Microflow Imaging Analyses Reflect Mechanisms of Aggregate Formation: Comparing Protein Particle Data Sets Using the Kullback-Leibler Divergence.

Subvisible particles in therapeutic protein formulations are an increasing manufacturing and regulatory concern because of their potential to cause adverse immune responses. Flow imaging microscopy is used extensively to detect subvisible particles and investigate product deviations, typically by comparing imaging data using histograms of particle descriptors. Such an approach discards much information and requires effort to interpret differences, which is problematic when comparing many data sets.

High throughput prediction of the long-term stability of pharmaceutical macromolecules from short-term multi-instrument spectroscopic data.

Changes in the measurements of a macromolecular biopharmaceutical's physical form are often used to predict changes in the drug's long-term stability. These can in turn be used as important markers of changes to a drug's efficacy and safety. Such stability estimates traditionally require human judgment and are frequently tentative.

Studies of the aggregation of RNase Sa.

Thirty-eight mutants of RNase Sa (ribonuclease from Streptomyces aureofaciens) were examined for their structure, thermal sensitivity, and tendency to aggregate. Although a biphasic correlation was seen between the effect of temperature on structure and the free energy of transfer changes in many of the mutants, little correlation was seen between the time at which aggregation is initiated or the rate of aggregation and the thermal sensitivity of the mutants. It is hypothesized that the nature of contacts between protein molecules in the associated (aggregated) phase rather than structural changes dominates the aggregation process for these series of mutants.

Comparative signature diagrams to evaluate biophysical data for differences in protein structure across various formulations.

A solution to the problem of being able to show statistically significant differences in the measurements of various levels of higher-order protein structure has been an elusive one. We propose the use of comparative signature diagrams (CSDs) to this end. CSDs compare datasets from different biophysical techniques that fingerprint the secondary, tertiary, and quaternary structures of a protein molecule and display statistically significant differences in these datasets.

An improved methodology for multidimensional high-throughput preformulation characterization of protein conformational stability.

The empirical phase diagram (EPD) technique is a vector-based multidimensional analysis method for summarizing large data sets from a variety of biophysical techniques. It can be used to provide comprehensive preformulation characterization of a macromolecule's higher-order structural integrity and conformational stability. In its most common mode, it represents a type of stimulus-response diagram using environmental variables such as temperature, pH, and ionic strength as the stimulus, with alterations in macromolecular structure being the response.

Investigation of protein conformational stability employing a multimodal spectrometer.

The conformational stability of proteins is typically investigated by use of a variety of biophysical measurements as a function of environmental stresses such as pH and temperature. Thus, multiple experiments are required on a variety of instruments, each providing information on a particular aspect of a protein's higher order structural integrity. These measurements typically require large sample quantities and long experimental times.

Multidimensional methods for the formulation of biopharmaceuticals and vaccines.

Determining and preserving the higher order structural integrity and conformational stability of proteins, plasmid DNA, and macromolecular complexes such as viruses, virus-like particles, and adjuvanted antigens are often a significant barrier to the successful stabilization and formulation of biopharmaceutical drugs and vaccines. These properties typically must be investigated with multiple lower resolution experimental methods because each technique monitors only a narrow aspect of the overall conformational state of a macromolecular system. This review describes the use of empirical phase diagrams (EPDs) to combine large amounts of data from multiple high-throughput instruments and construct a map of a target macromolecule's physical state as a function of temperature, solvent conditions, and other stress variables.

Differences in fibrinolysis and complement inactivation by venom from different northern blacktailed rattlesnakes (Crotalus molossus molossus).

Venom from 72 different Crotalus molossus molossus rattlesnakes was examined for fibrinolysis and for their ability to inactivate human complement. The fibrinolytic activity of the venoms was variable, but smaller (younger) snakes had less fibrinolytic activity than larger (older) snakes. Major differences between the venoms was detected by isoelectric focusing, and reflected in the number and pI of the proteins with fibrinolytic activity.

Detection of alkaline phosphatase in venom by blotting methods.

Venom alkaline phosphatase was detected using a blotting method following electrophoresis. The enzyme gave strong reactions in some venoms, but was absent in other venoms, some within the same species. The mol.

Hemorrhagic and Mojave toxins in the venoms of the offspring of two Mojave rattlesnakes (Crotalus scutulatus scutulatus).

1. The venoms of two Mojave rattlesnakes and those of their offsprings were analyzed for Mojave toxin and hemorrhagic toxin. 2.

Isolation of a hemorrhagic toxin from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom.

A hemorrhagic toxin was isolated from Mojave rattlesnake venom. The isoelectric point of the toxin was 4.7 and its mol.

Effect of prostaglandins on interferon synthesis in murine macrophage-like cell lines.

Murine macrophage-like cell lines were used to determine whether exogenously added prostaglandins and endogenous prostaglandins suppress interferon (IFN) synthesis in macrophages. The amount of IFN produced by J774A.1 cells induced with bacterial lipopolysaccharide (LPS) was reduced by 0.