Analysis of complex protein elution behavior in preparative ion exchange processes using a colloidal particle adsorption model.

A fundamental understanding of the protein retention mechanism in preparative ion exchange (IEX) chromatography columns is essential for a model-based process development approach. For the past three decades, the mechanistic description of protein retention has been based predominantly on the steric mass action (SMA) model. In recent years, however, retention profiles of proteins have been reported more frequently for preparative processes that are not consistent with the mechanistic understanding relying on the SMA model. In this work, complex elution behavior of proteins in preparative IEX processes is analyzed using a colloidal particle adsorption (CPA) model. The CPA model is found to be capable of reproducing elution profiles that cannot be described by the traditional SMA model. According to the CPA model, the reported complex behavior can be ascribed to a strong compression and concentration of the elution front in the lower unsaturated part of the chromatography column. As the unsaturated part of the column decreases with increasing protein load density, exceeding a critical load density can lead to the formation of a shoulder in the peak front. The general applicability of the model in describing preparative IEX processes is demonstrated using several industrial case studies including multiple monoclonal antibodies on different IEX adsorber systems. In this context, the work covers both salt controlled and pH-controlled protein elution.

Multiple functions of caprylic acid-induced impurity precipitation for process intensification in monoclonal antibody purification.

New emerging technologies delivering benefits in terms of process robustness and economy are an inevitable prerequisite for monoclonal antibody purification processes intensification. Caprylic acid was proven as an effective precipitating agent enabling efficient precipitaton of product- and process-related impurities while leaving the antibody in solution. This purification step at mild acidic pH was therefore introduced in generic antibody platform approaches after Protein A capture and evaluated for its impact regarding process robustness and antibody stability. Comparison of 13 different monoclonal antibodies showed significant differences in antibody recovery between 65-95% during caprylic acid-induced impurity precipitation. Among six compared physicochemical properties, isoelectric point of the antibody domains was figured out to correlate with yield. Antibodies with mild acidic pI of the light chain were significantly susceptible to caprylic acid-induced precipitation resulting in lower yields. Virus clearance studies revealed that caprylic acid provided complete virus inactivation of an enveloped virus. Multiple process relevant factors such as pH range, caprylic acid concentration and antibody stability were investigated in this study to enable an intensified purification process including caprylic acid precipitation for HCP removal of up to 2 log10 reduction values at mAb yields >90% while also contributing to the virus safety of the process.

Effects of corticotropin-releasing hormone (CRH) on endothelin-1 and NO release, mediated by CRH receptor subtype R2: a potential link between stress and endothelial dysfunction?

OBJECTIVE: Psychosocial factors, associated with elevated corticotropin releasing hormone (CRH) concentrations, have been reported to be independently associated with coronary heart disease. METHODS: Endothelin-1 and NO release of human endothelial cells were quantified via ELISA or fluorometrically after treatment with CRH. CRH-receptor subtype 2 (CRH-R2) was visualized on endothelial cells by immunohistochemistry and confirmed by polymerase chain reaction using CRH-R2 primers. RESULTS: CRH induced a significant increase of ET-1 release, and the effect was abolished by the CRH-receptor antagonist astressin. The effect was mediated by CRH-R2. In contrast, NO release was not affected. CONCLUSION: CRH-R2 is expressed on human endothelial cells, mediating the CRH-induced stimulation of ET-1 release, whereas NO release is not affected. Thus, peripherally circulating CRH may offset the balance between endothelial vasoconstrictor and vasodilator release with unopposed vasoconstriction. Our data may provide a new concept on how CRH-receptor antagonists may prevent CRH-induced disorders of vascular biology.

Effects of corticotropin-releasing hormone (CRH) on monocyte function, mediated by CRH-receptor subtype R1 and R2: a potential link between mood disorders and endothelial dysfunction?

Psychosocial factors have been reported to be independently associated with coronary heart disease (CHD). Though corticotropin-releasing hormone (CRH) is the major hormone activated during adaptive responses to stressful stimuli, the undergoing pathophysiological mechanism related to stress-induced endothelial dysfunction is still poorly understood. This study sought to investigate the effects of extrahypothalamic CRH on monocyte/endothelium adhesion. Second we elucidate the influence of CRH on monocytic endothelin-1 (ET-1) and nitric oxide (NO) release and the receptors involved. Cell adhesion was determined using an adhesion assay, MAC-1 expression by flow cytometry. ET-1/NO release were quantified via ELISA or fluorometrically, monocytic CRH-receptors were confirmed by mRNA. Corticotropin-releasing hormone induced a significant time- and concentration-dependent increase of cell adhesion as well as monocytic MAC-1 expression; endothelial ICAM-1 and VCAM-1 expression was not altered. In addition, corticotropin-releasing hormone significantly increased monocytic ET-1 release whereas nitric oxide release was decreased. The effect was abolished by the selective CRH-receptor antagonist astressin. Our findings support the importance of peripherally circulating corticotropin-releasing hormones, by influencing specific homeostatic properties of monocytes. Our data may provide a novel concept of how specific CRH-receptor antagonists may prevent CRH (stress)-related endothelial dysfunction up to cardiovascular complications.

The impact of immunosuppression on endothelial function.

Endothelial dysfunction is an early marker for transplant atherosclerosis. Potential mechanisms for allograft endothelial dysfunction include stimulation of alloimmune-dependent pathways, ischemia/reperfusion injury, metabolic alterations, chronic infections, as well as direct endothelial cell activation by immunosuppressive drugs. Thus far, no study has directly compared different immunosuppressive drugs with respect to their potential to modulate endothelial function under normoxic and hypoxic conditions. We examined human microvascular endothelial cells (HMEC-1) in vitro after stimulation with therapeutic concentrations of methylprednisolone (MP), mycophenolic acid (MMF), cyclosporine A (CS), rapamycin (Rapa), and tacrolimus (Tac) to designate the corresponding induction of oxidative stress, apoptosis, metabolic activity, proliferation, endothelin (ET-1) release, and nitric oxide (NO) production. HMEC-1 stimulation with CS, MMF, and Rapa resulted in a stronger induction of oxidative stress compared with MP and Tac. Induction of oxidative stress by immunosuppressives correlated with metabolic activity and apoptosis. Low- and high-dose MMF significantly inhibited cell proliferation under hypoxic conditions, whereas low-dose CS and MP increased endothelial cell proliferation. ET-1 release was significantly elevated by Rapa, Tac, and MP. NO production was significantly enhanced by all immunosuppressive drugs except Tac. Quality and quantity of immunosuppression modify endothelial function and lead to a dose-dependent and oxygenation-state-related endothelial activation. MP and MMF induced minor changes in endothelial function compared with CS, Rapa, and Tac.