We recently published our latest paper in Drug Discovery Today. Our authors Fernando Garcia and Eva Gefroh introduce a type of flexible biomanufacturing facility called J.POD®, capable of capitalizing on Intensified Continuous Biomanufacturing (ICB) to deliver against current cost and speed needs in the biopharmaceutical industry. We are pleased to share this featured review with industry and partners as we continue to work together to expand access to medicines that matter.
Abstract
In this work, process models were developed to capture the impact of biomanufacturing costs on a commercial scale and emphasize the way in which facility design and operation must balance meeting product demand while minimizing production costs. Using a scenario-based modeling approach, several facility design strategies were evaluated, including a traditional large stainless-steel facility and a small footprint, portable-on-demand (POD)-based facility. Bioprocessing platforms were compared by estimating their total production costs across different facility types and specifically illustrating how continuous bioprocessing has gained in popularity as a novel and cost-effective approach to manufacture high-quality biopharmaceuticals. The analysis showed how fluctuations in market demand have a dramatic effect on manufacturing costs and plant utilization, with far-reaching implications on the total cost to patients. Read the full article in Drug Discovery Today.
Tags: Articles & Whitepapers, Biologics
The importance of viral filtration studies
Biological therapeutics need to meet strict safety criteria. Virus safety is ensured through complementary manufacturing and quality control measures. Virus filtration is a critical element in this process, and viral filtration studies have become a key step in bioprocessing over the past decades. They are required by most regulators to bring a biologic to the market. The purpose is to assure that the final medicinal product is safe from the potential risk of viral contamination.
The quality of the design of such studies is key: when inappropriately designed, they may lead to undesired results such as non-representative filter fouling or virus breakthrough.
The characteristics of end-to-end continuous filtration
Continuous processing is a modern manufacturing method for biologicals, e.g. monoclonal antibodies (mAb). Consequently, also continuous viral clearance has to be investigated. Compared to batch filtration, continuous end-to-end processes present new challenges to the operation and validation of the viral filter:
Consequences for assessing viral clearance in continuous end-to-end filtration
This, in turn, means that assessment of viral clearance in continuous viral filtration may require studies spanning over several days and also novel virus-spiking strategies. This type of assessment is needed to adequately demonstrate a high level of viral clearance while ensuring the filters are not overloaded with virus and are consistent with real-life manufacturing conditions.
A team of scientists at Just- Evotec Biologics has performed studies to identify a robust virus filter that retains virus despite high load challenges and low operation pressures. Key findings were:
Summary and outlook
While bioprocessing technologies have evolved rapidly during the past decades, multiple factors such as increased cost, quality and production pressures are calling for further advances. At the same time, regulatory requirements are evolving and sophisticated safety testing has become a key prerequisite for market approval of novel biologics.
The team at JUST - Evotec Biologics has therefore kicked off an initiative to spearhead the development of next-generation bioprocessing technologies such as continuous end-to-end viral filtration.
Future work of the group will include optimization of the surrogate virus spiking and testing techniques as well as testing other virus filters for robustness of virus clearance at low flux/pressure conditions.
Interested in further information about Just-Evotec Biologics? Please visit our site.
For more information download the full poster.
Chinese Ovary Hamster (CHO) cells are the most common mammalian cell lines used for the mass manufacturing of therapeutic proteins as they can produce recombinant proteins on the scale of 3-10 grams per liter of culture. However, the expression of these recombinant proteins rely on random genomic integration events, which typically result in a widely heterogeneous cell population. Therefore, cell line development is taking up much time for the extensive pooling and clone screening to identify clones with high expression, growth, and product quality.
Also, the random integration precludes experiments such as variant library screening as this screening method needs stably overexpressing pools or libraries of molecules in a single cell culture. With random integration, the assessment of yield, degree of library enrichment, etc. is very complicated as it is difficult to determine whether resulting library members differ due to inherent characteristics of each variant or merely to due to variable genomic integration site(s).
To address this problem and to provide a time- and cost-efficient solution, Just-Evotec Biologics’ scientists have developed two targeted integration systems by generating two clonal CHO cell lines stably expressing enhanced green fluorescent protein (eGFP) reporter landing pads in genomic hot spots. The goal of this study was to compare two options to evaluate the one that might be most suitable for in-house usage.
For the study, the team combined several approaches to circumvent random chromosomal insertions, resulting in the precision and reproducibility associated with site-specific recombinases as well as the biased selection of genomic hotspots associated with a certain transposon.
Recombination was carried out either by Cre or PhiC31 recombinase. Subsequently, genes for the expression of three therapeutic protein molecules were used to test targeted integration. The cell lines were then assayed for yield and productivity as well as characterized for landing pad copy number and integration fidelity by targeted locus amplification (TLA) and PCR. Both cell lines expressed high levels of the respective recombinant protein. The scientists additionally tested for enrichment of cell subpopulations with fully saturated landing pads with ganciclovir (GCV) counterselection.
The results of these experiments were quite compelling: Genetic characterization of the altered cell lines showed correct targeting of landing pads. Post‐integration enrichment for fully saturated landing pads using GCV counterselection increased recombinant protein titer by 2–2.5‐fold and specific productivity by ∼3.4‐fold.
Finally, the team developed a small antibody library of ~100 variants through random pairing of 10 unique light chains and 10 unique heavy chains by transfecting this library into a cell line containing a single copy landing pad wherein each cell line would express a single variant. Puromycin selection was used to identify cells that had successfully taken up one of the variants and cell-sorting for variants that successfully paired and expressed. Finally, the identity of successful chain pairs was determined with next generation sequencing.
As a result, Just-Evotec Biologics has demonstrated proof-of-principle of targeted integration systems in the CHO host cell line, with consistent genome integration into expected landing pad sites and high productivity. Moreover, test cases using three antibody or antibody-fusion therapeutic molecules showed similar levels of productivity. Finally, the team demonstrated that library screening or CHO display is feasible with the 100-member variant library. The study also reveals preliminary data from ongoing work to build upon these targeted integration systems, which includes isolating a single-copy landing pad cell line and the development of a CHO display platform.
Although additional work and optimization is still needed, the great advantage of this approach lies in the predictability with regards to the chromosomal integration of transgenes of interest. Among others, a key advantage of this approach is its ability to combine eGFP as a reporter gene and transposon-mediated integration to establish high-expression landing pad cell lines. With a significant decrease in heterogeneity between clones, it is possible to be able to reduce extensive pool screening, large scale cloning, and clone screening efforts.
All in all, defining and targeting predefined locations that promote high expression of an exogenous protein allows to develop cell lines expressing different recombinant protein therapeutics with a high degree of specificity and reproducibility - which, in turn, and will save a lot of time and costs.
For more information, please read the study or have a look at our poster.
Full poster title: Development of recombinase-based targeted integration systems for production of exogenous proteins using transposon-mediated landing pads
Summary of the poster:
 |
Thierry Wurch SVP, Integrated Biologics Discovery | Evotec Thierry joined Evotec in May 2022 as SVP Integrated Biologics Discovery. He has more than 22 years of expertise in the antibody R&D space primarily in Oncology and immune-oncology. Thierry held positions of growing strategic importance from head of an antibody discovery and engineering department (Pierre Fabre, 2003-2011), head of immunotherapy discovery research activities (Servier, 2011-2017) then moving to BD-oriented activities and heading external innovation for Oncology (Servier, 2017-2020 and Ipsen 2020-2022). Thierry is also Chairman of the antibody sub-committee at NC-IUPHAR, member of the Editorial board of mAbs Journal and Distinguished Advisor of The Antibody Society. |
[hs-form id="052874a9-75e5-4960-9b82-931f9dd753b7" show="hide on login" url="https://player.vimeo.com/video/719092289"]
Tags: Presentations, Videos & Webinars, Biologics
Antibodies generated in the lab are important as potential treatments for a broad spectrum of diseases, in particular infectious diseases caused by viruses. They can be obtained either by animal-derived B cells or from antibody library display platforms. Evotec’s strategy for the optimal path to obtain lead candidates is offering access to both sources of antibodies for discovery, coupled with the exploitation of state-of- the-art technologies to ensure success for a broad range of targets and disease states. In addition, selected lead candidates can be further optimized using powerful computational platforms to enhance productivity, manufacturability, and formulation stability. This is the end-to-end J.Design biologics platform, which is fueled by the front-end discovery platform, J.HAL™ (Just Humanoid Antibody Library) and associated data-driven, company-wide machine learning methodology.
By using artificial intelligence (AI) and machine learning (ML), J.HAL can generate novel, humanoid antibody sequences that both represent natural repertoires and are biased towards desirable features. To enable properties such as broad target and epitope engagement, focused efficacy, and suitable developability, Just-Evotec Biologics has devised an Antibody-GAN (Generative Adversarial Network), a new synthetic approach to designing a novel class of antibody therapeutics, which is termed humanoid antibodies.
At the conferences International Conference on Antiviral Research (ICAR) 2021 and Antibody Engineering & Therapeutics Europe 2022, researchers from Evotec and Just-Evotec Biologics introduced results obtained by using GAN to generate novel sequences, which mimic natural human response and provide the necessary diversity and developability features.
Competing Neural Networks
GAN is based on competing, deep layer neural networks that learn and produce the features of the mature human antibody repertoire, including sequence characteristics and structure properties, allowing for the encoding of key properties of interest into diverse libraries for a feature-biased discovery platform. It works to:
The GAN network is trained by using hundreds of thousands of human antibody sequences to recognize legitimate human v-genes. The generator network generates random sequences to fool the discriminator while continually receiving feedback from the discriminator on sequence validity. Over time, the two networks get progressively better at their tasks. After full training, the Antibody-GAN generator is eventually able to produce fully human, novel antibody sequences for the germline for which the GAN was trained.
Antibodies targeting SARS-CoV-2
To demonstrate the usefulness of this platform, the researchers used their newly constructed, 1 billion theoretical diversity phage Fab library with the intent to discover antibodies to the SARS-CoV-2 spike protein. Candidates that specifically bound SARS-CoV-2 spike protein and did not bind an irrelevant antigen were further characterized for dose-dependent binding using AlphaLISA technology. In the primary “yes/no” binding screen a total of 73 unique antibody sequences specific for SARS-CoV-2 spike protein were identified. The researchers then performed binding assays using unpurified transfection supernatants and later reproduced the results with purified material. The candidate antibody supernatants that specifically bound SARS-CoV-2 spike protein were subsequently tested for their ability to block binding of this protein to human ACE-2 receptor. The team identified multiple antibodies that effectively blocked spike human ACE2 receptor interaction, demonstrating the feasibility to screen unpurified transfection supernatants for functional activity. After further rounds of panning, the top candidates expressed at flask scale were purified and tested for SARS-CoV-2 neutralization ability across multiple strains. The researchers identified multiple candidates with neutralizing activity against several strains of SARS-CoV-2. Nine of these antibodies exhibited blocking activity of the spike protein to the ACE2 receptor in an in vitro functional assay. Of note, all antibody data shown here were from native library candidates without any affinity maturation.
The presentation demonstrates that applying machine learning algorithms in antibody discovery “promotes efficient learning from the least expensive and most abundant data encoded in the DNA of antibodies, to validation of this learning through less abundant, more expensive, but most relevant data from GMP manufacturing at full commercial scale,” stated James N. Thomas, retired Executive Vice President, Global Head of Biotherapeutics and President U.S. Operations at Just - Evotec Biologics. “This is a systems approach to platform definition and continuous improvement, and it is unique in the industry, made possible by a number of factors that will be difficult for others to replicate."
To learn more about Evotec's capabilities read our related poster.
Key Takeaways:
Key Takeaways:
Improving virus clearing studies in recombinant protein production
Chinese hamster ovary (CHO) cells are the most frequently used mammalian host cells for the industrial manufacturing of recombinant protein therapeutics. They can produce recombinant proteins on the scale of up to 10 gram per liter of culture. However, they are also known to contain type‐C endogenous retrovirus (ERV) sequences in their genome and to release retroviral‐like particles. Although evidence for their infectivity is missing, this has raised safety concerns, and regulatory agencies require demonstration that the purification process removes or inactivates viruses.
Viral clearance validation is assessed through “spiking studies”, whereby model mammalian viruses are introduced into process material which then undergoes the purification technique to be tested. Viral quantity before and after processing is determined through infectivity or qPCR assay. As these studies use live viruses, they require specialized Biological Safety Level laboratories (BSL) and experienced personnel and can create a substantial bottleneck because typically only 3rd party facilities are qualified to perform these studies.
As an alternative, Just - Evotec Biologics is in the early stages of establishing a high-throughput process using commercially available purified retrovirus-like particles from Cygnus Technologies LLC. These particles are non-infectious and mimic the physicochemical properties of live infectious viruses. By using these particles as spiking agents, the retroviral clearance capability of downstream unit operations can be studied, assessed, and quantified by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Usually, this is performed at bench scale using chromatography columns.
In a poster presented at this year’s ACS spring conference entitled High throughput optimization of chromatography steps for viral clearance using retrovirus-like particles (RVLPs), researchers from Just-Evotec Biologics detailed the high-throughput workflow for the analysis of RVLP content for rapid analysis of in-process samples.
The research team compared common bench scale chromatography runs with a plate-based screen using resin-loaded filter plates and a liquid handling robot. While at bench scale, only a single set of run conditions can be tested at a time, the plate-based screening can examine up to 24 different run conditions simultaneously. It also uses less RVLP stock solution. The researchers expect that plate-based screening of RVLPs will not only save time and costs, but also allows for better evaluation and confidence before formal viral clearance studies.
iPSC and age-related diseases: Case study AMD
Age-related macular degeneration (AMD) is a leading cause of vision loss in people over the age of 50, accounting for 90% of blindness in this population. There are dry and wet forms of the disease and to date, there are limited treatment options for the wet form and none for dry AMD. The exact cause of the disease is unknown, but it is suspected that it results from a combination of hereditary and environmental factors, with smoking and diet being implicated. Globally, more than 190 million people are affected by AMD and this figure is expected to increase to more than 285 million by the year 2040. The estimated global cost of the disease currently stands at $343bn, with $255bn in direct healthcare costs.
Significant research effort has been targeted towards identification of the genes involved in permanent vision loss through photoreceptor and/or retinal pigment epithelium (RPE) cell dysfunction or cell death. So far, mutations in over 250 genes are known to be involved. Despite a good understanding of the genotype-phenotype relationship in AMD, this has not translated into predictive in vitro and in vivo models to understand disease mechanisms.
Partnership on iPSC-derived RPE cells
To drive the search for treatments for this devastating disease, it is vital to identify and develop new models which enable elucidation of retinal disease mechanisms and reliably predict the efficacy of therapeutic compounds. To address these challenges, Evotec has teamed up with the Centre for Regenerative Therapies (CRTD) in Dresden, Germany. The CRTD has a longstanding interest in degenerative processes and has contributed significantly to our understanding of retinal diseases in recent years. The joint TargetRD project is based on Evotec’s know-how in induced pluripotent stem cell (iPSC) technology, generating iPSC-derived RPE cells from AMD patients.
RPE cells are essential for visual function and a key component for light detection by photoreceptor neurons. They also are crucial for maintaining the blood-retina-barrier, for the transport of diverse biomolecules, ions, and fluids in and out of the retinal tissue, and recycling of the visual chromophore retinal molecule.
Even more important is their ability for phagocytosis: A single RPE cell is in contact with about 30 photoreceptor cells and responsible for the phagocytosis and removal of the distal portions of the photoreceptor outer segments that are phagocytozed in the course of a day. This is an important process with up to 10% eliminated daily, meaning the entire population of photoreceptor outer segments is turned over every 2 weeks. Maintaining, repairing, or replacing RPE cells therefore is crucial for the management of AMD, but also for other retinal degenerative diseases.
So far, research has been hampered by limited access to RPE cells. There are immortalized retinal cell lines available, but they lack the typical cell morphology or function of their in vivo counterparts, e.g. pigmentation, polarization, and expression of certain proteins. Likewise, artificial organoids, so-called 3D-cups, are not ideal since they are difficult to grow and differentiate and not suited for high-throughput profiling of compounds.
This situation is now improving as Evotec and CRTD succeeded in developing a protocol to efficiently and robustly produce high quality human iPSC-derived RPE cells at industrial scale from patient cells. This means that partners can not only investigate disease pathology directly in this highly relevant retinal cell type but for the first time also study disease phenotypes and mechanisms within the context of a patient’s genome. Moreover, the TargetRD platform enables the study of individual, overlapping functional and morphological changes from iPSC-RPE cells derived from patients with different genetic backgrounds, providing an opportunity to unravel complex AMD disease phenotypes.
Promising first results
Already, the partners were able to show the utility of the TargetRD platform by analyzing iPSC-RPE cells carrying a patient mutation resulting a lysosomal storage disorder. Amongst other symptoms, patients with this type of mutation develop severe retinal degeneration, leading to complete blindness early in life. Using the various phenotypic and functional assays established, it was possible to demonstrate impaired trans-epithelial resistance in patient cells. This indicates that in this case, patient RPE cells are unable to form the tight monolayer required for normal RPE cell function. Furthermore, patient RPE cells have, as expected, impaired lysosomal activity and are unable to phagocytose photo- receptor outer segments (POS) at the same rate as control RPE cells. Since all assays are able to support high-throughput screening, the partners can use the TargetRD platform to identify phenotypic and functional disease phenotypes from patient cells, enabling novel drug discovery approaches.
Furthermore, the project combines Evotec’s drug discovery expertise and the academic excellence of the CRTD to achieve significant progress towards developing therapies much needed by the patients.
Both partners believe this is a very promising approach that enables successful drug discovery programs for retinal degenerations coupled to a high likelihood of successful translation into the clinic.
Tags: Blog, Biologics, Age-Related Diseases