During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non-electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science-based process validation strategies to ensure viral safety of biotechnology products.

The innate immune recognition of bacterial lipopolysaccharide (LPS) is mediated by Toll-like receptor 4 (TLR4) and results in activation of proinflammatory signaling including NF-B and MAPK pathways. Heterotrimeric G proteins have been previously implicated in LPS signaling in macrophages and monocytes. In the present study, we show that pertussis toxin sensitive heterotrimeric G proteins (G␣i/o) are involved in the activation of MAPK and Akt downstream of TLR2, TLR3, and TLR4 in endothelial cells. G␣ i/o are also required for full activation of interferon signaling downstream of TLR3 and TLR4 but are not required for the activation of NF-B. We find that G␣i/o-mediated activation of the MAPK is independent of the canonical MyD88, interleukin-1 receptor-associated kinase, and tumor necrosis factor receptor-associated factor 6 signaling cascade in LPS-stimulated cells. Taken together, the data presented here suggest that heterotrimeric G proteins are widely involved in TLR pathways along a signaling cascade that is distinct from MyD88-TRAF6.lipopolysaccharide; innate immunity; toll-like receptors; heterotrimeric G proteins; myeloid differentiation factor THE INNATE IMMUNE RECOGNITION of bacterial and viral products is mediated by a family of transmembrane receptors known as Toll-like receptors (TLRs). Lipopolysaccharide (LPS), a key component of the outer wall of gram-negative bacteria, initiates endothelial activation through a receptor complex consisting of TLR4, CD14, and MD2 (9). Recruitment of the adaptor proteins TIR-containing adaptor molecule (TIRAP) and myeloid differentiation factor (MyD88) initiates a MyD88-dependent pathway that culminates in the activation of NF-B and MAPK. Activation of these downstream targets requires recruitment of interleukin-1 receptor-associated kinase (IRAK4) and IRAK1 through interaction between the death domains of MyD88 and the IRAKs. The autophosphorylation and activation of IRAK1 results in the ability to bind tumor necrosis factor receptor-associated factor 6 (TRAF6) (7), which leads to oligomerization and polyubiquitination of the TRAF6 molecule (11). This facilitates activation of transforming growth factor-␤ (TGF-␤)-activated kinase 1, which leads to activation of the IKK-NF-B pathway and the three MAPK: p38, ERK, and JNK (39). In addition to the MyD88-dependent pathway, LPS stimulation also results in the activation of a MyD88-independent pathway, through recruitment of the adaptor molecules TIR-containing adaptor inducing IFN-␤ (TRIF) and TRIF-related adaptor molecule. This leads to the late-phase activation of NF-B and interferon regulatory factor 3 (IRF3), as well as activation of the MAPKs and phosphatidylinositol 3-kinase (PI3K) (10).The endothelium plays a major role in the pathogenesis of sepsis. Under normal conditions, the endothelium functions to maintain organ homeostasis through vasoregulation, selective vascular permeability, and providing an anticoagulant surface. During bacterial infection, the normal physiological functions of the endothelium are pert...

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