A general approach to site-specific antibody drug conjugates.

Using an expanded genetic code, antibodies with site-specifically incorporated nonnative amino acids were produced in stable cell lines derived from a CHO cell line with titers over 1 g/L. Using anti-5T4 and anti-Her2 antibodies as model systems, site-specific antibody drug conjugates (NDCs) were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized monomethyl auristatin D with either protease-cleavable or noncleavable linkers. When noncleavable linkers were used, these conjugates were highly stable and displayed improved in vitro efficacy as well as in vivo efficacy and pharmacokinetic stability in rodent models relative to conventional antibody drug conjugates conjugated through either engineered surface-exposed or reduced interchain disulfide bond cysteine residues. The advantages of the oxime-bonded, site-specific NDCs were even more apparent when low-antigen-expressing (2+) target cell lines were used in the comparative studies. NDCs generated with protease-cleavable linkers demonstrated that the site of conjugation had a significant impact on the stability of these rationally designed prodrug linkers. In a single-dose rat toxicology study, a site-specific anti-Her2 NDC was well tolerated at dose levels up to 90 mg/kg. These experiments support the notion that chemically defined antibody conjugates can be synthesized in commercially relevant yields and can lead to antibody drug conjugates with improved properties relative to the heterogeneous conjugates formed by nonspecific chemical modification.

Solar and tidal modulations of fecal indicator bacteria in coastal waters at Huntington Beach, California.

The coastal waters at many beaches in California and the United States are afflicted with fecal pollution, which poses a health risk for people exposed to the water through recreational activities such as swimming, surfing, and diving. Identifying sources of pollution is complicated by oceanographic transport/mixing processes and the nonconservative behavior of microorganisms exposed to sunlight and hostile marine conditions. This article investigates the variation of fecal indicator bacteria (FIB) concentrations in the surf zone and the adjacent coastal marsh by applying autocorrelation and cross-correlation analyses that illustrate solar and tidal modulations. A steady state bioreactor model was developed to explain solar inactivation in the surf zone, whereas a dynamic model was applied to explain tidally influenced disturbances in the coastal marsh. These models applied to intensive monitoring datasets on FIB and environmental variables have provided insights into the biologic and physical processes controlling coastal water quality, specifically the influence of sunlight and tides on bacterial levels.

Application of fractional factorial designs to screen active factors for antibody production by chinese hamster ovary cells.

With ever increasing need for cost-effective large-scale production of monoclonal antibodies, it is essential to develop highly productive and commercially viable processes. Previous research showed that growth and production capacity of the culture media can be improved by micronutrient supplements, such as insulin, vitamins, and growth factors. Since these micronutrients may not act independently of one another, factorial designs can expose critical interactions between nutrients as opposed to a serial approach of changing one factor at a time. In this study, fractional factorial designs were applied to observe the effect of several micronutrients on antibody production and culture longevity in shake flasks. Response surface designs were used to investigate the factors in depth and confirm the results of the fractional factorial study. The results demonstrate that fractional factorial design is an effective tool for rapid development of antibody-producing Chinese hamster ovary (CHO) cells.

Heuristic optimization of antibody production by Chinese hamster ovary cells.

Large-scale production of monoclonal antibodies necessitates the development of a commercially viable process using the appropriate bioreactors, culture medium, and optimal feeding strategies. In the development of feeding strategies for higher antibody titers it is critical to assess the effects of limiting substrates on cell culture longevity and antibody production. In this study, glucose and L-glutamine were identified as limiting substrates and their effects on culture longevity and antibody production were evaluated in small-scale experiments. The results suggested that an optimal feeding strategy should account for the osmolality profile of the culture. The heuristic approach taken to optimize the antibody production showed that the fed-batch cultivation is superior to batch culture and maintaining low osmolality during growth phase increases cumulative viable cell density and thus leads to higher final antibody titer.

Use of fecal steroids to infer the sources of fecal indicator bacteria in the Lower Santa Ana River Watershed, California: sewage is unlikely a significant source.

The Santa Ana River (SAR), CA and adjacent wetlands have been identified as potential sources of fecal indicator bacteria (FIB) to the surf zone at Huntington Beach, CA. A suite of fecal steroids, including coprostanol (COP), epicoprostanol (eCOP), cholesterol (CHOE), cholestanol (CHOA), alpha-cholestanone (aONE), beta-cholestanone (bONE), beta-sitosterol (bSIT), stigmasterol (STIG), stigmastanol (STAN), and campesterol (CAM), were used as chemical markers to examine whether sewage was a significant source of FIB within the lower Santa Ana River watershed. A total of 54 water samples were collected from three locations in the intertidal zone near the mouth of the Santa Ana River at different tidal stages. Steroid ratios in SAR samples were different from those found in raw and treated sewage from a local wastewater treatment plant or in nearby effluent plume and did not appear to be influenced by the sampling location, daily tides, and spring/neap tidal cycle. The characteristics of steroid ratios suggested a diagenetic ratherthan a biogenic source forthe COP content of the samples. The log-based concentrations of COP and FIB in the SAR samples were not significantly correlated, inconsistent with sewage being the source of FIB in the study area. In addition, multivariate statistical analysis showed that the concentrations of FIB were better correlated with bird fecal steroids than with the typical sewage sterols. The results implied that sewage was not a significant source of fecal steroids, and therefore perhaps FIB to the study area. Instead, birds may be one possible source of the intermittently high levels of FIB observed in the lower Santa Ana River watershed and the nearby surf zone.

Unstructured model for L-lysine fermentation under controlled dissolved oxygen.

An unstructured model was developed for batch cultivation of Corynebacterium lactofermentum (ATCC 21799) under controlled dissolved oxygen. The model is capable of predicting batch experiments performed at various initial substrate concentrations. By extending the batch culture model to a fed-batch model and using a heuristic approach to optimize the fed-batch cultivation, it is shown that fed-batch cultivation is superior to batch operation due to increased productivity at high substrate concentrations.