Proteomic analysis of host cell protein fouling during bioreactor harvesting.

Chinese hamster ovary (CHO) cells are among the most common cell lines used for therapeutic protein production. Membrane fouling during bioreactor harvesting is a major limitation for the downstream purification of therapeutic proteins. Host cell proteins (HCP) are the most challenging impurities during downstream purification processes. The present work focuses on identification of HCP foulants during CHO bioreactor harvesting using reverse asymmetrical commercial membrane BioOptimal™ MF-SL. In order to investigate foulants and fouling behavior during cell clarification, for the first time a novel backwash process was developed to effectively elute almost all the HCP and DNA from the fouled membrane filter. The isoelectric points (pIs) and molecular weights (MWs) of major HCP in the bioreactor harvest and fouled on the membrane were successfully characterized using two-dimensional gel electrophoresis (2D SDS-PAGE). In addition, a total of 8 HCP were identified using matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS). The majority of these HCP are enzymes or associated with exosomes, both of which can form submicron-sized particles which could lead to the plugging of the filters.

Evaluation of a single-use disk stack centrifuge for improved efficiency and sustainability at 1000 L GMP manufacturing scale.

Direct depth filtration is an established technology for single-use harvest operation. Advantages of direct depth filtration include familiarity with depth filtration in downstream processes and simplicity of the operation. Drawbacks include low capacity, large footprint, labor-intensive set-up, high water use, and high waste in the form of discarded filters. Single-use centrifugation is emerging as an alternative to depth filtration for the single-use harvest step. Within the single-use centrifugation space, disc stack centrifugation represents the newest entrant. In this study, we evaluated the performance of the GEA kytero single-use disc stack centrifuge to clarify two monoclonal antibody-producing cell culture fluids. The separation performance of the GEA kytero centrifuge varied between the two cell culture fluids, with differences in centrate turbidity and centrate filterability measured. A comparison was then performed to determine resource savings, compared to direct two-stage depth filtration, when using a GEA kytero centrifuge to harvest a 1000 L bioreactor. The analysis concluded that replacement of the first stage of depth filters with a GEA kytero centrifuge has the potential to decrease the required second stage depth filtration area by up to 80%. The decrease in depth filter area resulting from the use of the GEA kytero would result in a decrease in the harvest step footprint, a decrease in buffer volume required to prime and rinse depth filters, and a decrease in the volume of plastic waste. An economic comparison of the GEA kytero single-use centrifuge against a direct depth filtration step found that for a 1000 L harvest step, the GEA kytero centrifuge may reduce costs by up to 20% compared with two-stage direct depth filtration.

Glucose monitoring and adaptive feeding of mammalian cell culture in the presence of strong autofluorescence by near infrared Raman spectroscopy.

Raman spectroscopy offers an attractive platform for real-time monitoring and control of metabolites and feeds in cell culture processes, including mammalian cell culture for biopharmaceutical production. However, specific cell culture processes may generate substantial concentrations of chemical species and byproducts with high levels of autofluorescence when excited with the standard 785 nm wavelength. Shifting excitation further toward the near-infrared allows reduction or elimination of process autofluorescence. We demonstrate such a reduction in a highly autofluorescent mammalian cell culture process. Using the Kaiser RXN2-1000 platform, which utilizes excitation at 993 nm, we developed multivariate glucose models in a cell culture process which was previously impossible using 785 nm excitation. Additionally, the glucose level in the production bioreactor was controlled entirely by Raman adaptive feeding, allowing for maintenance of glucose levels at an arbitrary set point for the duration of the culture. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1574-1580, 2018.