Overcoming obstacles in biotherapeutic development

A high-quality cell line with stable production qualities is critical in the manufacture of biopharmaceuticals. Achieving such a cell line, however, can be challenging given the variety of variables that might affect its quality, the complexity of biologics as they get more sophisticated, and the expanding regulatory requirements requiring verification of clonality.

Dr. Louis Boon, Chief Scientific Officer of Polpharma Biologics Utrecht, discusses some of the methods they employ to overcome challenges in cell line selection and optimize biotherapeutic production.

Optimizing biotherapeutic production

The novel strategy presented takes into account:

  • A technique intended to alter the charge profiles of biological systems to increase cell-specific productivity
  • A method designed to accurately alter the biological system’s essential qualities to enhance effector functions
  • An automated, high throughput method of finding and choosing the best, highly effective monoclonal cell line

Increasing cell-specific productivity

Increasing the viable cell density (VCD) during upstream processing is a frequent strategy for achieving high productivity. While high VCD results in higher product titers, it introduces problems during the later downstream processing (DSP) phase.

An exciting area of development focuses on enhancing specific cell productivity with the addition of citrulline. The technology is based on increasing the cellular volume rather than using the traditional increasing VCD method.

This unique use of citrulline in the culture medium increases the volume of mammalian cells more than 2-fold whilst decreasing VCD by 60%, which leaves total biomass and productivity unchanged.

Furthermore, they observed that citrulline was able to modulate the charged profile of the product by decreasing the product’s percentage of acidic species.

Due to the increased cell size and thus enhanced cell-specific productivity, the addition of citrulline can also lower the protein content of host cells by 40% and increase the filterability for clarity by 40%/g of output.

The latter has significant effects on the GMP footprint of the cell culture clarification stage since it reduces the amount of cell debris that needs to be removed during clarification, which lowers product loss and costs.

Improving process efficiency

Biologics, such as mAbs, are complex molecules with defined tertiary structures, including an N-linked glycan on the heavy chain constant domain. they require specific post-translational modifications (PTMs) and a cellular system to perform complex protein folding and glycosylation.

This intricacy, together with the intrinsic diversity of the cellular factories that produce them, tends to lead to various difficulties.

Although certain aspects regarding the quality of the product may be related to the nature of the cell line, by far, most PTMs are introduced in the upstream process. Protein breakdown or cell death can come from misfolded recombinant biotherapeutic proteins that stress the secretory system.

The structure and bio-functional properties of the biologic could be compromised by incorrect multi-chain biotherapeutic protein pairing and insufficient glycosylation.

The effector processes known as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC), which are related to therapeutic effectiveness, could be impacted by variations in N-glycan species.

Another significant development has been the creation of an upstream process modulation toolbox for enhancing process efficiency and reliability. This has been made possible by combining the comprehensive route platform expertise and metabolic understanding.

To maximize the effector functions for unique biological entities, the techniques established in this modulation toolbox can specifically adjust crucial quality characteristics on the biological, such as terminal galactosylation, core-fucosylation, and mannosylation. The charge profile of the product can be modified by using molecules like the citrulline molecule mentioned above.

Researchers can employ the modulation toolbox to fine-tune a novel product’s anticipated biological efficiency since low core-fucosylation can increase ADCC effector function, and high terminal galactosylation on antibodies can improve CDC effector function.

Researchers can adjust the upstream process to high terminal galactosylation or low core-fucosylation for a therapeutic antibody mode of action that requires effector activities. In contrast, the opposite can be created in the upstream processing pathway for a new antagonist antibody to remove effector functions.

Culture media optimization is another important factor. Even though researchers should customize the culture media for the desired clone, starting with off-the-shelf media can help researchers prevent supply chain issues and reliance on a single provider. By bypassing these possible issues, users can increase process efficiency and minimize downstream process concerns.

Accelerating cell line selection and monoclonality assurance

The ideal production cell line will have high cell-specific productivity and titers, proven scalability and stability, and monoclonality, which confirms that the cell line originates from a distinct single cell. The cell line selection procedure necessitates screening thousands of clones to find one with these optimal characteristics.

Traditionally, cell line selection has been carried out manually in microtiter plates up to small bioreactors across progressively larger volume scales. The time required to create a monoclonal cell line using this conventional method is quite long—more than 50 weeks.

However, with substantial advancements in microfluidics and automation, scientists can now use automated instruments like Cyto-Mine® to replace this traditionally time-consuming and multi-step method.

Overcoming obstacles in biotherapeutic development

A high-throughput screening workflow. Image Credit: Sphere Fluidics

Cyto-Mine® integrates isolation, screening, sorting, imaging, and dispensing into a single benchtop system that streamlines workflows and significantly reduces timelines.

The technology quickly separates millions of single cells into picodroplets, each with a volume of one picoliter. Each cell can be tested in its own microenvironment when it is enclosed in picodroplets, allowing for selective screening of the single cell that demonstrates the highest specific productivity.

The platform’s integration of high-speed imaging into the single-cell dispensing process is noteworthy because it enables researchers to recover top clones with visual evidence of monoclonality without the need for further screening or apparatus.


In conclusion, the combination of a comprehensive understanding of the intracellular mechanism by which the therapeutic protein of interest is produced and its quality is modified with automated, fully integrated upstream processing technology can enhance the effectiveness of biotherapeutic production.

Using this approach during cell line selection, researchers can obtain stronger candidates for downstream processing through significantly faster timelines.

Overcoming obstacles in biotherapeutic development

Sphere Fluidics’ Cyto-Mine®. Image Credit: Sphere Fluidics

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We initially focused on producing novel biochip systems and providing R&D services. We have since extended our expertise and are developing a technology platform that enables discovery in a range of growing markets through single cell analysis. Our systems make the development of new biopharmaceuticals faster and more cost-effective, improve monoclonal antibody screening, cell line development, and overall research efficiency in a number of other applications including synthetic biology, single cell diagnostics, prognostics and single cell genome editing.

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Last updated: May 23, 2023 at 8:35 AM


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