In recent years, researchers have turned to transient gene expression (TGE) as an alternative to CHO stable cell line generation for early-stage antibody development. Despite advances in transfection methods and culture optimization, the majority of CHO-based TGE systems produce insufficient antibody titers for extensive use within biotherapeutic development pipelines. Flow electroporation using the MaxCyte STX Scalable Transfection System is a highly efficient, scalable means of CHO-based TGE for gram-level production of antibodies without the need for specialized expression vectors or genetically engineered CHO cell lines. CHO cell flow electroporation is easily scaled from milligram to multigram quantities without protocol reoptimization while maintaining transfection performance and antibody productivity. In this article, data are presented that demonstrate the reproducibility, scalability, and antibody production capabilities of CHO-based TGE using the MaxCyte STX. Data show optimization of posttransfection parameters such as cell density, media composition, and feed strategy that result in secreted antibody titers >1 g/L and production of multiple grams of antibody within 2 weeks of a single CHO-S cell transfection. In addition, data are presented to demonstrate the application of scalable electroporation for the rapid generation of high-yield stable CHO cell lines to bridge the gap between early- and late-stage antibody development activities.

No abstract

Regulation of protein phosphorylation is a primary cellular signaling mechanism. Many cellular responses to internal and external events are mitigated by protein kinase signaling cascades. Dysfunction of protein kinase activity has been linked to a variety of human pathologies, in the areas of cancer, inflammation, metabolism, cell cycle, apoptosis, as well as cardiovascular, neurodegenerative and autoimmune diseases [1-3]. As such, there is an important need for protein kinase activity detection methodologies for researchers engaged in Drug Discovery. A number of different technologies have been employed for the measurement of protein kinase activity, including radioactive methods, luminescent methods, and fluorescent methods. More recently, Homogeneous Time Resolved Fluorescence technology (HTRF®), based on the principle of time-resolved fluorescent resonance energy transfer (TR-FRET), has been developed and applied for the measurement of protein kinase activity in vitro. This technology note describes the development of an HTRF® assay for detection of Syk enzyme activity in a format consistent with the requirements of High-Throughput Screening (HTS) campaigns currently used in drug discovery.

Rat virus (RV) is a common parvovirus of laboratory rodents which can disrupt rat-based research. Prenatal or perinatal infection can be pathogenic or lead to persistent infection, whereas infection of adult rats is typically self-limiting. Effects on the host immune system have been documented during RV infection, but little is known about immune responses necessary for viral clearance. Our studies were conducted to identify humoral and cellular responses to the predominant capsid protein, VP2, during experimental infection of adult rats. We observed VP2-specific proliferation, gamma interferon production, and an immunoglobulin G2a humoral response that is maintained for at least 35 days following RV infection. These results strongly suggest the induction of virus-specific Th1-mediated immunity.Parvoviruses are nonenveloped viruses with a singlestranded genome of approximately 5 kb. Viral replication is dependent on many cellular functions, particularly those expressed during S phase of the cell cycle (7). Rodent parvoviruses encode two classes of proteins: nonstructural (NS) proteins and structural or capsid proteins (VP). NS proteins are involved in viral replication, transcription, and cytotoxicity. VP1 comprises 15% of the icosahedral virion, and its coding sequence contains all of VP2 plus 140 additional N-terminal amino acids. VP2 is the predominant capsid protein, accounting for approximately 85% of the virion. VP3, a cleavage product of VP2, is present in small, varying amounts in DNAcontaining virions.A recent national survey found that parvovirus infections are highly prevalent among laboratory rats and mice (17). Infection with some of these agents can be lethal in fetal or perinatal animals, probably due to high numbers of mitotically active cells that serve as targets for cytolytic viral replication. Larger impacts on biomedical research result from clinically silent infections in infant or adult rodents in which biological processes and immune responses are altered. Specifically, infections with murine parvoviruses such as mouse minute virus and mouse parvovirus are known to inhibit several T-cell effector functions in vitro (2, 8); suppress antigen-induced proliferation of specific cloned T cells (18); augment the rate of tumor, allogeneic, and syngeneic graft rejection; and modulate other T-cell effector functions (19,21). In addition, infection with rat virus (RV), the prototypic parvovirus of rats, can decrease lymphocyte viability and suppress proliferative responses to alloantigens (4), diminish responses of mixed lymphocyte culture and cytolytic T cells from peripheral and mesenteric lymph nodes (20), alter humoral responses to ovalbumin (5), and provoke autoimmune diabetes in the diabetes-resistant strain of Biobreeding/Worcester (BB/WOR) rats (3,5,13).Despite the impact of these agents on host immunity, little is known about immune responses to rodent parvoviruses. It is known that rats develop antiviral antibodies and perivascular mononuclear cell infiltrates in infected tissues (10,14)....

Simian-human immunodeficiency virus (SHIV) infection in macaques provides a convenient model for testing vaccine efficacy and for understanding viral pathogenesis in AIDS. We immunized macaques with recombinant, Salmonella typhimurium (expressing Gag) or soluble Gag in adjuvant to generate T-cell-dependent lymphoproliferative or serum antibody responses. Immunized animals were challenged by intrarectal inoculation with SHIV89.6PD. Virus infection was accompanied by rapid losses of lymphoproliferative responses to Gag or phytohemagglutinin. By 8 weeks, mitogen responses recovered to near normal levels but antigen-specific immunity remained at low or undetectable levels. Serum antibody levels were elevated initially by virus exposure but soon dropped well below levels achieved by immunization. Our studies show a rapid depletion of preexisting Gag-specific CD4+ T cells that prevent or limit subsequent antiviral cellular and humoral immune responses during acute SHIV infection.