EPM: Cell line development strategies fit for a new era of Bispecific Antibodies

26th June 2023

Nicole Wakes, VP of Scientific Operations, and Simon Keen, Vice President Cell Line Development at Abzena, outline the strategies that developers must consider to overcome the challenges of developing BsAbs under increasing pressure for speed.

Complex molecule production challenges

BsAbs are difficult to express due to their heterogeneity and multiple specific activities so there is no “one-size-fits-all” approach to their development and manufacturing. As a result, cell line development (CLD), upstream development (USP), and downstream development (DSP) must be carefully optimised to ensure maximum yield and quality for each project. The growing demand for these molecules from patients, investors, and developers means that rapid timelines are required. This can be achieved through building a robust CLD strategy, identifying molecule liabilities, adopting technologies for optimal speed, and working with experienced partners.

Building a robust CLD strategy

The right CLD approach can shorten timelines for investigational new drugs (IND) considerably. As CLD impacts every process, decisions made from the outset could proactively prevent delays. Developers should therefore consider the following parameters when designing their CLD strategy:

Analytics and validation: Critical quality attributes should be determined and monitored throughout the development process.

Scalability: Developers should consider how conditions such as oxygen transfer capacity and shear stress may change throughout scaling, as these may impact cell growth and target protein expression. It is essential to ensure variations do not impact the scale up to manufacture.

USP methods: Developers should aim for USP methods that allow for rapid progression of products throughout development. This should involve selecting clones that are a good platform fit, and considering productivity and product quality.

DSP methods: For efficient DSP, developers should consider the physicochemical properties of the product to ensure product consistency. Lead candidates should have minimal heterogeneity and production clones should consistently express in platform USP conditions.

Identifying potential molecule liabilities

Resolving issues that have not been considered proactively from the outset of CLD can be costly and time consuming for development and manufacturing, with possible strategy redesign requirements.

With antibody-based molecules such as BsAbs, molecular structures and sequences — such as sites for isomerisation, oxidation, glycation, and deamidation — can present liabilities. Reactions at any of these sites could result in changes to the molecule’s physical properties, leading to high risks of product heterogeneity and batch-to-batch variation.

To overcome these challenges, developers should conduct a risk assessment at the early design stage, determining where these sites of liability are, and how they may impact the functionality and stability of the molecule. At this stage, the molecule could be redesigned. Alternatively, if engineering out liability is too great a risk, development can continue, with the acceptance that potential issues further down the production chain may occur.

Building a robust CLD strategy requires the adoption of technologies such as vector selection, integration, and fast stable pools that can accelerate timelines without being detrimental to product quality. Developers should carefully consider the pros and cons of different integration techniques to determine which will produce the most stable cell line.

Vector selection

To accelerate CLD for the delivery of the target genes, choose a vector that encodes the desired biologic, with a library of carefully designed plasmids, alongside a standard backbone. The method employed for gene integration should also be carefully considered to select the approach that will produce a stable cell line that consistently expresses the right quality of the product.

Integration and transparency

Traditionally, integration of transgenes in antibody-based CLD into the host genome has been achieved via random non-homologous recombination. Although effective, this is time-consuming. Targeted integration can increase productivity, where the site for integration into the genome has been predetermined and can be achieved using site-specific recombinases.

Another approach is transposase-mediated integration. Transposases recognise common sequences within the genome and will preferentially insert transposable elements into these sites. Cell line stability is improved as these sites are often associated with high transcription activity and are less prone to gene silencing effects. This method can reduce the screening efforts needed to identify highly productive clones.

Fast stable pools

Another technique that can significantly reduce timelines for IND filing is the generation of fast stable pools. Following molecule design and development, small amounts of protein can be rapidly generated through transient expression for basic screening of a primary characteristic. However, transiently produced material does come with a risk of altered product quality attributes. Bulk fast stable pools of potential drug candidates can be generated in a matter of a few weeks for more in- depth analysis.

Process support from partners with experience and expertise

There is a clear need for developers to adapt to the rapidly changing antibody-based biologics market as these drugs will be essential in targeting diseases of growing prevalence. Delivering these vital biologics to patients at speed will require developers to employ a robust strategy, with CLD at the core.

A robust CLD strategy will incorporate a proactive approach to determining potential challenges and avoiding delays and employ methods to enable development activities to be initiated in parallel, such as generating fast stable pools. Seeking support from a trusted and proficient partner with years of experience and a thorough understanding of the market can help ease the burdens involved in successfully delivering these critical biologics to patients.

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