Managing the key challenges facing human health and the global environment is an increasingly complex task. Fortunately, bioprocessing – the creation of useful products using biological materials, such as microorganisms – holds the potential to address many of these issues. Bioprocessing bridges the gap between scientific discoveries and real-world applications, enabling the large-scale production of increasingly advanced biobased products, from vaccines and cell therapies to food and biofuels.
Ingenza’s bioprocessing pipeline involves a series of intricate steps split into two main segments: upstream processing (USP) and downstream processing (DSP). While initial upstream activities include fermentation and the optimisation of cellular processes to maximise product formation, DSP occurs in the later stages of production, and is carried out to purify and concentrate a final protein product, while meeting stringent quality standards. In this blog, Dr Scott Baxter – the head of the DSP team at Ingenza – discusses how the company develops scalable downstream bioprocesses that adhere to the principles of GMP.
Process development
The DSP team at Ingenza is responsible for designing processes to isolate a recombinant protein of interest from biological material generated by the molecular biology and fermentation departments. This is an essential stage in any project aiming to create a protein product, such as a biopharmaceutical. Our customers working in the field of human health are typically seeking an isolated protein that they can progress through preclinical testing to the bulk production phase, and Ingenza is responsible for accelerating them on their journey to clinical trials. We produce their recombinant protein of interest – usually after a cell line development stage – in quantities sufficient for preclinical testing and formulation development. However, our predominant output is the development of a process to enable them to continue to generate their product in a GMP-compliant manner for phase 1 testing. Less frequently, a customer may come to us with a semi-established bioprocess, and ask us to test and optimise a specific step within their protocol. Our ability to scale down (as well as scale up) bioprocesses is critical here, as we can mimic processes at a fraction of their true scale to generate representative data and material that the customer can use.
The isolation of specific proteins and enzymes is a less important requirement for customers working in the sustainability sector, who are targeting biochemical production from a specific host organism. However, DSP can still be used as a tool to evaluate the ability of a specific molecule within their biochemical pathway to perform a particular reaction. Our ability to isolate key enzymes within biochemical pathways, and then individually assay them for reactions of interest in vitro, has been of great benefit in many projects and, in some cases, has transformed our understanding of what is happening in vivo.
Sequential steps to purify recombinant proteins
Every bioprocessing pathway is unique, in the same way that every car is unique. Just like a car – which will always have four wheels and a steering mechanism – each bioprocess will also consist of the same basic unit operations: a harvest step to obtain the crude product from the growth culture, a clarification protocol to effectively remove large impurities, further chromatography steps to purify the desired protein, and a filtration step to further polish and concentrate the product. However, the methods that we use at each of those stages may differ significantly depending on the properties of the product that we are looking to purify – such as its charge, hydrophobicity and size.
For example, varying resins – including ion exchange, hydrophobic interaction and affinity resins – can be chosen to separate and purify proteins based on a specific property. A range of filtration techniques are also available, including depth and tangential flow filtration (TFF). We are even in the process of introducing a new cell disruptor to harvest intracellular products from large amounts of biomass more efficiently. It is essential that the methods used in each step are carefully selected and optimised to provide efficient purification, while maintaining product recovery and stability. Fortunately, our team at Ingenza has the knowledge, experience and expertise to adapt and tailor our selection of methods to suit the needs of each customer’s product.
Our recent SARS-CoV-2 vaccine project was a testament to our DSP capabilities. While a typical bioprocessing workflow only requires one protein to be isolated and purified, we needed to manufacture and combine nine separate proteins of interest to produce this vaccine. Our DSP process therefore had to be evaluated for each individual protein, to find a combination of processing methodologies that worked across the board . At the same time, we needed to ensure that each step met certain quality and regulatory standards. The resulting broad-spectrum vaccine offered highly effective protection against multiple coronavirus strains in preclinical animal models, and progression to phase 1 trials is underway. As the processes developed by Ingenza for preclinical safety toxicology evaluation are based on the principles of GMP, they will be directly transferable to our collaborators responsible for production of the clinical trial material.
Ensuring high performing, scalable and GMP-compliant processes
Part of Ingenza’s DSP strategy as a CDMO is to ensure that all processes developed – and the products they generate – are ready to enter the next steps in the bioprocessing pipeline. For example, it is crucial that proteins are prepared with the further preclinical steps of product characterisation and analysis in mind. Samples of all the material that we generate will be fed to our analytical team, where key performance indicators – such as the clearance of common impurities, including host cell proteins, host cell DNA and endotoxins – are assessed to determine the quality of the protein product. The analytical team also develops a range of bespoke in-process controls to allow us to monitor the performance of the bioprocess. The process itself, and its performance – measured by its robustness, reproducibility and predictability – is equally as important as the quality of the final product.
Scalability is also a key consideration in the design process. Many of our customers come to us with existing processes developed in a research environment, but the techniques and equipment used in an academic laboratory are typically not suitable for scales larger than a few litres. Our role is to convert these academic techniques into industrial processes. For example, separation techniques based on high speed centrifugation cannot be scaled effectively, so we replace these with scalable filtration methodologies. Size exclusion chromatography (SEC) is also a very efficient purification technique that is commonly used in academic labs as a final polishing step, but it cannot be used to effectively produce large quantities of material in a scalable manner, so we avoid this technique unless it is absolutely essential. We typically work at laboratory scale – with some projects reaching pilot scale – but all the processes developed can be scaled up to purify protein from large volumes of fermentation harvest.
We also ensure that all the processes we develop adhere to the principles of GMP, so that the technology can be transferred to a GMP-compliant laboratory in future. For example, we replace histidine tag purification – a common academic approach to protein isolation – with an alternative method more suitable for future GMP manufacturing and regulatory acceptance.
Collaboration to drive success
A key benefit of Ingenza’s DSP team is that we all have varied backgrounds. Some of us are trained molecular biologists, whereas others come from an analytical chemistry background. Our unique selling point is that we can call on overlapping experiences and expertise from the fermentation, molecular biology and chemistry departments to carry out all aspects of bioprocessing. This allows us to understand and appreciate the bioengineering steps that precede and follow the DSP stage. As a result, DSP is not a standalone unit at Ingenza; it is considered throughout the formation of a project proposal, to ensure that the process and products that we generate are representative of a future GMP process. This collaborative approach allows us to effectively manage complex multi-disciplinary projects – such as our recent vaccine project – and sets us up to keep supporting customers as demand for novel biobased products continues to grow.