Peptones are protein digests composed mainly of amino acids and small peptides. Peptones have been used in mammalian cell culture medium as a serum substitute to enhance cell growth and product formation. The first part of this study describes our evaluation of peptones from different sources (soy, wheat, yeast, and casein) on the cell growth and productivity of Sp2/0 myeloma cells expressing recombinant prourokinase (r-ProUK). The results of these studies demonstrated that wheat peptone was the most effective plant peptone to increase r-ProUK yield. Addition of 2 g/L wheat peptone to the culture medium increased batch r-ProUK production between 28-67% compared to cells grown in the absence of peptone supplements. Peptones did not increase cell productivity, but increases r-ProUK yield through increased culture longevity…
Category: <span>Biologics Production</span>
Electron microscopy (EM) provides data for viral clearance studies, information on the presence and quantitation of endogenous retroviruses, and the detection and characterization of other potential contaminants. The technique is favored in this field because it is simple, reliable, and can give reliable quantitation for risk assessments. This article describes the main EM techniques currently used for testing cell cultures, culture supernatants, and bulk harvests. It also includes an in-depth description of a thin sectioning technique used to estimate virus titre in culture supernatants and bulk harvests…
Testing for parallelism is a fundamental requirement for assessing the validity of bioassay data used to calculate relative potency. When a test sample and the reference material are diluted and assayed, the assumption is that their dose response curves have an identical shape. The standard statistical approach to demonstrate that two curves are parallel involves testing the null hypothesis for equality. This article explains why that approach is not appropriate and presents an argument for testing the null hypothesis for a specified difference to statistically ascertain parallelism…
Glycosylation, a posttranslational modification that adds sugars to proteins, is required by many proteins to function properly. Glycosylation can modulate the biological activities of monoclonal antibodies (MAbs), including certain effector functions in the Fc region of IgG antibodies. Monoclonal antibodies produced at higher expression levels by mammalian cell culture may contain small amounts of nonglycosylated heavy chain (NGHC). Recent cell culture mini-reactor studies have shed light on the process parameters that most affect the occurrence of NGHC, and have greatly minimized NGHC levels in IgG MAb products…
The Gel Microdrop (GMD) Secretion Assay involves encapsulating cells within a biotinylated agarose matrix, followed by capture and detection of cell-secreted molecules with fluorescent markers. This technology differs from other encapsulation methods in that the small size of the microdrop (<50 ?m diameter) creates a defined microenvironment around the cell without impeding the fusion of nutrients, antibodies, or nucleic acid probes into the GMDs, or the diffusion of secreted products out of the GMDs. Large numbers of GMDs can be readily analyzed using flow cytometry, and sub-populations of rare or high-secreting cells, as small as 0.1%, can be detected and recovered in one day. This assay format is a rapid alternative to limited dilution cloning (LDC)...
It is well known that the characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. There are numerous selective and adaptive culture processes, in addition to genetic instability, that may promote phenotypic changes in cell growth, virus susceptibility, gene expression, et cetera. Similar detrimental effects of long term passaging of insect cells have also been reported for continuous cell lines. In this paper, we describe the isolation of cell clones from low passage BTI Tn5B1-4 cells (High FiveTM Cells), and report their growth characteristics and high level of recombinant protein production…
The characterization of a batch cell culture process to produce a monoclonal antibody from a GS-NS0 mouse myeloma cell line is described. Productivity and cellular metabolism were monitored during scale-up to both characterize the process and aid in assessing cell culture stability. During fermentation scale-up studies, it was found that as culture generation number increased, productivity declined. In both flask and bioreactor cultures, declining production started abruptly at approximately generation 60. In this study, we assessed whether the decline in productivity was due to genetic instability of the cell line, which resulted in the generation of a non-producer sub-population, or a shift to a less productive state of cellular metabolism…