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The enrichment of trace proteins from human fluid samples is of great importance in diverse clinical and industrial applications. In clinical diagnostics, such enrichment may enable detection of trace proteins that serve as biomarkers of disease. Affinity-based approaches, such as immunoaffinity pulldown, are widely used to enrich trace proteins, but this strategy relies on the availability and performance of antibodies that act on all proteoforms in an unbiased manner. Our prior work to characterize MUC16 (the mucin protein that carries the ovarian cancer biomarker CA125) by mass spectrometry successfully overcame the reliance on affinity-based enrichment and was used to enrich this biomarker from ascites of individual ovarian cancer patients, however, this strategy was not demonstrated on clinically relevant volumes of serum, a biofluid that is more accessible than ascites. The present work developed a non-affinity-based chromatographic method to enrich MUC16 from serum. The enriched MUC16 sample was further processed using a Midi Top 14 abundant protein depletion column. Peptides identified using bottom-up proteomics yielded 1-8% coverage of MUC16. Additionally, MUC16 was detected in samples containing less than the clinical cut-off level of CA125 (35 U mL-1), suggesting that this strategy of enrichment and bottom-up proteomics can enable analysis of CA125 from the serum of individuals with early-stage ovarian cancer and those whose tumors express CA125 (MUC16) at low levels.

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Protein-polysaccharide glycates are food ingredients that use the Maillard reaction to form a Schiff base linkage between the carbonyl of a polysaccharide and the free amino moiety of a protein. Glycates are excellent emulsification, foaming, and gelling agents in foods and improve protein solubility and heat stability. The present work examined if glycates dissociate by hydrolysis, returning to free un-glycated protein and dextran due to the reversibility of the Schiff base linkage. Hydrolysis of glycates made from whey protein isolate and dextran was measured versus time and temperature, allowing determination of the rate constants and equilibrium constants for glycate hydrolysis. Glycates underwent hydrolysis when placed into aqueous solutions at common food processing temperatures. For example, during hot food storage (60 °C), equilibrium fractional hydrolysis was 44%, whereas at ambient temperature (22 °C), it was 8%. The present work aims to increase the successful use of glycates in new foods by knowing what foods and conditions avoid glycate hydrolysis.

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Glycation of proteins by polysaccharides via the Maillard reaction improves the functional properties of proteins in foods, such as solubility, heat stability, emulsification, foaming, and gelation. Glycation is achieved by either the dry heating or the wet heating method, and considerable research has been reported on the functionality of the reaction mixture as tested in foods. While the characteristics of the glycates in foods have been well studied, the kinetics and equilibrium yield of the protein-polysaccharide glycation reaction has received little attention. Industrial manufacture of the glycates will require understanding the kinetics and yield of the glycation reaction. This work examined the glycation of whey protein isolate (WPI) and glycomacropeptide (GMP) by using dextran and the dry-heating method at 70 °C and 80% relative humidity. The disappearance of un-glycated protein and the creation of glycated protein were observed using chromatographic analysis and fluorescence laser densitometry of sodium dodecyl sulfate-polyacrylamide gels. Data were fit using a first-order reversible kinetic model. The rate constants measured for the disappearance of un-glycated protein by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) (k = 0.33 h-1) and by chromatographic analysis (k = 0.38 h-1) were not statistically different from each other for WPI-dextran glycation. Dextran glycation of GMP was slower than for WPI (k = 0.13 h-1). The slower rate of glycation of GMP was attributed to the 50% lower Lys content of GMP compared to WPI. Yield for the dry-heating dextran glycation method was 89% for WPI and 87% for GMP. The present work is useful to the food industry to expand the use of glycated proteins in creating new food products.

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Fractionation of the bovine glycomacropeptide (GMP) from the other proteins in cheese whey was examined using ultrafiltration membranes surface modified to contain positively charged polymer brushes made of polyhexamethylene biguanide. By placing a strong positive charge on a 1000 kDa ultrafiltration membrane and adjusting the pH of whey close to the isoelectric point of GMP, a 14-fold increase in selectivity was observed compared to unmodified membranes. A one stage membrane system gave 90% pure GMP and a three-stage rectification system gave 97% pure GMP. The charged membrane was salt-tolerant up to 40 mS cm-1 conductivity, allowing fractionation of GMP directly from cheese whey without first lowering the whey conductivity by water dilution. Thus, similarly sized proteins that differed somewhat in isoelectric points and were 50⁻100 fold smaller than the membrane molecular weight cut-off (MWCO), were cleanly fractionated using charged ultrafiltration membranes without water addition. This is the first study to report on the use of salt-tolerant charged ultrafiltration membranes to produce chromatographically pure protein fractions from whey, making ultrafiltration an attractive alternative to chromatography for dairy protein fractionation.

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In this work, milk protein concentrate (MPC) was made using wide-pore negatively charged ultrafiltration membranes. The charged membranes were used for a six-fold volume concentration of skim milk and subsequent diafiltration to mimic the industrial MPC process. The charged 100 kDa membranes had at least a four-fold higher permeate flux at the same protein recovery as unmodified 30 kDa membranes, which are currently used in the dairy industry to make MPC. By placing a negative charge on the surface of an ultrafiltration membrane, the negatively charged proteins were rejected by electrostatic repulsion and not simply size-based sieving. Mass balance models of concentration and diafiltration were developed and the calculations matched the experimental observations. This is the first study to use wide-pore charged tangential-flow membranes for MPC manufacturing. Additionally, a unique mass balance model was applied, which accurately predicted experimental results.

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In the present work, we derive a theoretical framework to determine the adsorbed layer thickness from pressure drop measurements for convective-based media without any assumptions about the geometry of the pore structure of the stationary phase matrix. Equations are presented to calculate accuracy of the estimated adsorbed layer thickness as a consequence of measurement error and approximations of the mathematical model. We discovered that there is a minimum in the error for certain pressure drops that results in optimal experimental conditions for determining the adsorbed layer thickness. We demonstrate that the adsorbed layer thickness can be determined with less than 10% error using a wide range of experimental conditions simply from pressure drop data. By careful selection of porous bed dimensions and flow rates, the adsorbed layer thicknesses from subnanometer dimensions up to several hundred nanometers can be determined by measurement of the pressure drop in a range of several bars. The method was experimentally tested on methacrylate monolithic columns using monodisperse latex nanoparticles as a reference standard and two different proteins as unknowns demonstrating close agreement with calculations.

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Thermal inactivation kinetics for single strains of Shiga toxin-producing Escherichia coli (STEC), Listeria monocytogenes, and Salmonella enterica were measured in acidified tryptic soy broth (TSB; pH 4.5) heated at 54°C. Inactivation curves also were measured for single-pathogen five-strain cocktails of E. coli O157:H7, L. monocytogenes, and S. enterica heated in tomato purée (pH 4.5) at 52, 54, 56, and 58°C. Inactivation curves were fit using log-linear and nonlinear (Weibull) models. The Weibull model yields the time for a 5-log reduction (t*) and a curve shape parameter (ß). Decimal reduction times (D-values) and thermal resistance constants (z-values) from the two models were compared by defining t* = 5D* for the Weibull model. When the log-linear and Weibull models match at the 5-log reduction time, then t* = 5D* = 5D and D = D*. In 18 of 20 strains heated in acidified TSB, D and D* for the two models were not significantly different, although nonlinearity was observed in 35 of 60 trials. Similarly, in 51 of 52 trials for pathogen cocktails heated in tomato purée, D and D* were not significantly different, although nonlinearity was observed in 31% of trials. At a given temperature, D-values for S. enterica << L. monocytogenes < E. coli O157:H7 in tomato purée (pH 4.5). When using the two models, z-values calculated from the D-values were not significantly different for a given pathogen. Across all pathogens, z-values for E. coli O157:H7 and S. enterica were not different but were significantly lower than the z-values for L. monocytogenes. These results are useful for supporting process filings for tomato-based acidified food products with pH 4.5 and below and are relevant to small processors of tomato-based acidified canned foods who do not have the resources to conduct research on and validate pathogen lethality.

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We processed applesauce, tomato juice, and cranberries in pint jars in a boiling water canner to test thermal processing theories against home canning of high-acid foods. For each product, thermocouples were placed at various heights in the jar. Values for f h (heating), f cl (cooling), and F 82.2°C (lethality) were determined for each thermocouple location, and did not depend substantially on thermocouple location in accordance with heat transfer theory. There was a cold spot in the jar, but the cold spot during heating became the hot spot during cooling. During heating, the geometric center was the last to heat, and remained coldest the longest, but during coooling, it was also the last to cool, and remained hottest the longest. The net effect was that calculated lethality in home canning was not affected by thermocouple location. Most of the lethality during home canning occurred during air cooling, making cooling of home canned foods of great importance. Calculated lethality was far greater than the required 5-log reduction of spores in tomato juice and vegetative cells in cranberries, suggesting a wide margin of safety for approved home-canning processes for high-acid foods.

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A chromatographic method for the analysis of whey protein isolate (WPI)-dextran glycates was developed in this work that is useful for quantification of sample purity and concentration, and as a sample-preparation method for subsequent analysis by gel electrophoresis (SDS-PAGE) and laser-light scattering. Glycation was by the Maillard reaction between WPI and dextran of 3 different sizes. Glycate fractions from each dextran were collected and analyzed by fluorescent and glycoprotein staining of gels, bicinchoinic acid protein assay, and static and dynamic laser light scattering. The weight-average molecular mass of the glycates was 27-34 kDa (from 3.5 kDa dextran), 32-39 kDa (from 10 kDa dextran), and 250-270 kDa (from 150 kDa dextran). The new method was used to characterize the kinetics of the glycation reaction, which followed a reversible pseudo first-order model. The kinetics of decomposition of the purified glycate by hydrolysis was also examined. The new method is rapid (25 min) and quantitative, and is the first chromatographic method for direct analysis of WPI-dextran glycation products.

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A process for the food-grade preparative-scale production and chromatographic purification of whey protein isolate (WPI)-dextran glycates was developed in this work. The Maillard reaction was used to produce the glycates in aqueous solution. A 5 mL cation exchange column and salt step gradients were utilized to elute the glycated protein at low salt and unreacted protein at high salt. The process was scaled-up 160-fold to an 800 mL column. Glycated products were analyzed by SDS-PAGE, BCA protein assay and glycoprotein carbohydrate estimation kit, MALDI-TOF and static and dynamic light scattering. Glycated protein was relatively pure, containing only traces of beta-lactoglobulin, and it was heterogeneous due to oligo-glycation. It had a molecular mass of 26-35 kDa by static light scattering, and 22-67 kDa by MALDI-TOF. Mono-glycated protein would have been 23.8 kDa from beta-lactoglobulin (18.6 kDa) and dextran (5.2 kDa). This work demonstrated the utility of cation exchange chromatography for the large-scale purification of glycated proteins using food-grade chemicals and procedures.

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Proteins conjugated to neutral biopolymers are of keen interest to the food and pharmaceutical industries. Conjugated proteins are larger and more charge shielded than un-reacted proteins, making purification difficult using conventional beaded chromatographic supports because of slow mass transfer rates, weak binding, and viscous solutions. Past methods developed for pharmaceuticals are unsuitable for foods. In this work, a food-grade whey protein-dextran conjugate was purified from a feed solution also containing un-reacted protein and dextran using either a column packed with 800 mL of a beaded support that was specifically designed for purification of conjugated proteins or an 8 mL tube monolith. The monolith gave a similar dynamic binding capacity as the beaded support (4-6 g/L), at a 42-fold greater mass productivity, and 48-fold higher flow rate, albeit at somewhat lower conjugate purity. Performance of the monolith did not depend on flow rate. In conclusion, monoliths were found to be well suited for the purification of whey protein-dextran conjugates.

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A challenge of shelf stable beverages that contain whey protein is that a small portion of protein can be denatured and aggregated during thermal processing, resulting in a turbid solution or white precipitate that consumers perceive as a defect. In this study, 3 approaches were taken to reduce turbidity in heat-treated beverages that contain whey protein: (1) centrifugation to remove insoluble protein aggregates, (2) addition of ingredients, and (3) alteration of pH in the range from 3.0 to 4.0. At pH 3.6 and below, all samples were essentially clear both before and after heating for all ingredients. At a pH of 3.8 and above, ingredient selection was crucial to solution clarity after heat treatment. At a pH of 4.0, addition of salts at both 10 and 50 mM increased the turbidity significantly compared to the control, which contained only whey protein in water. Neither addition of sugars at 25, 50, and 100 g/L, nor addition of sugar alcohols at 25 g/L significantly affected turbidity after heat treatment compared to the control. However, sugar alcohols added at 50 or 100 g/L significantly reduced turbidity after heat treatment compared to the control. Removal of insoluble protein aggregates by centrifugation prior to heat treatment resulted in a statistically significant decrease in turbidity after heat treatment. Understanding these results at the molecular level will assist food scientists in selecting processing treatments, ingredients, and pH in the development of shelf stable clear beverages that contain whey protein.

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During storage of heat-treated acidic (pH

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Clearance of impurities such as viruses, host cell protein (HCP), and DNA is a critical purification design consideration for manufacture of monoclonal antibody therapeutics. Anion exchange chromatography has frequently been utilized to accomplish this goal; however, anion exchange adsorbents based on the traditional quaternary amine (Q) ligand are sensitive to salt concentration, leading to reduced clearance levels of impurities at moderate salt concentrations (50-150 mM). In this report, membrane adsorbers incorporating four alternative salt tolerant anion exchange ligands were examined for impurity clearance: agmatine, tris-2-aminoethyl amine, polyhexamethylene biguanide (PHMB), and polyethyleneimine. Each of these ligands provided greater than 5 log reduction value (LRV) for viral clearance of phage phiX174 (pI approximately 6.7) at pH 7.5 and phage PR772 (pI approximately 4) at pH 4.2 in the presence of salt. Under these same conditions, the commercial Q membrane adsorber provided no clearance (zero LRV). Clearance of host-cell protein at pH 7.5 was the most challenging test case; only PHMB maintained 1.5 LRV in 150 mM salt. The salt tolerance of PHMB was attributed to its large positive net charge through the presence of multiple biguanide groups that participated in electrostatic and hydrogen bonding interactions with the impurity molecules. On the basis of the results of this study, membrane adsorbers that incorporate salt tolerant anion exchange ligands provide a robust approach to impurity clearance during the purification of monoclonal antibodies.

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Individuals with phenylketonuria (PKU) cannot metabolize phenylalanine (Phe) and must adhere to a low-Phe diet in which most dietary protein is provided by a Phe-free amino acid formula. Glycomacropeptide (GMP) is the only naturally occurring protein that does not contain Phe, and is of interest as a source of protein for dietary management of PKU. However, commercially available GMP contains too much Phe from residual whey proteins and does not contain adequate levels of all the indispensable amino acids to provide a nutritionally complete protein. The aim of this study was to increase purity of GMP and develop a mass balance calculation for indispensable amino acid supplementation of GMP foods. Cation exchange chromatography, ultrafiltration/diafiltration, and lyophilization were used at the pilot plant scale to decrease Phe. Enough purified GMP (5 kg) was manufactured to provide 15 PKU subjects with a 4-d diet in which the majority of protein was from GMP foods. A mass balance was used to supplement GMP foods so that all indispensable amino acids met or exceeded the daily recommended intake. GMP foods were tested in a human clinical trial as a replacement for the traditional amino acid formula. Nutritionally complete GMP foods created with high purity GMP provide individuals with PKU with more options to manage PKU, which may lead to improved compliance and quality of life.

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BACKGROUND: Phenylketonuria (PKU) requires a lifelong low-phenylalanine diet that provides the majority of protein from a phenylalanine-free amino acid (AA) formula. Glycomacropeptide (GMP), an intact protein formed during cheese production, contains minimal phenylalanine. OBJECTIVE: The objective was to investigate the effects of substituting GMP food products for the AA formula on acceptability, safety, plasma AA concentrations, and measures of protein utilization in subjects with PKU. DESIGN: Eleven subjects participated in an inpatient metabolic study with two 4-d treatments: a current AA diet (AA diet) followed by a diet that replaced the AA formula with GMP (GMP diet) supplemented with limiting AAs. Plasma concentrations of AAs, blood chemistries, and insulin were measured and compared in AA (day 4) and GMP diets (day 8). RESULTS: The GMP diet was preferred to the AA diet in 10 of 11 subjects with PKU, and there were no adverse reactions to GMP. There was no significant difference in phenylalanine concentration in postprandial plasma with the GMP diet compared with the AA diet. When comparing fasting with postprandial plasma, plasma phenalyalanine concentration increased significantly with the AA but not with the GMP diet. Blood urea nitrogen was significantly lower, which suggests decreased ureagenesis, and plasma insulin was higher with the GMP diet than with the AA diet. CONCLUSIONS: GMP, when supplemented with limiting AAs, is a safe and highly acceptable alternative to synthetic AAs as the primary protein source in the nutritional management of PKU. As an intact protein source, GMP improves protein retention and phenylalanine utilization compared with AAs.

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Clearance of biological impurities is an essential part of the manufacture of biotechnology-derived products such as monoclonal antibodies (mAbs). Salt is required during manufacture to solubilize the mAb product and stabilize it against aggregation, but salt can be a problem later during impurity clearance operations. In this work, the use of a traditional quaternary amine (Q) monolith, and a new salt-tolerant monolith were evaluated for the clearance of pathogenic impurities including viruses, DNA, and host-cell protein (HCP). The impact of flow rate, salt concentration, and presence of mixtures of impurities in the feed stream were evaluated. Both monoliths cleared DNA to the limit of detection at all salt concentrations, and both cleared virus and HCP equally well at no salt. At intermediate salt, clearance of HCP was greater for the salt-tolerant monolith, and only the salt-tolerant monolith cleared virus at elevated salt. In conclusion, monoliths successfully trapped impurities such as DNA, host-cell protein, and viruses, and at flow rates far greater than traditional chromatography columns packed with beads.

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Phenylketonuria (PKU) is a genetic disorder caused by deficiency of phenylalanine hydroxylase (PAH) that requires life-long adherence to a low-phenylalanine (Phe) diet. Glycomacropeptide (GMP) is uniquely suited to the nutritional management of PKU, because pure GMP contains no Phe. Our aim was to assess how ingestion of diets containing GMP support growth and affect the concentrations of amino acids in plasma and brains of mice with a deficiency of PAH, the Pah(enu2) mouse (PKU mouse). Experiments were conducted in 4- to 6-wk-old wild-type (WT) (C57Bl/6) and PKU mice fed diets containing 20% protein from casein, amino acids, or GMP supplemented with limiting indispensable amino acids (IAA). PKU mice fed the GMP diet showed gains in body weight, feed efficiency, and a protein efficiency ratio that did not differ from the amino acid diet. The concentrations of isoleucine and threonine in plasma showed a significant 2- to 3-fold increase for WT and PKU mice fed GMP compared with casein or amino acid diets, respectively. PKU mice fed the GMP diet had decreased concentrations of Phe in plasma (11% decrease) and in 5 regions of the brain (20% decrease) compared with the amino acid diet. The concentration of Phe in the brain was inversely correlated with the concentrations of isoleucine, threonine, and valine in plasma (R2 = 0.74; P < 0.0001), suggesting competitive inhibition of Phe transport into the brain. In summary, PKU mice fed GMP showed comparable growth and reduced concentrations of Phe in plasma and the brain compared with an amino acid diet. These data support the use of GMP supplemented with IAA as an alternative source of dietary protein for individuals with PKU.

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The kinetics of lysozyme crystallization under seeded isothermal batch conditions was followed by measurement of the decline in solution concentration versus time. Kinetics were measured for five different values of the seed crystal mass. The data were analyzed using a recently proposed mathematical model. For each seed mass, the model fit the kinetic data well. Growth rate constants determined using the model were approximately constant over a sixfold increase in the seed crystal mass, and fell well within the range of values reported in the literature, but obtained using entirely different experimental techniques. These results confirmed the utility of the proposed model. The proposed model can be used to analyze crystallization kinetics using absorbance measurements only, without the need to characterize the crystal size, thus avoiding the need for expensive laser light scattering and digital microscopy instrumentation. Thus, the model offers a low-cost straightforward method to analyze and simulate the effects of changes in operating parameters such as the seed crystal mass, solution volume, initial protein concentration, pH, temperature, salt concentration, and time.

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Bulk protein crystallization, unlike small molecule crystallization, has found very limited use in biopharmaceutical manufacture. Most work in this area targets obtaining single large crystals for molecular structure determination by crystallography. Design and optimization of bulk crystallization for protein recovery and purification is much less common, and requires a mathematical model for analysis of laboratory data suitable for scale-up purposes. Traditionally, the crystal size distribution and method of moments is used to characterize the crystallization process. A simpler method is presented in this paper that utilizes the desupersaturation curve. The method uses an approach that does not require expensive instrumentation or characterization of the seed crystal size distribution. The method is extended to allow determination of both the mass deposition rate constant and the growth rate order from a single desuperaturation curve. Experimental data for the bulk crystallization of ovalbumin are used to validate the method. The rate constants and rate order obtained using the new method compare well with literature values. Scale-up is illustrated by prediction of the impact of changes in seed mass on protein crystallization. This new method offers a straightforward and low-cost alternative to traditional methods for the analysis and scale-up of protein crystallization data.

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