Investigation of fouling mechanisms of virus filters during the filtration of protein solutions using a high throughput filtration screening device.

The downstream process development of novel antibodies (Abs) is often challenged by virus filter fouling making a better understanding of the underlying mechanisms highly desirable. The present study combines the protein characterization of different feedstreams with their virus filtration performance using a novel high throughput filtration screening system. Filtration experiments with Ab concentrations of up to 20 g/L using either low interacting or hydrophobically interacting pre-filters indicate the existence of two different fouling mechanisms, an irreversible and a reversible one. At the molecular level, size exclusion chromatography revealed that the presence of large amount of high molecular weight species-considered as irreversible aggregates-correlates with irreversible fouling that caused reduced Ab throughput. Results using dynamic light scattering show that a concentration dependent increase of the mean hydrodynamic diameter to the range of dimers (17 nm at 20 g/L) together with a negative DLS interaction parameter kD (-18 mL/g) correlate with the propensity to form reversible aggregates and to cause reversible fouling, probably by a decelerated Ab transport velocity within the virus filter. The two fouling mechanisms are further supported by buffer flush experiments. Finally, concepts for reversible and irreversible fouling mechanisms are discussed together with strategies for respective fouling mitigation. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2776, 2019.

Prediction uncertainty assessment of chromatography models using Bayesian inference.

Mechanistic modeling of chromatography has been around in academia for decades and has gained increased support in pharmaceutical companies in recent years. Despite the large number of published successful applications, process development in the pharmaceutical industry today still does not fully benefit from a systematic mechanistic model-based approach. The hesitation on the part of industry to systematically apply mechanistic models can often be attributed to the absence of a general approach for determining if a model is qualified to support decision making in process development. In this work a Bayesian framework for the calibration and quality assessment of mechanistic chromatography models is introduced. Bayesian Markov Chain Monte Carlo is used to assess parameter uncertainty by generating samples from the parameter posterior distribution. Once the parameter posterior distribution has been estimated, it can be used to propagate the parameter uncertainty to model predictions, allowing a prediction-based uncertainty assessment of the model. The benefit of this uncertainty assessment is demonstrated using the example of a mechanistic model describing the separation of an antibody from its impurities on a strong cation exchanger. The mechanistic model was calibrated at moderate column load density and used to make extrapolations at high load conditions. Using the Bayesian framework, it could be shown that despite significant parameter uncertainty, the model can extrapolate beyond observed process conditions with high accuracy and is qualified to support process development.

Assessment of Preclinical Liver and Skeletal Muscle Biomarkers Following Clofibrate Administration in Wistar Rats.

Clofibrate is a known rodent hepatotoxicant classically associated with hepatocellular hypertrophy and increased serum activities of cellular alanine aminotransferase/aspartate aminotransferase (ALT/AST) in the absence of microscopic hepatocellular degeneration. At toxic dose, clofibrate induces liver and skeletal muscle injury. The objective of this study was to assess novel liver and skeletal muscle biomarkers following clofibrate administration in Wistar rats at different dose levels for 7 days. In addition to classical biomarkers, liver injury was assessed by cytokeratin 18 (CK18) cleaved form, high-mobility group box 1, arginase 1 (ARG1), microRNA 122 (miR-122), and glutamate dehydrogenase. Skeletal muscle injury was evaluated with fatty acid binding protein 3 (Fabp3) and myosin light chain 3 (Myl3). Clofibrate-induced hepatocellular hypertrophy and skeletal muscle degeneration (type I rich muscles) were noted microscopically. CK, Fabp3, and Myl3 elevations correlated to myofiber degeneration. Fabp3 and Myl3 outperformed CK for detection of myofiber degeneration of minimal severity. miR-122 and ARG1 results were significantly correlated and indicated the absence of liver toxicity at low doses of clofibrate, despite increased ALT/AST activities. Moreover, combining classical and novel biomarkers (Fabp3, Myl3, ARG1, and miR-122) can be considered a valuable strategy for differentiating increased transaminases due to liver toxicity from skeletal muscle toxicity.

IKKα controls ATG16L1 degradation to prevent ER stress during inflammation.

Inhibition of the IκB kinase complex (IKK) has been implicated in the therapy of several chronic inflammatory diseases including inflammatory bowel diseases. In this study, using mice with an inactivatable IKKα kinase (IkkαAA/AA), we show that loss of IKKα function markedly impairs epithelial regeneration in a model of acute colitis. Mechanistically, this is caused by compromised secretion of cytoprotective IL-18 from IKKα-mutant intestinal epithelial cells because of elevated caspase 12 activation during an enhanced unfolded protein response (UPR). Induction of the UPR is linked to decreased ATG16L1 stabilization in IkkαAA/AA mice. We demonstrate that both TNF-R and nucleotide-binding oligomerization domain stimulation promote ATG16L1 stabilization via IKKα-dependent phosphorylation of ATG16L1 at Ser278. Thus, we propose IKKα as a central mediator sensing both cytokine and microbial stimulation to suppress endoplasmic reticulum stress, thereby assuring antiinflammatory function during acute intestinal inflammation.

IKKα promotes intestinal tumorigenesis by limiting recruitment of M1-like polarized myeloid cells.

The recruitment of immune cells into solid tumors is an essential prerequisite of tumor development. Depending on the prevailing polarization profile of these infiltrating leucocytes, tumorigenesis is either promoted or blocked. Here, we identify IκB kinase α (IKKα) as a central regulator of a tumoricidal microenvironment during intestinal carcinogenesis. Mice deficient in IKKα kinase activity are largely protected from intestinal tumor development that is dependent on the enhanced recruitment of interferon γ (IFNγ)-expressing M1-like myeloid cells. In IKKα mutant mice, M1-like polarization is not controlled in a cell-autonomous manner but, rather, depends on the interplay of both IKKα mutant tumor epithelia and immune cells. Because therapies aiming at the tumor microenvironment rather than directly at the mutated cancer cell may circumvent resistance development, we suggest IKKα as a promising target for colorectal cancer (CRC) therapy.

Ink4a/Arf and oncogene-induced senescence prevent tumor progression during alternative colorectal tumorigenesis.

Colonic cancers with a serrated morphology have been proposed to comprise a molecularly distinct tumor entity following an alternative pathway of genetic alterations independently of APC mutations. We demonstrate that intestinal epithelial cell specific expression of oncogenic K-ras(G12D) in mice induces serrated hyperplasia, which is characterized by p16(ink4a) overexpression and induction of senescence. Deletion of Ink4a/Arf in K-ras(G12D) expressing mice prevents senescence and leads to invasive, metastasizing carcinomas with morphological and molecular alterations comparable to human KRAS mutated serrated tumors. Thus, we suggest that oncogenic K-ras represents a key player during an alternative, serrated pathway to colorectal cancer and hence propose RAS-RAF-MEK signaling apart from APC as an additional gatekeeper in colorectal tumor development.

gp130-mediated Stat3 activation in enterocytes regulates cell survival and cell-cycle progression during colitis-associated tumorigenesis.

Although gastrointestinal cancers are frequently associated with chronic inflammation, the underlying molecular links have not been comprehensively deciphered. Using loss- and gain-of-function mice in a colitis-associated cancer model, we establish here a link comprising the gp130/Stat3 transcription factor signaling axis. Mutagen-induced tumor growth and multiplicity are reduced following intestinal epithelial cell (IEC)-specific Stat3 ablation, while its hyperactivation promotes tumor incidence and growth. Conversely, IEC-specific Stat3 deficiency enhances susceptibility to chemically induced epithelial damage and subsequent mucosal inflammation, while excessive Stat3 activation confers resistance to colitis. Stat3 has the capacity to mediate IL-6- and IL-11-dependent IEC survival and to promote proliferation through G1 and G2/M cell-cycle progression as the common tumor cell-autonomous mechanism that bridges chronic inflammation to tumor promotion.