Design Considerations for the Batch Capture of Proteins from Raw Whole Milk by Ion Exchange Chromatography

Batch extraction of proteins directly from raw, whole milk is described with a focus on design considerations for on-farm implementation. A demonstration of single-stage stirred tank extraction of bovine lactoferrin onto cation exchangers shows that processing can be achieved, with no pre-treatment of raw milk, within the timeframe required to milk an individual animal. The extraction rates of two cation exchange media, SP Sepharose Big Beads™ and SP Sepharose FF™ (GE Healthcare, Uppsala, Sweden), are compared, with the smaller FF media (average diameter 90 μm) having a faster adsorption rate than the larger BB media (155 μm). Maximum percentages of original protein extracted by SP Sepharose FF were 67%, 80%, 88% and 90% for extraction times of 5, 10, 20 and 30 minutes, respectively. The composite nonlinear kinetic model of Rowe et al [1] was found to fit the batch adsorption of LF from raw milk as a function of chromatography media volume to milk volume ratio, Φ. At extraction times of 30 minutes and longer, extraction % was almost independent of Φ over the range of values tested. On-farm extraction of proteins shows promise for both the production of minor, high-value proteins from conventional milk and for production of recombinant proteins from the milk of transgenic animals. The main advantage of the stirred tank process is that protein extraction is rapid and is achieved in a single step without the need for pre-treatment of milk. The process is not limited to ion exchange but could be implemented using other chromatographic techniques, such as affinity or reverse-phase chromatography

Prediction of the Viscosity Radius and the Size Exclusion Chromatography Behavior of PEGylated Proteins

Size exclusion chromatography (SEC) was used to determine the viscosity radii of equivalent spheres for proteins covalently grafted with poly(ethylene glycol) (PEG). The viscosity radius of such PEGylated proteins was found to depend on the molecular weight of the native protein and the total weight of grafted PEG but not on PEG molecular weight, or PEG-to-protein molar grafting ratio. Results suggest grafted PEG's form a dynamic layer over the surface of proteins. The geometry of this layer results in a surface area-to-volume ratio approximately equal to that of a randomly coiled PEG molecule of equivalent total molecular weight. Two simple methods are given to predict the viscosity radius of PEGylated proteins. Both methods accurately predicted (3% absolute error) the viscosity radii of various PEG-proteins produced using three native proteins, -lactalbumin (14.2 kDa MW), -lactoglobulin dimer (37.4 kDa MW), and bovine serum albumin (66.7 kDa MW), three PEG reagents (2400, 5600, and 22500 MW), and molar grafting ratios of 0 to 8. Accurate viscosity radius prediction allows calculation of the distribution coefficient, Kav, for PEG-proteins in SEC. The suitability of a given SEC step for the analytical or preparative fractionation of different PEGylated protein mixtures may therefore be assessed mathematically. The methods and results offer insight to several factors related to the production, purification, and uses of PEGylated proteins

Continuous radial flow chromatography of proteins

A continuous radial flow chromatography (CRFC) system consisting of an annular packed bed between two rotating concentric porous stainless steel cylinders was constructed and tested. The protein feed, wash buffer and elution buffer are applied simultaneously at different fixed angular positions at the periphery of the rotating bed and flow radially inwards. The target protein is bound in the feed zone and eluted in the elution zone while unbound protein flows through the feed zone. Continuity equations for fixed bed radial flow chromatography were extended to model the CRFC by including angular displacement terms, and solved using finite difference methods. Film diffusion at the resin particle boundary and pore diffusion within the resin particle was described by an overall mass transfer coefficient and a multicomponent Langmuir isotherm was used to describe protein absorption onto the resin particle. To verify the model, a solution containing 0.53 mg ml21 lactoferrin and 1.6 mg ml21 bovine serum albumin (BSA) was applied at 5 ml min21 to a 220-ml CRFC system packed with DEAE Sepharose Fast Flow ion exchange resin, with abed speed of 0.02 rpm. The proteins were separated into lactoferrin of 68% purity (74% recovery) and BSA of 94%purity (85% recovery). Experimental data were used to find values for the parameters in the model proposed

Application of peptide chromatography for the isolation of antibodies from bovine skim milk, acid whey and colostrum

Protein A mimetic peptide ligands have several benefits over conventional Protein A/G ligands, namely that they are small in size, have low production costs, are stable over a wide range of pH values and can withstand cleaning by harsh sanitization agents such as sodium hydroxide. In this paper, a hexamer peptide (HWRGWV) affinity matrix was used for the isolation of bovine immunoglobulins from various dairy streams (skim milk, acid whey and colostrum). Bound immunoglobulins were recovered in elution buffer (0.2 M sodium acetate buffer, pH 4.0) fractions with a purity of >85% in a single step. The peptide resin has achieved a maximum equilibrium adsorption capacity of 23 ± 0.58 mg mL-1 of resin for bovine IgG and had a dynamic binding capacity of 11.8 ± 0.03 mg mL-1 at residence time of 2 min. These results suggest that the hexamer peptide chromatography could potentially be used for the selective purification of bovine immunoglobulins from dairy streams. This method has promise as an alternative to conventional Protein A/G chromatography for direct capture of immunoglobulins from streams containing relatively high immunoglobulin concentrations such as colostrum, transgenic or hyper-immune milk

Erratum: "A Gravitational-wave Measurement of the Hubble Constant Following the Second Observing Run of Advanced LIGO and Virgo" (2021, ApJ, 909, 218)

[no abstract available

Search for gravitational waves from Scorpius X-1 in the second Advanced LIGO observing run with an improved hidden Markov model

We present results from a semicoherent search for continuous gravitational waves from the low-mass x-ray binary Scorpius X-1, using a hidden Markov model (HMM) to track spin wandering. This search improves on previous HMM-based searches of LIGO data by using an improved frequency domain matched filter, the J-statistic, and by analyzing data from Advanced LIGO's second observing run. In the frequency range searched, from 60 to 650 Hz, we find no evidence of gravitational radiation. At 194.6 Hz, the most sensitive search frequency, we report an upper limit on gravitational wave strain (at 95% confidence) of h095%=3.47×10-25 when marginalizing over source inclination angle. This is the most sensitive search for Scorpius X-1, to date, that is specifically designed to be robust in the presence of spin wandering. © 2019 American Physical Society

Search for Tensor, Vector, and Scalar Polarizations in the Stochastic Gravitational-Wave Background

The detection of gravitational waves with Advanced LIGO and Advanced Virgo has enabled novel tests of general relativity, including direct study of the polarization of gravitational waves. While general relativity allows for only two tensor gravitational-wave polarizations, general metric theories can additionally predict two vector and two scalar polarizations. The polarization of gravitational waves is encoded in the spectral shape of the stochastic gravitational-wave background, formed by the superposition of cosmological and individually unresolved astrophysical sources. Using data recorded by Advanced LIGO during its first observing run, we search for a stochastic background of generically polarized gravitational waves. We find no evidence for a background of any polarization, and place the first direct bounds on the contributions of vector and scalar polarizations to the stochastic background. Under log-uniform priors for the energy in each polarization, we limit the energy densities of tensor, vector, and scalar modes at 95% credibility to Ω0T<5.58×10-8, Ω0V<6.35×10-8, and Ω0S<1.08×10-7 at a reference frequency f0=25 Hz. © 2018 American Physical Society

Design Considerations for the Batch Capture of Proteins from Raw Whole Milk by Ion Exchange Chromatography

Batch extraction of proteins directly from raw, whole milk is described with a focus on design considerations for on-farm implementation. A demonstration of single-stage stirred tank extraction of bovine lactoferrin onto cation exchangers shows that processing can be achieved, with no pre-treatment of raw milk, within the timeframe required to milk an individual animal. The extraction rates of two cation exchange media, SP Sepharose Big Beads™ and SP Sepharose FF™ (GE Healthcare, Uppsala, Sweden), are compared, with the smaller FF media (average diameter 90 μm) having a faster adsorption rate than the larger BB media (155 μm). Maximum percentages of original protein extracted by SP Sepharose FF were 67%, 80%, 88% and 90% for extraction times of 5, 10, 20 and 30 minutes, respectively. The composite nonlinear kinetic model of Rowe et al [1] was found to fit the batch adsorption of LF from raw milk as a function of chromatography media volume to milk volume ratio, Φ. At extraction times of 30 minutes and longer, extraction % was almost independent of Φ over the range of values tested. On-farm extraction of proteins shows promise for both the production of minor, high-value proteins from conventional milk and for production of recombinant proteins from the milk of transgenic animals. The main advantage of the stirred tank process is that protein extraction is rapid and is achieved in a single step without the need for pre-treatment of milk. The process is not limited to ion exchange but could be implemented using other chromatographic techniques, such as affinity or reverse-phase chromatography

Intensification of minor milk protein purification processes

The performance of a robotic system that extracts proteins directly from the milk of individual cows on-farm is described. Batch extraction of proteins is demonstrated and extraction yields modelled, indicating that on-farm extraction can significantly outperform centralised, large-scale processing