The significance of tissue culture is that an understanding of animal physiology will come from a study of the basic elements of the animal (the cell) in isolated, pure form (clonal culture), under defined conditions (hormonally defined media). This should be the fundamental approach to animal physiology for the foreseeable future. The problem with this formulation in 1958, when I began work on tissue culture, was that cells in culture did not display the differentiated properties of the tissue of origin…
BioProcessing Journal Posts
For more than a decade, transgenic plants have been investigated as alternatives to microbial, mammalian cell, and transgenic animal systems for recombinant protein production. The main advantages of using plants as “bioreactors” are that the cost of upstream production (i.e. biomass creation) is low; plants do not carry viruses and other pathogens dangerous to humans such as human immunodeficiency virus (HIV), prions, hepatitis viruses and so on; and as eukaryotes, plants are capable of producing bioactive proteins. Numerous recombinant proteins have been expressed in various plant hosts, and some recombinant proteins are in various stages of clinical trials…
The baculovirus expression vector system, which is based on infecting insect cells with recombinant Autographa californica nuclear polyhedrosis virus (AcNPV), is one of the most commonly used eukaryotic expression systems aimed at producing functionally active mammalian proteins. It offers advantages such as high-level protein expression and post-translational processing capabilities that are extremely important to the biological activity of certain proteins. This system utilizes a strong promoter of the very late gene, polyhedrin, to drive heterologous protein overexpression. Nevertheless, in order to generate milligram amounts of recombinant proteins, cell culture often needs to be scaled up to as much as 25 liters….
The outstanding success and safety record of first generation monoclonal products has created an immense increase in the number of product candidates that need to be evaluated clinically. The concept of platform purification has emerged in response to this need. A platform is a semigeneric, multistep purification procedure that can be applied to a wide range of monoclonal antibodies without extensive method-scouting and optimization. This approach can substantially accelerate process development and hasten inception of clinical trials…
As human immunodeficiency virus type-1 (HIV-1) continues to spread around the world, scientists are actively pursuing effective vaccines against the infectious disease that results in AIDS. A number of vaccine designs have been developed, including plasmid DNA constructs encoding HIV proteins. One advantage of DNA vaccination is that after the uptake of the plasmid by the host cells, the encoded antigens are expressed in the native conformation and allow authentic immunological processing of the antigen. Another advantage of DNA vaccines is that they can be repeatedly administered without vector-directed immunity limiting the efficacy of the boost. DNA vaccines alone can induce both humoral and cellular immune responses and provide modest protection against disease progression in the preclinical, nonhuman primate model when challenged with simian immunodeficiency virus (SIV)…
Of the available on-line biomass assays, the radio-frequency (RF) impedance method has a clear advantage for current good manufacturing process (cGMP) because it is an unambiguous reflection of viable cell bio-volume rather than the total number of cells. Although other more approximate methods are available for cells in suspension, RF impedance is practically the only on-line method available for cells in suspension, attached to microcarriers and immobilized cells at high cell densities. Data are presented to show how live cell concentrations are derived from an RF impedance-derived instrument…
The enzyme-linked immunospot (ELISpot) assay is one of the most useful techniques for the immunological monitoring of vaccine trials and has increasing application as a measure of specific T-cell activation. Recently, we developed, optimized, and validated a customized ELISpot kit for the detection of interferon gamma (IFNγ) positive cells. The precision of the ELISpot was good and it varied over the range of the assay values, independent of the stimulus. Here we describe the development of a library of donors with characterized responses to the CEF peptide pool: cytomegalovirus (CMV), Epstein-Barr virus (EBV), and influenza (Flu); pool of 32 peptides which can be used as controls for IFNγ ELISpot and multiple immune monitoring assay validations for use in clinical trials…
The quantitative and qualitative analysis of proteins and their amino acid sequence composition is a critical operation in many research laboratories and operations. Much protein analysis is performed using high-pressure liquid chromatography (HPLC) with ultraviolet (UV) or fluorescence detection. Although these methods are robust and widely used, certain issues limit their utility. For example, some proteins or peptides may have a poor UV response and can be difficult to detect. Direct comparisons of protein levels when quantitated directly by UV can also be problematic due to differences in extinction coefficients of various proteins…