There is renewed interest in the possibility of using precipitation for initial capture of high‐value therapeutic proteins as part of an integrated continuous downstream process. Precipitation is greatly facilitated by the high product titers now achieved in most cell culture processes, in sharp contrast to chromatographic processes whose performance is reduced at high titers. The current study used a combination of reversible cross‐linking (zinc chloride, ZnCl2) and volume exclusion (polyethylene glycol) agents to precipitate a monoclonal antibody product directly from harvested cell culture fluid using a continuous tubular precipitation reactor. The precipitates were then dewatered and continuously washed using tangential flow filtration, with a countercurrent‐staged configuration used to reduce the amount of wash buffer required and increase host cell protein removal. Long‐term operation was achieved by operating the membrane modules below the critical filtrate flux to avoid fouling. Experimental results demonstrate the feasibility of this fully continuous integrated precipitation process at bench scale, with design calculations used to explore the key factors affecting the performance of this system for initial antibody capture.
(5-12). TGF-1, a factor that is important for Th17 differentiation in mice, is also critical for the initiation of EAE (13,14). Factors inhibiting Th17 cells, such as IL-25 and IL-27, inhibit EAE (15-17). In addition, Act1, a molecule within the IL-17 signal transduction pathway is required for EAE (18,19). Therefore, autoreactive Th17 cells are important for EAE.Th17 cells make both 20). Recently, an IL-17-IL-17F heterodimer was found to be expressed in Th17 cells together with IL-17 and IL-17F homodimers (21, 22). IL-17 binds and signals through IL-17 receptor A (IL-17RA) (23). IL-17F also functions through IL-17RA because a specific anti-IL17RA Ab neutralizes its function (24) and IL17RA Ϫ/Ϫ cells do not respond to IL17F in vitro (25). Therefore, studying IL-17RA Ϫ/Ϫ mice should allow us to assess the effect of the combined loss of function of IL-17 and IL-17F.Despite the appreciation of a critical role of Th17 cells in causing CNS autoimmunity, the pathology of a Th17-dominated inflammation in experimental models differs from that in most clinical specimens (26). Therefore, it is important to study a model system when IL-17 function is deficient. In this study, we have found that the IL-17RA is important for the development of EAE and a small number of IL-17RA Ϫ/Ϫ mice developed very mild clinical signs of EAE. In addition, autoreactive Th1 responses were drastically dampened in IL-17RA Ϫ/Ϫ mice. Interestingly, systemically blockade of TGF- in IL-17RA Ϫ/Ϫ mice resulted in exacerbated EAE and disease severity correlated with increased Th1 responses. These data suggest IL-17RA-independent EAE is likely mediated by Th1 immune response and is suppressed by TGF-. Materials and Methods MiceThe IL-17RA Ϫ/Ϫ mice were obtained from Dr. J. J. Peschon (Amgen) as previously described (27) and bred in the University of Pittsburgh Animal Care Facility. The IL-17RA Ϫ/Ϫ mice were backcrossed to C57BL/6 mice for more than 10 generations. The IL-17RA Ϫ/Ϫ mice were generated by crossing IL-17RA ϩ/Ϫ (het) with IL-17RA Ϫ/Ϫ . Littermate IL-17RA ϩ/Ϫ and wild-type (WT) C57BL/6 mice were used as controls. All mice were housed under specific pathogen-free conditions under a protocol approved by the Institutional Animal Care and Use Committee. EAE inductionMice were used for EAE induction by s.c. injection of 150 g MOG (MEVGWYRSPFSRVVHLYRNGK) peptide in IFA and 500 g of heatinactivated Mycobacterium tuberculosis. One hundred nanograms of pertussis toxin was injected i.v. on days 0 and 2. To block TGF- systemically in vivo, mice were injected i.p. at 5 days and 9 days post-EAE elicitation with 400 g of anti-TGF- Ab (clone 1D11) or 400 g of rat IgG (ICN Biomedicals). Mice were monitored for clinical signs of EAE, scored as follows: 0, normal; 1, flaccid tail; 2, hind limb weakness or abnormal gait with poor ability to right from supine; 3, partial hind limb paralysis; 4, both hind limbs paralyzed with or without forelimb paralysis and incontinence; 5, moribund state. The experiments were performed in accordance with the regulatio...
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