we present a robust analytical chromatographic tool, called LNP switcher. It is equipped with two analytical columns of different modalities, independent pumps and multiple detector setup. This configuration enables us to monitor key quality attributes of LNPs production process.

Event: mRNA Process Development & Manufacturing Summit USA
Date: September 27, 2023
Presenter: Aleš Štrancar, Sartorius BIA Separations

Event: Cell and Gene Therapy Insights Webinar
Date: July 26, 2023
Presenter: Andreja Gramc Livk, Sartorius BIA Separations

Adeno-associated viruses (AAVs) are widely used vectors for gene therapies mainly due to their favorable safety profile and high transduction efficiency to various target tissues. With increasing demand for faster process development of advanced therapies using AAVs, rapid at-line analytical chromatographic solutions are becoming indispensable. 
PATfix analytical system with multiple detectors seamlessly delivers coherent process information, considerably speeding up process development and enabling a fast-paced delivery of the product to the market. 
Analytical methods, embedded in the PATfix system that enables at-line monitoring of common product and process related impurities, provide a critical platform for the rapid development and characterization of AAV-based gene therapy vectors.

Event: mRNA Applications in Discovery & Development
Date: June 20, 2023
Presenter: Rok Sekirnik, Sartorius BIA Separations

This talk presents case studies of leveraging the power of at-line HPLC monitoring for process development to increase purity and yield of mRNA production. It describes approaches to optimize plasmid purification and linearization, IVT reaction and mRNA purification, including purification methods to achieve more than 95% supercoiled plasmid purity, optimize linearization reaction, and increase of IVT reaction yield from 3-5 mg/mL to 10-12 mg/mL. Further, use of at-line analytics to monitor automated, continuous production in single use bioreactors to produce multi-gram amounts of mRNA are demonstrated. Oligo dT-based affinity purification is described with approaches to optimize dynamic binding capacity to reach up to 6 g/L. Multimodal chromatography and reverse-phase chromatography cases are discussed for capture of non-polyadenylated RNA, and removal of dsRNA.

Event: Cell and Gene Therapy Insights webinar
Date: April 5, 2023
Presenter: Rok Žigon, Head of Product-Application Area (AAV/Adeno), Process Development Viruses

Adeno-associated virus (AAV) is go-to vector for gene therapy treatments. In each AAV downstream process, one of the key and often a bottleneck step is enrichment of full capsids.  Along with scalability and capsid’s separation challenges one must account for sample heterogeneity and asses which analytics are adequate to distinguish between AAV sub-populations to deliver only the potent AAV product.
Chromatography on monolith columns offers efficient and scalable downstream processes.  Case study using Design-of-experiment approach of buffer selection and robust preparation for improved separation of empty and full capsids will be presented along with paramount considerations for scaling up. In addition, residual host cell proteins, host cell DNA, plasmid DNA, and endotoxin removal efficiency will be discussed.

Event: Cell and Gene Therapy Insights webinar
Date: March 1, 2023
Presenter: Blaž Bakalar, Product Manager PATfix platform

mRNA-based therapeutics represent a promising new modality for therapies and vaccines. However, industry-wide process understanding of the CQA and CPP interplay is still developing. As such, the lack of immediate feedback on the process state during process optimization, sometimes delayed by weeks and limited by throughput, represents a big bottleneck in mRNA commercialization. The PATfix mRNA analytical platform enables reliable at-line insight during process development and production of mRNA-based therapeutics. With an ability to resolve key IVT reaction components (NTPs, capping reagent, template and mRNA), both USP and DSP process insight can be gained, with a single analytical tool. The PATfix mRNA platform has been upgraded to include LNP encapsulation analytics, enabling at-line monitoring of a crucial process step on the path from mRNA drug substance to LNP drug product.

Presenter: Blaž Bakalar, Product Manager PATfix platform

Date: February 15, 2023

Event: 2nd mRNA Analytical Development Summit 2023

Abstract:

• PATfix mRNA analytical platform is a perfect tool for process developers looking to optimize their mRNA production and purification
• Three orthogonal analytical methods based on mix-mode, affinity and reverse phase allow for precise quantification and mRNA sample characterization
• The newly developed method for characterization of encapsulated mRNA molecules covers the last crucial part in mRNA therapeutic production

Speaker: Rok Sekirnik, Head Process Development for mRNA and pDNA

The recently demonstrated efficacy of mRNA-based Covid-19 vaccines has shown the promise of this therapeutic format, but also highlighted the need for higher efficiency of mRNA production to meet enormous needs for global vaccine supply. The production process typically involves 10-15 steps including plasmid production, plasmid linearization, an in vitro transcription (IVT) reaction, mRNA purification, and lipid nanoparticle (LNP) production. This webinar will discuss the versatility of chromatography as applied to mRNA production, starting with the purification of the key raw material (plasmid DNA) to final polishing of mRNA drug substance.

Speaker: Katja Vrabec, Project Manager

The development of fast and efficient processes for the isolation of extracellular vesicles (EVs) depends on the availability of chromatography media that meet the special fractionation needs of these products and analytical methods for process monitoring. Convective Interaction Media (CIM®) monolithic columns offer high binding capacities and low shear stress conditions for the purification of large biologics such as EVs. Multiple-detector PATfix™ technology was developed for monitoring sample composition and product detection in upstream and downstream processes. We will present the EV isolation process from clarified conditioned media and fractionation using CIMmultus™ chromatography columns. Testing of the raw materials, analysis of upstream samples produced in different cell lines, and analysis of downstream samples were performed using PATfix™ analytical approaches. The composition of EV populations was monitored through the detection of tetraspanins using immunofluorescent labelling and SEC analytics. High-throughput analysis of in-process samples and impurity composition was monitored using CIMac™ anion exchange columns.

Speaker: Mojca Tajnik Sbaizero, Project Manager 

rAAV downstream process using monoliths contains a series of steps that are mutually dependent. Therefore each step should be optimized for sufficient outcome. Standard 96-well design offers a great advantage for screening many samples and conditions. It supports small sample intake and process automatization. We will present example of multi-parallel screening of different mobile phases for rAAVcapture step using CIM SO3 0.05 mL Monolithic 96-well Plates. Optimization of capture step leads to increased process productivity and product purity, as well as improves later polishing step. High vector recovery and greater reduction of impurities were confirmed on preparative scale chromatography resulting in successful implementation of screening tools for process development optimization.

Speaker: Rok Sekirnik, Head Process Development for mRNA and pDNA

The recently demonstrated efficacy of mRNA-based Covid-19 vaccines has shown the promise of this therapeutic format, but also highlighted the need for higher efficiency of mRNA production to meet enormous needs for global vaccine supply. The production process typically involves 10-15 steps including plasmid production, plasmid linearization, an in vitro transcription (IVT) reaction, mRNA purification, and lipid nanoparticle (LNP) production. This webinar will discuss the versatility of chromatography as applied to mRNA production, starting with the purification of the key raw material (plasmid DNA) to final polishing of mRNA drug substance.

Event: Cell and Gene Therapy Insights webinar
Date: June 7, 2022
Presenter: Nejc Pavlin 

Pre-developed and validated PATfix analytical methods using different column chemistries enable:
-    Effective control over linear plasmid upstream and downstream,
-    IVT reaction optimization for mRNA synthesis,
-    Control of yield, purity and downstream process of mRNA,
-    mRNA formulation and stability analysis,
-    Scale-up optimization.

In this webinar, will discuss a robust analytical tool PATfix™ platform, and its specific methods for pDNA and mRNA analytics that enable efficient monitoring of key upstream and downstream process steps will be showcased.

Event: Cell and Gene Therapy Insights webinar
Date: April 12, 2022
Presenter: Rok Sekirnik, Head of Process Development, mRNA/pDNA

Key learning points:

• Benefits of implementing HPLC-based analytics in mRNA production workflow
• Principles of monolith chromatography and its use for mRNA analytics and purification
• Principles of IVT reaction and factors affecting rate and yield
• Principles of conversion from batch to fed-batch process

Event: Informa 1-day digital event, highlighting mRNA Applications and Therapeutics
Date: March 23, 2022
Presenter: Tomas Kostelec, Technology & Business Development Manager

Key learning points:

• Ensuring consistent and robust IVT production of mRNA 
• Achieving high yield and high purity in mRNA purification processes 
• Chromatography solutions for in process analytics and purification

Event: LCGC Live Webcast

Date: March 17, 2022

Presenters:  

Rok Zigon, Project Manager; Process Development Dept.

Blaz Goricar, Project Manager; Process Analytical Technology Dept.

Key learning points:

• Rapid downstream process of AAV using monoliths, with emphasis on PATfix analytical tools
• Orthogonal analytics are paramount for AAV characterization
• Integration using PATfix enables fast evaluation of empty-full ratio

Event: BPI Ask the Expert
Date: March 3, 2022 
Speaker: Rok Sekirnik, Head Process Development for mRNA and pDNA

In this webinar, we will discuss an HPLC-based analytical method using a multimodal ligand based on anion exchange/hydrogen-bonding ligand (PrimaS) to monitor the IVT reaction, which allows for simultaneous quantification of NTPs, capping reagent, plasmid, and mRNA within 3.5 min.

Event: Cell and Gene Therapy Insights webinar

Date: February 17, 2022

Presenter: Ales Strancar, Managing Director

Key learning points:

• One of the key challenges in manufacturing viral vectors is to increase the ratio between empty and full capsids.
• When the expression in the cell line results in less than 10% full capsid it is quite impossible to reach better than 90% full in the final product. It is therefore mandatory to optimise the USP to result in better empty/full ratio.  
• This can be efficiently realised by using at-line PATfix to allow for analysis of the full and empty capsids ratio directly in the harvest.
• The residual empty capsids can be removed by polishing step using different anion exchange columns.