Recombinant adeno-associated virus (AAV) and lentiviral vectors (LV) are vehicles for direct delivery of therapeutic genes to patients' cells. In the coming years, the use of AAV and LV in clinical applications is projected to increase exponentially. Maximizing production of safe and effective viral vectors is essential to match this growing need. In this talk, we present optimization strategies for the transfection step of vector production to generate high titers of both AAV and LV using TransIT-VirusGEN® Transfection Reagent. The VirusGEN® AAV and LV Transfection Kits push the limits of high-titer virus production, which expand the manufacturing capabilities for gene and cell therapies.

Learning Objectives:

1. Identify critical parameters for optimizing viral vector production using VirusGEN®

2. Explain how VirusGEN® supports development and manufacturing of gene and cell therapies

The pre-packed disposable chromatography market is increasing rapidly. There is a growing need for manufacturing flexibility and single-use systems. You are invited to find out more about what Astrea are offering clients and customers in this field, from scouting columns through to pilot/process scale columns and their scalability.

One challenge pharmaceutical companies face, is moving promising biotherapeutic products into the clinic as fast as safely possible. Devoting the correct amount of time and resources to early stage products can streamline the late stage and commercial development activities. A cell line change between Phase I/II and Phase III can result in spending significant quality, and regulatory resources to demonstrate comparability between the 1st and 2nd generation products. Early DOE screening studies can select for cell lines that have the most robust manufacturability profile, perhaps negating the need for a cell line switch. As the project progresses, process characterization studies can be very labor intensive and are usually delayed until a product has proven itself worthy of investment through success in early clinical trials. Definitive Screening Designs1  have potential to be a one-stop shop for screening important parameters and optimizing settings with low resource costs. Advanced machine learning methods have revolutionized the analysis of large data sets where the data can be divided into portions for training, testing and validation. During bioprocessing development, the datasets are usually small and until now have not been suitable for portioning the data into different training and validation sets. The fractionally weighted bootstrapping method2 uses the same data set for training and validation.  This auto-validation technique has been employed to successfully de-risk model testing experiments that complete the DOE lifecycle.

1  Jones, Bradley and Nachtsheim, Christopher 2011 Definitive Screening Designs with Added Two-Level Categorical Factors  ASQ.org Vol. 45, No. 2, April 2013

2   Ramsey, Phil and Gotwalt, Chris 2017  Model Validation Strategies for Designed Experiments [Power point Slides] Fall Technical Conference https://falltechnicalconference.org/wp-content/uploads/2018_Presentations/3B-FTC-Ramsey-Final.pdf

Learning Objectives:

1. Identify phase appropriate DOE strategies to minimize development timelines and maximize product life-cycle efficiency

2. Recognize the importance of system variability on the DOE settings and levels explored

3. Understand how to use auto-validation to identify model terms

Although affinity tag methods for protein purification have greatly accelerated research in laboratories throughout the world, the presence of the tag on the purified target can modify the target and has prevented these methods from being used for clinical applications.  To address these limitations, we have spent over 20 years developing a truly practical self-cleaving tag technology.  In practice, once the tagged target is bound to its cognate affinity resin and washed, the tag is induced to release the tagless target from the bound tag by a small shift in pH.  This method thus allows the elution of a native target from an affinity resin, without the need for exogenous protease addition or the requirement for a downstream tag removal step.  We have now been able to demonstrate this system for proteins expressed in E. coli and mammalian cells, where we have been able to purify “tagless and traceless” targets that include enzymes, antigens, complex glycoproteins and potential biosimilar therapeutics.  In designing this system, we have focused carefully on aspects that will make it more attractive to the biopharmaceutical industry.  The result is a resin that is compatible with conventional buffers and chromatography systems, has a competitive price point at large scale, and can be reused for multiple manufacturing cycles.  It is our hope that this technology will provide a bridge between the research and manufacturing worlds of protein purification, where a single platform can now be used from the earliest high-throughput discovery efforts through full-scale clinical production.

Learning Objectives:

1. Understand the basic design of this self-cleaving tag technology

2. Evaluate the performance of the technology in different contexts using case studies

3. Clarify the limitations and caveats to the technology for proposed applications

Chinese hamster ovary (CHO) cells are the most widely used cells for producing biopharmaceuticals. Engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the Chinese hamster genome were computationally predicted and are frequently inaccurate. Here, we revised TSS annotations for 15,308 Chinese hamster genes and 4,478 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. The experimental realignment and discovery of TSSs now expose previously hidden motifs, such as the TATA box. We further demonstrate, by targeting the glycosyltransferase gene Mgat3, how accurate annotations readily facilitate activating silent genes by CRISPRa to obtain more human-like glycosylation. Together, we envision our annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

Learning Objectives:

1. The mapping of TSSs in Chinese Hamster Ovary cells is improved using nascent capped-RNA sequencing methods combined with samples across multiple hamster tissues.

2. The experimental TSSs is used for CRISPR activation of the silenced protein Mgat3 in the Chinese Hamster.

The scale-up and characterization of industrial bioprocesses is to a large extent still based on empirical knowledge. With advanced algorithms and graphic processing units in the computational fluid dynamics field the inner workings of these bioprocesses can be examined in detail for the first time. This lays the foundation for new apparatus designs as well as for the optimization of operating parameters and process characterization on the computer. Using the example of aerated stirred bioreactors, I will show how this new simulation tool integrates – and how it accelerates – your workflow from the lab to the market.

Learning objectives:

1. Explain new algorithms for the computational fluid dynamics

2. Define the usage of new CFD tools for accelerating the path from the lab to the market

Cell encapsulation within semi-permeable devices represents a local immunoisolation strategy for cell-based therapies without the need for systemic immunosuppression. The encapsulation system allows for the diffusion of nutrients into the device which are important for cell survival, and the therapeutic payload produced by the cells to diffuse out in order to regulate the disease of choice. The barrier provided by the semi-permeable membrane prevents the entrance of immune cells that would otherwise destroy the therapeutic cells. For some time, encapsulation strategies have been applied to insulin producing islets of Langerhans transplantation and have shown long-term diabetes correction in many rodent models. However, these same strategies have failed when applied to a small number of clinical trials with diabetic patients. The foreign body response (FBR) to the biomaterials comprising the devices has been the major issue when testing in larger animal models. FBR results in the deposition of a dense ECM and cellular network covering the devices that restricts nutrition of the cells inside and compromises viability. Our lab is developing novel biomaterials and drug delivery strategies that can resist the foreign body response and fibrosis of implantable materials. Without fibrosis covering these encapsulation devices, free nutritional exchange for the therapeutic cells inside ensures continued secretion of their therapeutic payload for regulation of the disease of interest. This talk will discuss the discovery of a number of immune modulatory materials that have been developed in the Anderson/Langer lab that can reduce fibrotic activation and can lead to the protection of allogeneic islets in non-human primates for up to 4 months without any immune suppression.

Learning Objectives:

1. What is the major contributor to the failure of immunoisolated cell therapy devices in patients

2. Identify key immune cell types for immunomodulatory biomaterials

The advancement of microbioreactor technology in recent years has transformed early- and mid-stage process development. Their monitoring and control capabilities not only promote the rapid accumulation of process knowledge, but also led to an increased scalability when compared to traditionally used systems such as shake flasks and microtiter plates. This study seeks to establish a framework for the micro-Matrix microbioreactor (Applikon-Biotechnology BV) as process development tool. Using experimental methodologies, the system was initially characterised for oxygen mass transfer and mixing time. A problem specific to pH-controlled small- and benchtop-scale bioreactors was highlighted - the efficient removal of CO2 at the investigated scales caused the pCO2 to drop to 0 mmHg during the cultivation. This was shown to negatively affect the maximum viable cell concentration. Therefore, a combination of matched mixing time and matched CO2 addition profile was proposed as a scale-down criterion between 5 L STR and micro-Matrix. Growth trends, maximum viable cell concentrations, final titre, and glycoprofiles were nearly identical at both scales. The scale-down model was then employed to optimise a bolus feeding regime using response surface methodology, which led to a 25% increase of the space-time yield and the final titre.

Learning Objectives:

1. Understand the methods used for characterisation of microbioreactors

2. Evaluate different methods for scale translation from shaken to stirred bioreactors

3. Learn about application of Design of Experiment methodology for process optimisation

Potency is a critical quality attribute of biological products, defined by the US FDA as the specific ability of the product to cause the required therapeutically clinical benefit. Potency assay will leverage the product's mechanism of action to define the biological activity. The absence of rigorous mechanistic data for the majority of cell and gene therapy therapeutics currently in the process research pipelines has impeded efforts to design and validate indices of product potency. The current talk tries to summarize the principal and unique challenges facing the development of functionally relevant potency assays for cell and gene therapy products.

Learning Objectives:

1. General guidance of FDA for developing potency assays

2. Understand unique challenges for developing in-vitro potency assays

Within the last decade, bioprocess modeling received increased attention. But what are actually the crucial things to consider to create value through process modeling? During this talk, the modeling circle (from data to digital twin) is highlighted for both upstream and downstream showcases. A hybrid modeling methodology is presented and it will be highlighted how  the modeling circle can be closed with an intuitive toolbox which you as a user can directly use to solve your own bioprocess related modeling tasks. The Toolbox can be used to apply model-based and intensified Design of Experiments (m&iDoE) to significantly accelerated bioprocess development. The models can further be used in a digital twin environment to  explore the process or exported to use the models online in-real time as soft-sensor and for process control.

Learning Objectives:

1. Understand the modeling circle (from data to digital twin)

2. Learn how hybrid modeling outperforms purely data-driven modeling

3. Understand how intensified and model-based DoE help to accelerate bioprocess development

Demand for high-value biologics, a rapidly growing pipeline, and pressure from competition, time-to-market and regulators, necessitate novel biomanufacturing approaches, including Quality by Design (QbD) principles and Process Analytical Technologies (PAT), to facilitate accelerated, efficient and effective process development platforms that ensure consistent product quality and reduced lot-to-lot variability. Herein, QbD and PAT principles were incorporated within an innovative in vitro-in silico integrated framework for upstream process development (UPD). The central component of the UPD framework is a mathematical model that predicts dynamic nutrient uptake and average intracellular ATP content, based on biochemical reaction networks, to quantify and characterize energy metabolism and its adaptive response, metabolic shifts, to maintain ATP homeostasis. The accuracy and flexibility of the model depends on critical cell type/product/clone-specific parameters, which are experimentally estimated. The integrated in vitro-in silico platform and the model’s predictive capacity reduced burden, time and expense of experimentation resulting in optimal medium design compared to commercially available culture media (80% amino acid reduction) and a fed-batch feeding strategy that increased productivity by 129%. The framework represents a flexible and efficient tool that transforms, improves and accelerates conventional process development in biomanufacturing with wide applications, including stem cell-based therapies.

Learning Objectives:

1. Learn metabolic requirements of mammalian cells; Metabolic Shifts

2. Learn about modelling of mammalian Cells

Biologics derived from mammalian sources are expected to undergo two orthogonal virus removal processes. One common processing step is a batch low-pH hold to inactivate enveloped viruses. Attempts to adapt this technique to continuous processing have involved cyclic batch operation, continuous flow tubular reactors, or continuous flow column-based reactors. None of the continuous flow reactors, however, address the control challenges with operating this system. This presentation describes a low-cost, column-based continuous viral inactivation system constructed with off-the-shelf components. Model-based, reaction-invariant pH controller is implemented to account for the nonlinearities with Bayesian estimation addressing variations in the operation. The residence time distribution is modeled as a plug flow reactor with axial dispersion in series with a continuously stirred tank reactor, and is periodically estimated during operation through inverse tracer experiments. The estimated residence time distribution quantifies the minimum residence time, which is used to adjust feed flow rates. Controller validation experiments demonstrate that pH and minimum residence time setpoint tracking and disturbance rejection are achieved with fast and accurate response and no instability.

Learning Objectives:

1. Discuss current trends in biopharmaceutical manufacturing operations and control

2. Describe a mathematical model that incorporates pH and residence time to more accurately control continuous bioprocessing

The past few decades have seen a substantial increase in the research based on nanocarrier based combinative drug therapies in various diseases such as cancer, tuberculosis, Alzheimer’s and, asthma. However, due to prolonged dosage regimens, increased doses of the drugs, side effects and lack of stability, their large-scale use and upscaling to industrial levels have been affected. Hence, there is a need to develop a nano drug delivery approach in order to tackle the above issues and provide a better therapy to the patients. Niosomes or non-ionic surfactant-based liposomes are promising bilayer nanocarriers with an ability to entrap drugs having different modes of action within a single vesicle. There have been many potential advancements in this specific field where lipid based nanocarriers have showcased excellent stability of the nanoformulation along with better therapeutic actions in comparison to free drug combinations.  This talk will specifically focus on the use and future potential of these bilayer niosomes as a combinative drug delivery option for future treatments. This presentation will also showcase our work on the use of dual drug loaded niosomes in regards to the treatment of Tuberculosis and Breast cancer.

Learning Objectives:

1. Provide budding researchers and students to come up with novel combinatorial drug delivery approaches using different lipidic and other nanocarriers.

2. Showcase the potential of niosomes as a robust alternative to liposomes in terms of drug effectiveness and stability.

3. Using this approach or technology targeting different diseases using FDA approved drugs and nanocarriers is envisioned for a faster clinical trial and market approval of the nanoformulation.

4. The study discussed will help in fostering novel ideas and applications in different areas of pharmaceutical and medical sciences.