Measuring the effects of macromolecular crowding on antibody function with biolayer interferometry.

Biotherapeutic proteins are commonly dosed at high concentrations into the blood, which is an inherently complex, crowded solution with substantial protein content. The effects of macromolecular crowding may lead to an appreciable level of non-specific hetero-association in this physiological environment. Therefore, developing a method to characterize the diverse consequences of non-specific interactions between proteins under such non-ideal, crowded conditions, which deviate substantially from those commonly employed for in vitro characterization, is vital to achieving a more complete picture of antibody function in a biological context. In this study, we investigated non-specific interactions between human serum albumin (HSA) and two monoclonal antibodies (mAbs) by static light scattering and determined these interactions are both ionic strength-dependent and mAb-dependent. Using biolayer interferometry (BLI), we assessed the effect of HSA on antigen binding by mAbs, demonstrating that these non-specific interactions have a functional impact on mAb:antigen interactions, particularly at low ionic strength. While this effect is mitigated at physiological ionic strength, our in vitro data support the notion that HSA in the blood may lead to non-specific interactions with mAbs in vivo, with a potential impact on their interactions with antigen. Furthermore, the BLI method offers a high-throughput advantage compared to orthogonal techniques such as analytical ultracentrifugation and is amenable to a greater variety of solution conditions compared to nuclear magnetic resonance spectroscopy. Our study demonstrates that BLI is a viable technology for examining the impact of non-specific interactions on specific biologically relevant interactions, providing a direct method to assess binding events in crowded conditions.

Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity.

An alternative method to traditional 2-dimensional gel electrophoresis (2D-PAGE) and its application in characterizing the inherent charge heterogeneity of chromatographically isolated monoclonal antibody heavy and light chains is described. This method, referred to as ChromiCE, utilizes analytical size-exclusion chromatography (SEC), performed under reducing and denaturing conditions, followed by imaged capillary isoelectric focusing (icIEF) of the chromatographically separated heavy and light chains. Under conditions suitable for the subsequent icIEF analysis, the absolute and relative SEC elution volumes of the heavy and light chains were found to be highly pH dependent, a phenomenon that can be exploited in optimizing chromatographic separation. Compared to 2D-PAGE, the ChromiCE method substantially decreases the time and labor needed to complete the analysis, improves reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown.

Alda-1 is an agonist and chemical chaperone for the common human aldehyde dehydrogenase 2 variant.

In approximately one billion people, a point mutation inactivates a key detoxifying enzyme, aldehyde dehydrogenase (ALDH2). This mitochondrial enzyme metabolizes toxic biogenic and environmental aldehydes, including the endogenously produced 4-hydroxynonenal (4HNE) and the environmental pollutant acrolein, and also bioactivates nitroglycerin. ALDH2 is best known, however, for its role in ethanol metabolism. The accumulation of acetaldehyde following the consumption of even a single alcoholic beverage leads to the Asian alcohol-induced flushing syndrome in ALDH2*2 homozygotes. The ALDH2*2 allele is semidominant, and heterozygotic individuals show a similar but less severe phenotype. We recently identified a small molecule, Alda-1, that activates wild-type ALDH2 and restores near-wild-type activity to ALDH2*2. The structures of Alda-1 bound to ALDH2 and ALDH2*2 reveal how Alda-1 activates the wild-type enzyme and how it restores the activity of ALDH2*2 by acting as a structural chaperone.

Suppression of insulin aggregation by heparin.

The aggregation of insulin near its isoelectric point and at low ionic strength was suppressed in the presence of heparin. To understand this effect, we used turbidimetry and stopped-flow to study the pH- and ionic strength ( I)-dependence of the aggregation of heparin-free insulin. The results supported the role of interprotein electrostatic interactions, contrary to the commonly held view that such forces are minimized at pH = pI. Electrostatic modeling of insulin (DelPhi) revealed that attractive interactions arise from the marked charge anisotropy of insulin near pI. We show how screening of the interprotein attractions by added salt lead to maximum aggregation near I = 0.01 M, corresponding to a Debye length nearly equal to the diameter of the insulin dimer, consistent with a dipole-like protein charge distribution. This analysis is also consistent with suppression of aggregation by heparin, a strong polyanion that by binding to the positive domain of one protein, inhibits its interaction with the negative domain of another.

Electrostatically driven protein aggregation: beta-lactoglobulin at low ionic strength.

The aggregation of beta-lactoglobulin (BLG) at ambient temperature was studied using turbidimetry and dynamic light scattering in the range 3.8

Effect of magnesium sulfate on contractile force and intracellular calcium concentration in pregnant human myometrium.

OBJECTIVE: This study was undertaken to evaluate the effects of magnesium sulfate (MgSO4) on contractile force and increases in free intracellular calcium concentration ([Ca2+]i) in human myometrial strips from pregnant women. STUDY DESIGN: Simultaneous measurements of isometric tension and [Ca2+]i were measured in myometrial strips obtained at the time of cesarean delivery from pregnant nonlaboring women at term with the use of a fluorescence spectrometer equipped with a displacement force transducer. Changes in [Ca2+]i were measured with fura-2, a Ca(2+)-sensitive fluorescent probe. Myometrial strips were exposed to MgSO4 (5 or 10 mmol/L) for 5, 10, 20, and 30 minutes and observed for spontaneous contractions or stimulated with either oxytocin (OT; 0.1 micromol/L) or potassium chloride (KCl; 90 mmol/L). RESULTS: MgSO4 reduced spontaneous, OT, and KCl-evoked contractions and increases in [Ca2+]i in a time and concentration-dependent manner. After 20 minutes exposure to 5 mmol/L MgSO4, the OT-elicited changes in contractile response and [Ca2+]i were significantly decreased. MgSO4 did not change [Ca2+]i/force relationship of the responses to OT or KCl, or during spontaneous activity. CONCLUSION: At a pharmacologic concentration (5 mmol/L), MgSO4 inhibits contractile response and [Ca2+]i in pregnant human myometrial strips by a pattern that is consistent with both extra- and intracellular mechanisms. At a suprapharmacologic concentration (10 mmol/L), the more immediate effect of MgSO4 is consistent with an extracellular mechanism. MgSO4 does not appear to interfere at the level of the calcium-calmodulin interface, since the [Ca2+]i/force relationship was not changed.

The role of estrogen in cardiovascular disease.

Cardiovascular disease is the number one cause of death among women, accounting for nearly 50% of female deaths. Statistics show that women on average develop cardiovascular disease 10 to 15 years later in life than men, and that the risk may increase after menopause. This observation has led to much speculation as to what physiological change(s) associated with menopause is responsible for the higher risk of atherosclerosis. Estrogen, with its potential as a cardioprotective agent and as an immunomodulator of the inflammatory response in atherosclerosis, has received the most attention. Understanding the mechanisms that lead to these differences may allow beneficial therapeutic intervention to enhance this effect in females and evoke this protection in males. This review will do the following: (1) characterize mechanisms of atherosclerosis, (2) explore the role of estrogen-replacement therapy, (3) define the effect of gender on inflammation, (4) compare and contrast the effects of estrogen and testosterone on endothelial functional, and (5) suggest mechanistic based therapeutic opportunities.