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Seminars & Webinars

2022

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™ HPLC 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.

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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.

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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.

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

Pre-developed and validated HPLC 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™ HPLC platform, and its specific methods for pDNA and mRNA analytics that enable efficient monitoring of key upstream and downstream process steps will be showcased.

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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

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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

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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 HPLC analytical tools
  • Orthogonal analytics are paramount for AAV characterization
  • Integration using PATfix enables fast evaluation of empty-full ratio

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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.

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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 HPLC 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.

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Event: mRNA-Based Therapeutics Summit Europe

Date: January 2022

Presenter: Rok Sekirnik, Head of Process development for mRNA and pDNA

Abstract:

In vitro-transcribed messenger RNA-based therapeutics represent a relatively novel and highly efficient class of drugs. Current clinical efforts encompassing mRNA – based drugs are directed to three categories: mRNA vaccine, mRNA therapy and protein replacement therapy. As mRNA vaccine became the front runner to fight the COVID-19 pandemic, challenges surrounding their process development and manufacturing became readily apparent:

  1. mRNA manufacturing is not yet a platform approach. Standardized products along the processes are needed
  2. mRNA is a relatively large molecule, which leads more challenges for downstream purification.
  3. mRNA is intrinsically unstable and prone to degradation.
  4. High raw material cost is limiting mRNA commercialization.

This presentation provides a detailed overview of current mRNA manufacturing approaches, highlights challenges and recent successes, and offers perspectives on the future of mRNA manufacturing.

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2021

Published: Cell & Gene Therapy Insights 2021; 7(12), 1661

Date: December 20, 2021

Presenter: Maja Leskovec, Head of Process Development Viruses (viral vectors and vaccines)

Watch the video or read the poster to learn:

  • Importance of sample pretreatment before first chromatographic step
  • Rapid downstream process of AAV with emphasis on chromatographic step of CIMmultus SO3 capture
  • Three different ligands full enrichment of AAV
  • Why orthogonal analytics is the key in downstream development and what are the options

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Published: Cell & Gene Therapy Insights 2021; 7(11), 1409

Date: December 2, 2021

Presenter: Rok Sekirnik, Head Process development for mRNA and pDNA

Watch the video or read the poster to learn:

  • Non-affinity chromatography with PrimaS provides purification for IVT processes with co-transcriptional polyadenylation
  • The Sartorius mRNA purification toolbox provides a scalable chromatographic solution for commercial manufacturing of mRNA therapeutics
  • CIMac analytical HPLC columns can resolve individual IVT reaction components in minutes, resolving the analytical bottleneck in optimization of IVT reaction conditions
  • Rapid HPLC analytics can improve in-process and quality control of the mRNA production process

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Published: Cell & Gene Therapy Insights 2021; 7(10), 1437

Date: November 15, 2021

Presenter: Nejc Pavlin, Project Manager in Process Analytics Development department

Watch the video or read the poster to learn:

  • The importance of pDNA as an enabling product in the production of mRNA, AAV, and other therapeutic vectors
  • How the PATfix™ pDNA platform using CIMac™ pDNA column offers fast and efficient solution for problems related to pDNA upstream and downstream processes
  • How the PATfix™ pDNA platform can be used to optimally adjust pDNA production and purification procedures of pDNA according to your needs, increasing pDNA yield and purity and reducing the cost of its production

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Presenter: Ivana Petrović Koshmak, PhD

Event: Late Stage Bioprocessing & Viral Vectors 2 11 November 2021

Date: November 3, 2021

Abstract:

Adeno associated virus (AAV) is the leading vector in the field of gene therapy because of its low toxicity, good overall safety profile, and ability to maintain stable expression for long periods of time. It is therefore crucial to develop a robust and high efficiency platform for its manufacturing.

One of the key challenges in manufacturing viral vectors is to increase the ratio between empty and full capsids. The most efficient way is to design the USP to result in less than 10% of empty capsids. This can be realised by process optimisation using at-line HPLC to allow for analysis of the full and empty capsids ratio during the virus expression.

Fast and reliable in HPLC methods to allow for process optimisation USP and DSP, and assessing the purity of the final product using PATfix system will be presented.

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Published: Cell & Gene Therapy Insights 2021; 7(8), 1125

Date: October 4, 2021

Presenter: Hana Jug, Project Manager in process development for viral vectors and vaccines

Watch the video or read the poster to learn: 

  • Why downstream processing remains one of the main bottlenecks in adenoviral vector manufacturing 
  • How an adenoviral vector purification platform using CIMmultus™ QA as the key purification step, secures a fast and robust process with better purity 
  • CIMmultus™ QA offers high capacity and high yields of adenovirus, results in cost reduction and overcomes raw material supply bottlenecks 

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Presenter: Aleš Štrancar

Event: BPI and Cell & Gene Therapy Bioprocessing & Commercialization 2021

Date: September 28, 2021

Abstract:

Adeno associated virus (AAV) is the leading vector in the field of gene therapy because of its low toxicity, good overall safety profile, and ability to maintain stable expression for long periods of time. It is therefore crucial to develop a robust and high efficiency platform for its manufacturing.

One of the key challenges in manufacturing viral vectors is to increase the ratio between empty and full capsids. The most efficient way is to design the USP to result in less than 10% of empty capsids. This can be realised by process optimisation using at-line HPLC to allow for analysis of the full and empty capsids ratio directly in the harvest.

The residual empty capsids can be further removed by polishing purification step using different anion exchange columns.

Fast and reliable in HPLC methods to allow for process optimisation and assessing the purity of the final product using PATfix system will be presented.

Attachments

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Presenter: Aleš Štrancar

Event: BPI and Cell & Gene Therapy Bioprocessing & Commercialization 2021

Date: September 30, 2021

Abstract:

Global demand for pDNA production is at an all time high, due to increased need from Gene Therapy ramp-up. pDNA, as an enabling product, is critical in production of mRNA, AAV and other therapeutic vectors. Increasing yield and purity in the production of pDNA is a vital step in meeting such demand. Supporting reliable in-process control during pDNA purification, PATfix pDNA analytical platform is enabling rapid process development and optimization while providing a reliable analytical platform for production runs.

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Presenter: Rok Sekirnik

Event: mRNA Therapeutics Summit

Date: July 14, 2021

Abstract:

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. Typical mRNA production process involves three key steps: 1) plasmid DNA (pDNA) production in supercoiled (sc) isoform, linearization and purification, 2) in-vitro transcription (IVT) reaction and 3) mRNA purification. Here we present a chromatographic toolbox for integrated mRNA production from pDNA to mRNA purification, including in-process analytics.

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Presenter: Aleš Štrancar

Event: TIDES mRNA Digital Week

Date: June 28, 2021

Abstract:

A new purification approach starting from E.Coli all through to mRNA production is presented here. This new approach integrates a pDNA linearisation step before polishing (removal of linear and open circular isoforms) of plasmid DNA. The polishing step, placed after enzymatic linearisation, purifies linear pDNA from enzyme and other unwanted process impurities. The linearised plasmid DNA is then used in IVT for production of mRNA. Fast in-process analytics allows for transcription number well above 100 in reproducible manner.

This approach further introduces mRNA purification tools for improved contaminant removal. The altered sequence of purification and linearisation reduces the overall number of purification steps required, improves recoveries, while the complete process results in extra low protein impurity and very efficient dsRNA removal.

The participants would get insight into the mRNA production steps, their optimisation and tools to allow for robust process. The analytical tools to allow for safe product will be highlighted, too.

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Presenter: Ivana Petrović Koshmak

Event: 2nd Annual Gene Therapy Analytical Development EU

Date: May 26, 2021

Abstract:

In-process analytics of vector capsid production is a critical optimization target in development of AAV-based gene therapy products. In this presentation we introduce a fast at-line HPLC based system that enables differentiation of vector capsid and empty capsid production in transfection mixtures. Case study results  demonstrating the effects of different process variables on capsid production and assembly are shown and insights about purification are also discussed.

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