BioPharm International: Optimizing Cell Line Development for Next Generation Biologics

26th June 2024

BioPharm International’s June 2024 issue features our VP of Cell Line Development, Simon Keen, discussing how improving the flexibility of cell line development through the utilization of platform approaches and suitable partnerships can reduce potential bottlenecks in the development pathway of novel biologics.

To rapidly develop the next generation of biological therapeutics, cell line developers need to build efficient platforms so that cell line development (CLD) does not become a bottleneck in the development pathway. To make allowance for the increasing array of novel antibody–based and non-antibody modalities, these platforms need to be flexible in terms of both molecule build and production method. Therefore, CLD unit operations need to be considered as modules, with predeveloped alternative modules that can be quickly and easily swapped in and out, plus defined application and predictable outcomes. Additionally, cell line developers need to work effectively across teams to leverage knowledge accumulated during lead candidate design and selection, while working with analytical scientists to apply a well-tuned analytical strategy.

When contracting out steps such as CLD, it is important to choose a contractor who is prepared to work collaboratively with the drug developer and apply a streamlined analytical approach. Working with an integrated contract development and manufacturing organization means that more complex programs can be tackled without significantly increasing CLD timelines or risk of failure.

Improved productivity feeds a growing sector

The rise of biological therapeutics looks set to continue, with recent forecasts predicting approximately a 7% compound annual growth rate over the next five years, a rate which would value the field at more than $600 billion by 2029 (1). The build-out of manufacturing volume has not kept pace with the increasing demand for biologics, but the long-touted “capacity crunch” has not yet occurred. Instead, there has been a maturing of the bioprocessing sector so that increases in productivity have been the focus, rather than building out volume (2).

These productivity improvements have come across the board, including the rise of single-use equipment (3), process intensification (4), and the adoption of platform processes. Mammalian cell line productivity in particular has seen some significant increases in titers. Average commercial monoclonal antibody (mAb) expression titers were 1.95 g/L in 2008; by 2020, this had increased to 3.5 g/L (2). However, these average figures don’t accurately reflect the true increases, with cell line developers for new products now routinely achieving mAbs titers above 5 g/L.

Trend toward more complex biologics

In parallel to these leaps in productivity, there has also been a trend toward more complex products. The production of bispecific antibodies, multiactive fusion proteins, nanoparticles, and other complex biologics is now possible because of advanced protein engineering technology and bioprocessing advances. These advances have made it feasible to express complex modalities, hitherto considered difficult to express, in economically viable quantities; however, challenges remain.

Cell line developers are required to generate manufacturing cell lines for an ever-widening pipeline of new modalities, with shortened timelines and without compromising on quality. Many of these molecules may only be required in small quantities with higher potency, to serve smaller patient populations for orphan and rare diseases or as ancillary materials for cell and gene therapy products. Therefore, there is a requirement for efficient, productive processes to support the economics of smaller batch sizes.

Additionally, many of these new modalities will come with unique critical quality attributes (CQAs) and more complex impurity profiles, challenging traditional mAb upstream and downstream platforms. These challenges can be alleviated by generating and selecting high-quality production clones. The right clone will generate more of the desired product, with CQAs that conform to the quality target product profile, and fewer of the impurities that might complicate downstream removal. Cell line developers need to balance the tension between the efficiency of a platform that delivers highly productive clones against the nuances of more complex proteins. Building flexible CLD platforms, where platform unit operation can be modified or swapped for more bespoke versions, while maintaining the backbone of the platform process is one way in which the competing needs of speed, quality, and cost can be balanced.

It starts with vectors

Having a robust expression vector, adaptable to a wide range of modalities, is critical to having a flexible CLD platform. Most vectors used in CLD have been optimized around mAbs, employing strong constitutive promoters like cytomegalovirus or elongation factor 1 alpha, with enhancer regions to maximize rates of gene transcription. Other technologies include elements to modify chromatin and gene accessibility to transcription complexes (such as ubiquitous chromatin-opening elements and matrix attachment regions) or utilize transposase mediated integration (such as Atum Bio’s Leap-in, Addgene’s PiggyBac, and Sleeping Beauty) to semitarget efficient gene integration. Premium vectors, such as ProteoNic’s 2G-UNic and Abzena’s AbZelectPRO , combine a powerful promoter to maximize transcription with optimized untranslated regions to generate more protein from each message copy, making them well-suited for mAb production as well as proteins that are more difficult to express.

All of these technologies are focused on maximizing gene expression, but when looking at more complex multichain proteins, it is important to consider flexible vectors and transfection strategies that can account for potential differential chain expression. To optimize productivity, expression of each chain needs to be balanced. Balancing the expression of each chain ensures they are produced in the correct ratios, maximizing the amount of desirable product and minimizing product-related impurities (PRI).

Balancing chain expression can be done using promoters of varying strength or through altering ratios of gene copy number. In either case, for any given pair (or more) of subunits, the optimal ratios or promoter combinations will need to be determined experimentally. To optimize quickly and without extending timelines, a vector system is required that allows rapid cloning of genes and a flexible process for rapidly generating multiple stable pools in parallel. This, aligned with a strong analytical strategy, can provide empirical data for analysis, which allows quick decisions without elongating timelines.

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