Advancements in upstream CLD for enhanced biotherapeutics production

Two scientists working in a lab

This article is sponsored by Revvity.

By Anis H Khimani, PhD, Senior Strategy Leader, Life Sciences strategy group, Revvity, Inc.

Across the biotherapeutics workflow

The landscape of large molecule drug development has evolved significantly in recent decades. As the significance of large molecules continues to broaden across diverse modalities, emphasis on bispecific and multispecific antibodies to treat various disorders such as rheumatologic diseases, cancer, diabetes, anaemia, and infectious diseases becomes paramount.

Technological innovations supporting discovery and development stages have concentrated on both upstream and downstream aspects of the workflow. Prioritising critical attributes in the upstream workflow is crucial for predicting and preventing challenges downstream, ultimately avoiding high development costs and delays in drug approvals.

A complex journey

Biotherapeutics discovery and development upstream encounters challenges in cell line development (CLD), clone screening and characterisation, scale up, and eventually production. However, technology advancements have significantly contributed to overcome bottlenecks and enable a streamlined process. Recently, the pandemic has accelerated the development process while still maintaining safety and efficacy standards. In addition, it has stimulated increased collaboration among drug developers, technology providers, supply chain organisations, and regulators.

Upstream cell line development has seen notable progress with recent advances in cell line engineering, particularly in enhancing gene of interest integration specificity, clone selection, and production efficiency. Next-generation engineering tools, such as gene editing have enabled knock-out cell lines with enhanced characteristics. For instance, the engineering of CHO cells, widely used protein expression system, for various metabolic pathways has resulted in improved expression levels of large molecule drugs and product quality1,2. After engineering, stable clones are screened, identified, and characterised. Stable clonal cell banks are used for GMP production of the final drug product.

Predictable milestones and future perspectives

Transposons (genetic elements) have been used to deliver a gene of interest into transcriptionally active sites of the cell line genome leading to rapid cell line development eventually resulting in high titers of the recombinant protein drug. Unlike the use of random integration expression vectors, an advantage of transposon system is that it enables intact and site-specific integration, therefore, eliminating any fragmentation, rearrangement, or concatamerisation.

A novel, two-component transposon system, is a vector system which enables multi-copy, non-fragmented gene cassette integration3. This strategy achieves a clonal stability of >95%, minimising clone screening efforts and streamlining scale up.

In summary, large molecule drug development has advanced along both upstream and downstream stages. Focus on upstream cell line development (CLD) is a fundamental step that not only enables directed and efficient clone selection of the large molecule drug but also facilitates both product titer yields and scale up. Furthermore, recent novel molecular tools such as gene editing and gene of interest delivery vector systems (e.g., transposon technologies mentioned above), have enabled targeted and consistent cell line engineering to simplify drug development and streamline manufacturing efforts.

In addition, adoption of new methods such as standardisation of cell counting and clone screening via automated cell evaluation technologies will further streamline the end-to-end CLD workflow. Integration of processes and protocols with automation, detection, and cell imaging technologies, as well as data analysis and management will redefine the process to scale upstream processes. Transformation with digitisation and adoption of machine learning will further facilitate advanced predictive and data models to establish best practices and eliminate process gaps.


  1. Mistry RK, Kelsall E, Sou SN, et al. A novel hydrogen peroxide evolved CHO host can improve the expression of difficult to express bispecific antibodies. Biotechnol Bioeng 2012;118:2326.
  2. Budge JD, Knight TJ, Povey J, et al. Engineering of Chinese hamster ovary cell lipid metabolism results in an expanded ER and enhanced recombinant biotherapeutic protein production. Metab Eng 2020;57:203.
  3. Patel T, Ingham C, Fleming J, et al. 2022. CHOSOURCE™ ADCC+ cell line for enhanced therapeutic potency.

About the author

Anis is within the strategy group in Life Sciences at Revvity, Inc. With more than 20 years of experience in business management, strategy, marketing, and product management, Anis also has research background from biotech, pharmaceutical, and premier academic organisations. In his previous roles, he was responsible for strategy and product management of biology informatics, drug discovery assay portfolios, genomics, proteomics, and was also the programme leader within R&D and applications.

Anis also serves on the scientific and editorial advisory board of Genetic Engineering News, Mary Ann Liebert, Inc. He has authored and co-authored numerous publications, chapters, review articles, abstracts, and has been invited to present talks at national and international conferences. He has a post-doctorate in virology, viral pathogenesis, viral vector development, and vaccines evaluation from Harvard Medical School and a PhD in Virology/Molecular Biology.

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