Abstracts

Highlights and abstracts of oral and poster presentations at AdBIOPRO Symposium 2024

A novel in-line sensor system for real-time bioprocess monitoring - Erik Martinsson, ArgusEye

Highlights:

In-line, real-time monitoring and control of bioprocesses
Accelerated process development using integrated sensors
Real-time detection of IgG aggregates

Economic modelling of magnetic bead-based mAb manufacturing - Nils Brechmann, MAGic Bioprocessing

Highlights:

Economic modelling
Magnetic beads-based mAb manufacturing
Step integration

Exploring bioprocess operation modes for the expansion of human Mesenchymal Stromal Cells - Johanna Pechan, Dept. of Industrial Biotechnology, KTH

Abstract:

Due to their immunomodulatory properties, human Mesenchymal Stromal Cells (hMSCs) offer high potential as allogenic cell therapy for the treatment of several medical conditions. For a single dose treatment, a high number of cells are required, putting demand on the expansion process. Current state-of-the-art production make use of multi-layered tissue culture flasks, a process that is labor intensive while lacking in-process controls and monitoring. This could in turn lead to batch-to-batch differences and ultimately poor reproducibility in clinical trials. Therefore, efforts are being made to develop automated and controlled bioreactor-based cultivations for a cost-effective, scalable, and robust expansion process.

In this work, bone-marrow derived hMSCs were cultured on microcarriers in 100 mL stirred-tank bioreactors to explore culture operation modes (fed-batch and perfusion) compared to conventional repeated media changes. Instead of passaging by enzymatic digestion, fresh beads were added to ongoing cultures to increase the surface area and extend the expansion process.

While both fed-batch and perfusion resulted in a high expansion factor, the ease of a perfusion process encouraged further exploration. Higher perfusion rate resulted in faster growth rate, and substrate consumption was kept steady. With an optimized protocol for bead addition and feed rate, the process was improved compared to the repeated media changes. This holds promise for industrial implementation to increase the availability of stem cell treatments on a global scale.

Authors: Johanna Pechan, Kristina Engström, Nora Mac Key, Lina Sörvik, Véronique Chotteau

Towards an Understanding of Donor-to-Donor Heterogeneity in In Vitro Natural Killer Cell Production - Brian Ladd, Dept. of Industrial Biotechnology, KTH

Abstract:

Introduction: Autologous NK cell therapies for cancer treatments have shown great promise in recent years. However, as with any autologous cell therapy the donor-to-donor heterogeneity is a significant hurdle for the robust production of these ground breaking treatments.

Methods/Approach: A feeder-cell free NK cell production process using Peripheral Blood Mononuclear Cells, after apheresis generating a mixed population as starting material, was studied with single cell transcriptomics. Samples were taken from multiple donors and at several timepoints during the production, which involves NK cell proliferation. The sc-mRNA data were used to study cell-to-cell interactions, pathway enrichment, and phenotype changes.

Results: Studying the cell-to-cell interactions and pathway enrichment of all donors together led to the development of a donor independent map. This map could be used to better understand which interactions are consistent throughout the NK cell production process regardless of donor. Cluster annotation on identified cell types revealed differences in the expansion of certain subtypes of cells between donors, possibly revealing the cause of the observed heterogeneity.

Conclusion: The use of sc-mRNA provided unique insight into the cultivation and proliferation of NK cells. These insights enabled a greater understanding of the underlying interactions necessary for NK cell proliferation and possibly highlighted the cause of the donor-to-donor heterogeneity in this process. This information also provided key insights to tune the process, such as, by adding signalling molecules to strengthen beneficial interactions.

Authors: Brian Ladd, Markella Zacharoulli

Metabolome data for improvement of CHO-cell based bioproduction process performance - Meeri Mäkinen, Dept. of Industrial Biotechnology, KTH

Model-based optimization of culture medium for CHO cell perfusion culture by Mirko Pasquini, Dept. of Decision and Control Systems, KTH.

Highlights:

Why model-based optimization? Framework, advantages and disadvantages
Dealing with model uncertainties: three different approaches
A software library for model-based approaches in bioprocessing
Take-home messages, discussion and future directions

Transcriptomics-guided genome scale modeling of the kinetics of CHO cells under perfusion culture - Kévin Colin, Dept. of Decision and Control Systems, KTH

Highlights:

Objective: complex macroscopic modeling of the reactions rates for mammalian cells
Issue: too large number of possible pathways for large metabolic network VS too few data
Approach: use transcriptomics data in order to compute the pathways used by the cells
Novelty: adaptation of the method to the genome scale metabolic network
Experimental validation on CHO cells with significant modeling improvements.

Heterogeneity in Adeno-Associated Virus Transfection-Based Production Process Limits the Production Efficiency - Sofia Tunmats, Dept. of Industrial Biotechnology, KTH

Abstract:

Impact/Novelty: The novelty of this work lies in the use of single cell transcriptomics to study the heterogeneity of rAAV production, supported as well by phenotypic evidence.

Introduction: Of the four approved and five pre-registration in-vivo viral vector gene therapies, six use rAAV, while many others are in the pipeline. These upcoming therapies are placing a burden on current production methodologies requiring improvements in overall titres and lowering of the cost-of-goods.

Methods/Approach: Cultures of a HEK293T cell line were sampled at various time points during rAAV9 production. A triple plasmid transient transfection system was used for rAAV9 production. The sampled cells were studied with single cell and bulk transcriptomics, intercellular rAAV9 capsids were stained and analysed by flow cytometry, and supernatant and cell lysate samples were analysed by qPCR and ELISA.

Results: The results showed that there is only a small fraction of cells producing rAAV9 in the culture. The single cell transcriptomic analysis highlighted that there was a significant proportion, 41%, of cells not expressing any genes on at least one of the plasmids. Among the 59% rest of the cells only roughly half of them showed high expression of all three plasmids. The intracellular staining of rAAV9 capsids confirmed these results, by showing that only ~3% of cells had significant levels of rAAV9.

Authors: Brian Ladd, Sofia Tunmats

Development of small binders for biotechnological applications - Sophia Hober, Dept. of Protein Technology, KTH

Novel affinity resin: bispecific and fragment antibody purification in focus - Moumita Ghosh Laskar, Cytiva

Abstract:

Background: Therapeutic antibody formats like bispecific (bsAb) and fragment antibodies such as antigen binding fragments (Fab), single-chain variable fragments (scFv), and heavy chain variable domains (VH) are increasing in the pipeline. Protein A-based affinity resins typically have an affinity for the fragment crystallizable region (Fc), or both Fc and Fab regions, of an antibody. For bioprocessing of this diverse class of antibody therapeutics, an affinity resin with a targeted interaction with only a Fab region could be advantageous over the dual interactor affinity resins for efficient separation.

Results and novelty: We present results for capture of bsAb and antibody fragments, e.g., Fab, and variable heavy domain of heavy chain (VHH), that contain the VH3 sequence family, with a novel protein A resin (MabSelect™ VH3 affinity resin) that only interacts with the VH3 region. This resin enables new separation capabilities in mAb capture to address challenges with mispaired species and half antibodies in purification of bsAb.

Characteristics such as binding capacity, purification performance, and tolerance to harsh cleaning conditions i.e., sodium hydroxide can have a significant impact on process efficiency and process robustness. Here we also show the effect of these parameters based on application data.

Conclusion: MabSelect VH3 affinity resin interacts only with the VH chain of the VH3 sequence family of the human antibody. The traditional protein A interaction with the antibody’s Fc region is knocked out and allows efficient separation of bsAb and antibody fragments. In addition, this resin provides high dynamic binding capacity (DBC), excellent alkaline stability, and is now part of the Cytiva antibody resin toolbox.

Authors: Moumita Ghosh Laskar

A scalable single-use two step plasmid purification process - Hans Blom, Cytiva

Highlights:

Plasmid supply and quality is of key importance for manufacturing of viral vectors, mRNA, and DNA vaccines.
Rapid capacity of plasmids can be deployed through integrated, modular biomanufacturing solutions
The intensified two-step downstream process shortens timelines, reduces buffer consumption and flexible scalability.
Process meets industry acceptance criteria of GMP grade pDNA.

Purification of biopharmaceuticals on electro-responsive brushes - Armaghan Romiani/Hanna Tranchell, Nyctea Technologies

The Mini, a small Any-Use centrifuge, it's challenges and possibilities - Peter Thorwid, Alfa Laval

Highlights:

A versatile device for academia, start-ups, and big pharma.
Scalability, scalability, scalability and some other (less) important attributes.
Scaling down a hermetic centrifugal separator, is there such a thing as “too small”?
From a real machine to a device on the desktop, can Alfa Laval make “a reverse blender”?

Bioprocess for human pluripotent stem cells culture, lineage-directed differentiation and application for heart repair - Robert Zweigerdt, Dept. of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School

Highlights:

Human pluripotent stem cells culture (hPSC) suspension culture in stirred tank bioreactors
High density hPSC culture by rational process optimization
Directed cardiac differentiation of hPSC
Upscaling cardiac differentiation to 2L scale in protein free media
Towards heart repair by hPSC-derived cardiomyocytes

Integration of stem cell-derived pancreatic aggregates into FN-silk network for in vitro maturation - Kelly Blust, Dept. of Protein Technology, KTH

Abstract:

Impact/Novelty: Incorporation of pancreatic aggregates derived from pluripotent stem cells FN-Silk networks may be a novel cell therapy to treat type 1 Diabetes.

Introduction: Diabetes type 1 is a life-threatening disease that accompanies a life-long insulin dependency and limits the quality of life. An efficient way to treat diabetes is pancreatic islet transplantation. However, pancreatic islet transplantation has significant disadvantages, e.g., massive loss of islets and donor shortage. These problems could be solved by using insulin-producing pancreatic aggregates differentiated from human pluripotent stem cells combined with a biomaterial based on a unique 3D spider silk (FN-Silk) scaffold to protect pancreatic aggregates during the transplantation. FN-Silk is recombinantly produced in E.coli and is functionalized with a fibronectin motif to promote cell adhesion.

Methods/Approach: We have analysed the viability of pancreatic aggregates incorporated in FN-Silk networks during a cultivation period of 3 weeks. Furthermore, we compared the functionality of free pancreatic aggregates to incorporated ones by measuring the expression of c-peptide (insulin) and glucagon expression. Specifically, the transcriptome was analyzed on a single-cell level to compare free and FN-silk aggregates gene expression profiles and heterogeneity.

Results: We observed high viability of pancreatic aggregates incorporated in FN-silk networks over 3 weeks of cultivation and enhanced pancreatic islet function by increasing insulin and glucagon expression. In particular, the maturation of beta cells in FN-silk networks seems to be improved.

Conclusion: FN-Silk is an excellent biomaterial to incorporate pancreatic aggregates and could be used for transplantation to diabetic patients.

Authors:

Cellevat3dTM nanofiber-based microcarriers designed for improved upstream productivity of viral vector biomanufacturing - Laura Chirica, Cellevate

Abstract:

Viral vector manufacturing needs to ensure scalability while upholding high product yield, quality and process robustness. Adherent cell cultures systems known for their ability to produce high yields of viral vectors are associated with scalability challenges. Cellevat3dTM nanofiber-based microcarriers, a novel microcarrier format, providing three-dimensional (3D) cell culturing of adherent cells readily scalable in stirred tank (suspension) bioreactor systems, offers promising avenues for scale up of adherent cell cultures. Data shows that HEK293T cells adhering to the Cellevat3dTM nanofiber-based microcarriers form homogenous 3D cultures with high cell viability. Importantly, the microcarriers large surface area and lightweight nature allow for achieving high cell densities and reduced shear forces in bioreactors. Importantly, transient transfection of adherent HEK293T cells growing on Cellevat3dTM nanofiber microcarriers shows high (>90%) transfection efficiency and volumetric productivity (1014 vg/L) of human adeno-associated virus serotype 2 (AAV2) vectors. These findings demonstrate the potential of Cellevat3dTM nanofiber-based microcarriers as a scalable platform for adherent cell cultures in gene therapy applications, advancing bioprocessing strategies for enhanced yield, reduced time and costs leading to improved upstream bioprocessing productivity.

Authors: Linda Mellby, Jessica Giacomoni, Laura Chirica

Environmental sustainability and bioprocessing - Alois Jungbauer, University of Natural Resources and Applied Life Sciences (BOKU)

POSTERS

Autonomous operation and advanced control of integrated continuous downstream processes

Bernt Nilsson¹, Julius Lorek¹, Niklas Andersson¹, Madeléne Isaksson¹, Daniel Espinoza¹, Maja Sondell¹

¹Lund University

There has been a lot of attention on integrated continuous downstream processing during the last decade, since they promise more efficient processing due to being based on a sequence of integrated purification steps. This enables process intensification, minimization of storage tanks and hold-up times, smaller footprints, decreased buffer consumption and increased sustainability. The result is a complex, integrated sequence of multiple unit operations that performs straight-through processing of the target protein, with minimal time from expression to formulation, often based on an upstream perfusion system. At the same time there is a pressure to decrease the use of resources in process and product development on these new efficient and complex downstream processes. This work addresses this question and presents a platform and methodology for efficient development of complex downstream processes including automation, real-time control, heterogenous data handling and on-line quality monitoring.

Complex downstream processes for lab-scale development studies can be configured and implemented using commonly available hardware, like ÄKTA and Agilent systems. The automation can be developed in the research software Orbit based on Python, which creates an open, flexible, extendable, and scalable control system. Orbit gives support for integration of multiple chromatography columns, operation of downstream processes on multiple parallel setups, integrated online analytics, use of advanced feedback control and batch-to-batch control.

A case study for continuous production of a monoclonal antibody is used to illustrate the platform and the methodology. The downstream process is based on continuous solvent/detergent based viral inactivation, continuous Protein A capture, and polishing based on mixed-mode, operated in flow-through mode. The automated, continuous downstream process is configured with support systems for automated buffer management, HPLC-based quality analysis and real-time database. The result is an illustration of a downstream process that can be operated (almost) autonomously.

Investigation of the metabolism and fed-batch culture development of Jurkat cells

Markella Zacharouli¹, Véronique Chotteau¹

¹Dept. of Industrial Biotechnology, KTH

Relevance to Theme Selected: This study aims to give insight into the metabolism of T-cells and develop a feeding strategy for their culture, where Jurkat cells are studied as a model.

Impact/Novelty: It is critical to understand the metabolism of Jurkat cells as T-cell models which has great importance in the immunology community.

Introduction: Treatment with T-cells is very important for fighting cancer. Optimal cultivation of these cells is important to ensure the cell expansion with high viability and quality. This requires understanding of the cell metabolism and process development, areas which are still poorly investigated.

Methods/Approach: We studied the effect of the feeding of the nutrients, glucose and glutamine, and the effect of the by-products, lactate and ammonia, in Jurkat cells culture using RPMI1640 medium supplemented with serum. We identified the feeding conditions optimal for cell growth, with maintained low levels of lactate and ammonia, and investigated the effects of these by-products on the cell growth and viability.

Results: It was observed that the cells can use lactate as an alternative carbon source when glucose was depleted. However the cells did not consume lactate when low glucose amounts were regularly provided. The cells actively consumed glutamine, indicating a low rate of its biosynthesis, to the contrary of other human cells, e.g. HEK293. A fed-batch strategy was developed leading to a 20-fold expansion of the cells, while maintaining a high viability.

Conclusion: Understanding the effects of the nutrients and the by-products is an important step towards ensuring satisfactory Jurkat cell expansion and enables the development of a strategy for fed-batch culture. This work provides a better understanding of the immune cell metabolism and provide strategies for the manufacturing process development.

AAV Purification using an Electrochemical Controllable Polyelectrolyte Brush

Hanna Tranchell¹

¹Nyctea Technologies

Adeno-associated Viruses (AAVs) are a promising gene therapy vector due to their low toxicity, long-term gene expression, versatility in transducing various cell types, and site-specific integration into the chromosome. However, several challenges remain in AAV production, such as low yield and difficulties in gene incorporation, which necessitate a thorough purification process that both is labor-intensive and expensive. Conventional techniques, that rely on chromatography column resins, face severe limitations, especially with diverse AAV serotypes.

These experiments explores a novel purification approach using an electrochemical controllable polyelectrolyte brush (PE) brush. The PE brush serves as an adhesive surface for biomolecules, with high protein-binding capacity, and electrochemical signals induce controlled release. The electrochemical method avoids the drawbacks of conventional pH changes and hence, offers the potential to resolve impurity challenges.

The objective was to validate the electrochemical PE brush system for AAV purification. Three milestones were established: confirming immobilization and elution, evaluating product purity and quantity, and optimizing the system. Results demonstrate successful immobilization and electrochemical elution using both PAA and PDEA brushes. The system shows the potential to up-concentrate capsids and enhance the filled-to-empty ratio. Recirculation further improves binding efficiency.

Exploring bioprocess operation modes for the expansion of human Mesenchymal Stromal Cells

Johanna Pechan¹, Kristina Engström², Nora Mac Key¹, Lina Sörvik², Véronique Chotteau¹

¹Dept. of Industrial Biotechnology, KTH

²Cellcolabs

New manufacturing method for therapeutic cells

Armaghan Romiani¹, Gustav Ferrand-Drake del Castillo¹, Maria Kyriakidou¹, Oliver Olsson¹

¹Nyctea Technologies

We aim to develop a cost-effective method for the purification and isolation of therapeutic cells by creating a bioreactor capable of producing isolated, free cells directly from the bioreactor. Current methods for cell isolation, including centrifugation, filtration, and bead-labeling, are resource-intensive and significantly increase production costs. Additionally, polymer-coated magnetic beads, commonly used for cell capture, pose challenges due to their strong binding to cell surfaces, necessitating chemical cleavage that can harm cell viability.

Nyctea has pioneered a patent-pending electrode and process for the non-invasive, reversible capture and release of biopharmaceuticals using electric signals, eliminating the need for chemical agents. Our technology has proven highly effective for capturing large non-living objects such as viral vectors and lipid particles. We are now extending this approach to build a solution for isolation of cells used in cell therapy treatments, using isolated chromaffin cells, specifically endocrine cells from adrenal glands as a model system to demonstrate that our electrochemically controlled polyelectrolyte (PE) brush can capture and release cells using electrochemical signals. Our ultimate goal is to apply this method to immune defense cells, such as CAR-T cells, for therapeutic purposes. This non-invasive method requires no chemicals to modulate binding and release, offering a promising solution for efficient and scalable cell purification.

Intensified in-line sensor-controlled magnetic separation of monoclonal antibodies at pilot scale

Ines Zimmermann¹, Friederike Eilts¹, Sophia Hober², Véronique Chotteau³, Sonja Berensmeier¹, Michaela Dölle³

¹Chair of Bioseparation Engineering, Technical University of Munich

²Dept. of Protein Technology, KTH

³Dept. of Industrial Biotechnology, KTH

With the growing monoclonal antibody (mAb) market comes the need for efficient manufacturing. However, current mAb purification in the downstream processing (DSP) is restricted by predominating Protein A chromatography capture steps. Despite excellent purities and yields, conventional packed-bed Protein A chromatography is limited in throughput and process integration prospects caused by diffusional mass transfer. Aiming to enhance mAb DSP, we work on magnetic separation processes based on self-synthesized cheap iron oxide nanoparticles (MNPs). These MNPs have a large accessible surface area for protein interactions and are freely dispersed in the fluidic feed stream, reducing mass transfer limitations. The general non-porosity of the MNP particle matrix also enables the processing of unclarified feed streams, thus enabling process integration and intensification. In our studies, we work with two different Protein A-based affinity ligands that we site-directly immobilize onto MNPs – the pH-dependent ligand rSpA and the calcium-dependent ligand ZCa that enables mAb processing at mild pH. Our developed particles reach IgG capacities above the state-of-the-art (0.1 – 0.3 g/g), high recoveries (85-89%), and can be reused in multiple purification cycles. After process development on a small scale (µL-mL scale), we scale up the processes into an automated pilot-scale high-gradient magnetic separator (HGMS). The HGMS is equipped with several in-line sensors (UV absorbance, conductivity, pH) that record comprehensive process data and help us control and optimize processes. Our data of a successful proof-of-concept HGMS run using MNP@rSpA validate the simple scalability of magnetic separation processes. So far, our work demonstrates that magnetic separation can help overcome bottlenecks in current mAb DSP.

Lentiviral vector production in HEK293-T suspension cells with single-use bioreactors

Johanna Viiliäinen¹, Josefin Thelander¹, Christine Sund-Lundström¹, Greta Hulting¹, Ann-Christin Magnusson¹

¹Cytiva

Introduction:
Lentivirus vectors (LVV) are extensively used in the growing cell and gene therapy field for the delivery of nucleic acids into target cells, both ex vivo and in vivo. Vector production is a major contributor to the cost of manufacturing cell and gene therapies. Improvements in vector production, such as increased vector yield per batch, are therefore paramount as they can reduce manufacturing costs and increase the availability of various therapies. We describe our scalable, robust process to produce LVV using suspension cells in single-use bioreactors.

Methods/Approach:
To produce LVV, we used a four-plasmid transfection system consisting of two packaging vectors, one envelope vector, and a transfer vector carrying the green fluorescent protein (GFP) reporter gene. PEI MAX (Polysciences) was used as the transfection reagent for HEK293-T cells. Three production runs were performed both in Xcellerex™ XDR-10 stirred-tank bioreactor and ReadyToProcess WAVE™ 25 rocking bioreactor. We describe the production process from cell inoculation and expansion in the single-use bioreactors to the final harvests. Our analysis of the harvested material included a transduction assay for infectious titer measured in transducing units (TU), and p24 assay for viral particles (VP) measured in VP/mL.

Results:
Data from our LVV production batches show that we reached our target for harvesting materials for infectious titers (10⁷ TU/mL) and viral particles (10¹⁰VP/mL). The consistency between batches indicates a robust and reproducible production process.

Conclusion:
The study confirms that our LVV production protocol is suitable for rocking motion as well as stirred-tank bioreactors.

N-1 perfusion process for intensification of AAV production

Thomas Falkman¹, Johanna Viiliäinen¹, Srdja Drakulic¹, Christine Sund-Lundström¹, Ann-Christin Magnusson¹

¹Cytiva

The upstream process is still one of the bottlenecks of AAV manufacturing with long and tedious cell expansion steps. To increase the efficacy of viral vector production, perfusion can be used in the N-1 step to intensify the seed train.

We achieved viable cell concentrations of up to 35 MVC/mL at a CSPR value of 80 pL/c/d at both 1.5-L and 50-L scales. These results make high-density perfusion processes suitable for inoculum preparation (N-1) of HEK293 cells for production in bioreactors up to 2000 L in scale. Consistent performance with respect to cell growth, cell viability, and volumetric productivity was achieved in our process.

We show the development of a bench-scale (1.5-L working volume) proof of concept model and an N-1 perfusion process at the 50-L scale with comparable cell growth, metabolic profiles, and volumetric productivity.