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For mammalian cell‐derived recombinant biotherapeutics, controlling host cell DNA levels below a threshold is a regulatory requirement to ensure patient safety. DNA removal during drug substance manufacture is accomplished by a series of chromatography‐based purification steps and a qPCR‐based analytical method is most used to measure DNA content in the purified drug substance to enable material disposition. While the qPCR approach is mature and its application to DNA measurement is widespread in the industry, it is susceptible to trace levels of process‐related contaminants that are carried forward. In this study, we observed failures in spike recovery studies that are an integral component of the qPCR‐based DNA testing, suggesting the presence of an inhibitory compound in the sample matrix. We generated hypotheses around the origin of the inhibitory compound and generated multiple sample matrices and deployed a suite of analytical techniques including Raman and NMR spectroscopy to determine the origin and identity of the inhibitory compound. The caustic wash step and depth filter extractables were ruled out as root causes after extensive experimentation and DNA testing. Subsequently, 2‐(N‐morpholino)ethanesulfonic acid (MES), a buffer used in the chromatography unit operations, was identified as the source of the contaminant. A 500‐fold concentration followed by Raman and NMR spectroscopy analysis revealed the identity of the inhibitory compound as polyvinyl sulfone (PVS), an impurity that originates in the MES manufacturing process. We have implemented PVS concentration controls for incoming MES raw material, and our work highlights the need for rigor in raw material qualification and control. [ABSTRACT FROM AUTHOR]

Continuous cultivation of mammalian cells originated in chemostats in the 1950s and, through a series of technological advancements, has evolved into modern‐day high‐density perfusion processes with volumetric productivities >5 g L–1 day–1 over extended cultivation periods. With appropriate process development, these productivities can be achieved with no adverse impact on cell line stability and product quality. Developments in downstream processing and automation have enabled the integration of drug substance manufacturing processes suitable for next‐generation plants that require lower capital deployment, with the potential for rapid construction and validation for clinical and commercial production. Perfusion also has been successfully used to generate high‐density cell banks (> 50 × 106 cells mL–1) and for seed‐train optimization. They have resulted in significant productivity increases (10‐fold increase in volumetric productivity) from fed‐batch bioreactors by providing a high‐density seeding culture that elevates the inoculation density in a production bioreactor (> 5 × 106 cells mL–1). Related advancements in cell culture medium formulations and cell line performance typically are independent of bioreactor format and have broad applicability for biotherapeutic manufacturing. The last decade has seen renewed interest in high‐density perfusion cultures and technological advances spanning the entire space of drug substance manufacturing – from cell line development to the final purification step. Continually evolving, these new technologies now are being introduced into clinical and commercial manufacturing. We anticipate that the general principles and knowledge gleaned from process intensification will extend well beyond classical perfusion cultivation and into all aspects of mammalian cell culture. © 2021 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

Techniques to incorporate non-natural amino acids (NNAAs) have enabled biosynthesis of proteins containing new building blocks with unique structures, chemistry, and reactivity that are not found in natural amino acids. It is crucial to understand how incorporation of NNAAs affects protein function because NNAA incorporation may perturb critical function of a target protein. This study investigates how the site-specific incorporation of NNAAs affects catalytic properties of an enzyme. A NNAA with a hydrophobic and bulky sidechain, 3-(2-naphthyl)-alanine (2Nal), was site-specifically incorporated at six different positions in the hydrophobic core of a model enzyme, murine dihydrofolate reductase (mDHFR). The mDHFR variants with a greater change in van der Waals volume upon 2Nal incorporation exhibited a greater reduction in the catalytic efficiency. Similarly, the steric incompatibility calculated using RosettaDesign, a protein stability calculation program, correlated with the changes in the catalytic efficiency.

Amyloid fibrils implicated in numerous human diseases are thermodynamically very stable. Stringent conditions that would not be possible in a physiological environment are often required to disrupt the stable fibrils. Recently, there is increasing evidence that small molecules can remodel amyloid fibrils in a physiologically relevant manner. In order to investigate possible fibril remodeling mechanisms using this approach, we performed comparative studies on the structural features of the different amyloid-β (Aβ) aggregates remodeled from Aβ fibrils by three biocompatible small molecules: methylene blue; brilliant blue G; and erythrosine B. Combined with circular dichroism (CD), immuno-blotting, transmission electron microscopy (TEM), and atomic force microscopy (AFM) results, it was found that brilliant blue G- and erythrosine B-treatment generate fragmented Aβ fibrils and protofibrils, respectively. In contrast, incubation of the Aβ fibrils with methylene blue perturbs fibrillar structure, leading to amorphous Aβ aggregates. Our findings provide insights on the molecular mechanism of amyloid fibril formation and remodeling and also illustrate the possibility of controlled changes in biomolecule nanostructures.