Determining the Impact of Genotype × Environment on Oat Protein Isolate Composition Using HPLC and LC-MS Techniques.

The effect of genotype and environment on oat protein composition was analyzed through size exclusion-high-performance liquid chromatography (SE-HPLC) and liquid chromatography-mass spectrometry (LC-MS) to characterize oat protein isolate (OPI) extracted from three genotypes grown at three locations in the Canadian Prairies. SE-HPLC identified four fractions in OPI, including polymeric globulins, avenins, glutelins, and albumins, and smaller proteins. The protein composition was dependent on the environment, rather than the genotype. The proteins identified through LC-MS were grouped into eight categories, including globulins, prolamins/avenins, glutelins, enzymes/albumins, enzyme inhibitors, heat shock proteins, grain softness proteins, and allergenic proteins. Three main globulin protein types were also identified, including the P14812|SSG2-12S seed storage globulin, the Q6UJY8_TRITU-globulin, and the M7ZQM3_TRIUA-Globulin-1 S. Principal component analysis indicated that samples from Manitoba showed a positive association with the M7ZQM3_TRIUA-Globulin-1 S allele and Q6UJY8_TRITU-globulin, while samples from Alberta and Saskatchewan had a negative association with them. The results show that the influence of G × E on oat protein fractions and their relative composition is crucial to understanding genotypes' behavior in response to different environments.

Stability assessment of Polatuzumab vedotin and Brentuximab vedotin using different analytical techniques.

Antibody-drug conjugates (ADC) are considered to be fast-growing innovative biopharmaceuticals. The science used for conjugating potent cytotoxic payload to the targeted monoclonal antibody through a chemical linker has played a great value in the area of oncology treatment. In this study; Polatuzumab vedotin (POLA) and Brentuximab vedotin (SGN-35) were subjected to various stress conditions enclosing different pH, thermal stress, agitation, and successive cycles of freeze and thaw in order to produce potential degradation by-products and guarantee the appropriateness of the applied testing protocol. Different analytical techniques were established and validated to be used in the quantitation of the degraded products from different perspectives. The formation of ADC aggregates and fragments was monitored using SE-HPLC as well as dynamic light scattering (DLS). The drug antibody ratio (DAR) and ADC conjugation profile were determined using hydrophobic interaction chromatography (HIC-HPLC). In addition to performing a statistical interpretation of HIC-HPLC results by principal component analysis (PCA) to explicate the obtained data. Also, the quantity of the unconjugated toxic drug was quantified using RP-HPLC. Testing the binding activity of ADC to their target receptor ADC was conducted using ELISA. Results presented that used assay protocol had worked as a complementary design for characterization and stability assessment of the used ADC. Variances in the stability profile of both products were observed which could be attributed to the usage of different formulation buffers. This highlighted the importance of using multiple techniques for the assessment of the quality attributes of such sophisticated products. The analytical assay protocol should be used for the evaluation of the quality and stability of several ADC.

Application progress of high performance liquid chromatography in detection of hemagglutinin content in influenza vaccine / 中国生物制品学杂志

@#At present,the most commonly used method for detecting hemagglutinin(HA)content in influenza vaccines is still single-radial immunodiffusion(SRID). However,the preparation of standards required by this method takes a long time,usually 2 ～ 3 months. Therefore,how to quantitatively analyze HA accurately has always been a difficult problem in the detection of HA content in the situation that reference products can not be obtained at the early stage of the pandemic influenza. High performance liquid chromatography(HPLC)has its own characteristics of rapidity,high sensitivity,good repeatability and high accuracy,which can rapidly determine HA content by using different separation principles and has been widely used in the detection of HA content in influenza vaccine. This paper reviewed the research progress of the application of HPLC in the determination of HA content in influenza vaccine.

Characterization of a novel bispecific antibody targeting tissue factor-positive tumors with T cell engagement.

T cell engaging bispecific antibody (TCB) is an effective immunotherapy for cancer treatment. Through co-targeting CD3 and tumor-associated antigen (TAA), TCB can redirect CD3+ T cells to eliminate tumor cells regardless of the specificity of T cell receptor. Tissue factor (TF) is a TAA that involved in tumor progression. Here, we designed and characterized a novel TCB targeting TF (TF-TCB) for the treatment of TF-positive tumors. In vitro, robust T cell activation, tumor cell lysis and T cell proliferation were induced by TF-TCB. The tumor cell lysis activity was dependent upon both CD3 and TF binding moieties of the TF-TCB, and was related to TF expression level of tumor cells. In vivo, in both tumor cell/human peripheral blood mononuclear cells (PBMC) co-grafting model and established tumor models with poor T cell infiltration, tumor growth was strongly inhibited by TF-TCB. T cell infiltration into tumors was induced during the treatment. Furthermore, efficacy of TF-TCB was further improved by combination with immune checkpoint inhibitors. For the first time, our results validated the feasibility of using TF as a target for TCB and highlighted the potential for TF-TCB to demonstrate efficacy in solid tumor treatment.

Comparison of High-Performance Liquid Chromatography with Sucrose Density Gradient Ultracentrifugation for the Quantification of Foot-and-Mouth Disease Vaccine Antigens.

Foot-and-mouth disease (FMD) causes substantial economic losses in the livestock industry. The protective immunizing component of the FMD virus (FMDV) is a ribonucleoprotein particle with a sedimentation coefficient of 146S. Size-exclusion high-performance liquid chromatography (SE-HPLC) was introduced to replace sucrose density gradient ultracentrifugation (SDG), which is the gold standard for the quantification of FMDV 146S particles. SE-HPLC showed a pattern similar to that of SDG; however, the two methods resulted in different quantities for the same amount of 146S particles. This study aimed to identify the reason for this disparity and adjust the difference between the two methods by employing a standard material. While SE-HPLC displayed all the virus particles in the peak fraction by SDS-PAGE and Western blotting, the virus particles were widely dispersed in multiple fractions, including peak fractions in the SDG. To adjust the difference between the two methods, a stable surrogate virus, bovine enterovirus, was devised to draw a standard curve, and the gap was reduced to <10%. To our knowledge, this is the first report to provide experimental evidence on the difference between SDG and SE-HPLC for the quantification of FMDV particles.

Validation of Pretreatment Methods for the In-Process Quantification of Foot-and-Mouth Disease Vaccine Antigens.

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is controlled by vaccine policy in many countries. For vaccine potency, the content of intact virus particles (146S antigens) is critical, and the sucrose density gradient (SDG) fractionation is the gold standard for the quantification of 146S antigens. However, this method has several drawbacks. Although size-exclusion high-performance liquid chromatography (SE-HPLC) was introduced to replace the classic method, its application is generally confined to purified samples owing to the interfering signals. Therefore, we aimed to develop optimal pretreatment methods for SE-HPLC quantification in less purified samples. Crude virus infection supernatant (CVIS) and semi-purified samples with PEG precipitation (PEG-P) were used. Chloroform pretreatment was essential to remove a high level of non-specific signals in CVIS, whereas it caused loss of 146S antigens without the distinctive removal of non-specific signals in PEG-P. Benzonase pretreatment was required to improve the resolution of the target peak in the chromatogram for both CVIS and PEG-P. Through spiking tests with pure 146S antigens, it was verified that the combined pretreatment with chloroform and benzonase was optimal for the CVIS, while the sole pretreatment of benzonase was beneficial for PEG-P.

Effect of extrusion and turmeric addition on phenolic compounds and kafirin properties in tannin and tannin-free sorghum.

Sorghum is a potential substitute for corn/wheat in cereal-based extruded products. Despite agronomic advantages and its rich diversity of phenolic compounds, sorghum kafirins group together and form complex with tannins, leading to a low digestibility. Phenolic content/profile by UPLC-ESI-QTOF-MSE and kafirins polymerization by SE-HPLC were evaluated in wholemeal sorghum extrudates; tannin-rich (#SC319) and tannin-free (#BRS330) genotypes with/without turmeric powder. Total phenolic, proantocyanidin and flavonoid contents were strongly correlated with antioxidant capacity (r > 0.9, p < 0.05). Extrusion increased free (+60%) and decreased bound phenolics (-40%) in #SC319, but reduced both (-40%; -90%, respectively) in #BRS330, which presented lower abundance after extrusion. Turmeric addition did not significantly impact antioxidant activity, phenolic content and profile and kafirins profile. Tannins presence/absence impacted phenolic profiles and polymerization of kafirins which appears related to the thermoplastic process. The extrusion improved proteins solubility and can positively enhance their digestibility (phenolic compounds-proteins interactions), making more accessible to proteolysis in sorghum extrudates.

Solvent Retention Capacity and Gluten Protein Composition of Durum Wheat Flour as Influenced by Drought and Heat Stress.

Drought and temperature stress can cause considerable gluten protein accumulation changes during grain-filling, resulting in variations in wheat quality. The contribution of functional polymeric components of flour to its overall functionality and quality can be measured using solvent retention capacity (SRC). The aim of this study was to determine the effect of moderate and severe drought and heat stress on SRC and swelling index of glutenin (SIG) in six durum wheat cultivars with the same glutenin subunit composition and its relation with gluten protein fractions from size exclusion high performance liquid chromatography. Distilled water, sodium carbonate and sucrose SRC reacted similarly to stress conditions, with moderate heat causing the lowest values. Lactic acid SRC and SIG reacted similarly, where severe heat stress highly significantly increased the values. SIG was significantly correlated with sodium dodecyl sulphate sedimentation (SDSS) and flour protein content (FPC) under all conditions. Lactic acid SRC was highly correlated with FPC under optimal and moderate heat stress and with SDSS under moderate drought and severe heat. SIG was negatively correlated with low molecular weight glutenins under optimal and drought conditions, and combined for all treatments. The relationship between SRC and gluten proteins was inconsistent under different stress conditions.

Isolation of Extracellular Vesicles (EVs) Using Size-Exclusion High-Performance Liquid Chromatography (SE-HPLC).

Extracellular vesicles (EVs), are membrane-bound nanoparticles of biological origin. These signature molecules of health and disease have raised remarkable attention of the biomedical arena due to its potential diagnostic and therapeutic applicability. Among the many different techniques available for EV isolation, size-exclusion chromatography (SEC) is widely accepted.In this chapter, we present a protocol of size-exclusion high-performance liquid chromatography (SE-HPLC) as a method of EV isolation. This method can be adapted as a low cost but a reliable and scalable method of EV isolation in those laboratories having access to the HPLC systems.

ANAVIP® interaction with western pygmy rattlesnake venom: In vitro assesment of reactivity using SE-HPLC.

Every year large numbers of venomous snake bites occur around the world, especially in tropical areas. The World Health Organization classifies venomous snake bites as one of their highest priority neglected tropical diseases, one of the reasons being the short supply of antivenom compared to the number of snake envenomations. The standard of care for snake envenomation is administration of antivenom. Many antivenoms are polyvalent, which are produced using venoms from multiple species of snakes. The polyvalent antivenoms can treat envenomation from snake venoms used in the production, but also show cross-reactivity against snake venoms with similar composition. Determining cross-reactivities of antivenoms could help improve the quality of treatment and provide a better understanding of venom-antivenom binding. One antivenom only has been available in the United States for treatment of North American Crotaline envenomation, with the recent introduction of an F(ab')2 antivenom (ANAVIP®). Size-exclusion high performance liquid chromatography (SE-HPLC) was used to assess cross-reactivity of the western pygmy rattlesnake, Sistrurus miliarius streckeri (S. m. streckeri), against ANAVIP®. Estimates of venom-antivenom reactivity was measured in reaction mixtures based on the increase in elution profile area of higher molecular weight complexes (region 1) and on the decrease in elution profile area of reactants (region 2). Reaction mixtures contained ANAVIP® (1.0 mg/ml) and S. m. streckeri venom (0.125, 0.25, 0.5, or 1.0 mg/ml). Controls were ANAVIP® and S. m. streckeri (1.0 mg/ml). Mixtures were incubated at 37 °C for 30 min, then stored at 4 °C (5 min) prior to SE-HPLC. Relative binding, estimated from the increase in region 1 (immune complexes) and decrease in region 2 (reactants) region areas, suggested saturation of reactive antivenom binding sites at 0.125 (and above) mg venom/mg antivenom. SE-HPLC data indicate that binding of ANAVIP® to S. m. streckeri venom does occur, consistent with protective effects observed clinically. Further studies are needed to compare the binding of S. m. streckeri venom to other commercially available antivenoms, and the binding of ANAVIP® to other venoms of clinical significance.

Identification and Quantification of Major Faba Bean Seed Proteins.

Faba bean (Vicia faba L.) holds great importance for human and animal nutrition for its high protein content. However, better understanding of its seed protein composition is required in order to develop cultivars that meet market demands for plant proteins with specific quality attributes. In this study, we screened 35 diverse Vicia faba genotypes by employing the one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE) method, and 35 major protein bands obtained from three genotypes with contrasting seed protein profiles were further analyzed by mass spectrometry (MS). Twenty-five of these protein bands (MW range: ∼ 9-107 kDa) had significant (p ≤ 0.05) matches to polypeptides in protein databases. MS analysis showed that most of the analyzed protein bands contained more than one protein type and, in total, over 100 proteins were identified. These included major seed storage proteins such as legumin, vicilin, and convicilin, as well as other protein classes like lipoxygenase, heat shock proteins, sucrose-binding proteins, albumin, and defensin. Furthermore, seed protein extracts were separated by size-exclusion high-performance liquid chromatography (SE-HPLC), and percentages of the major protein classes were determined. On average, legumin and vicilin/convicilin accounted for 50 and 27% of the total protein extract, respectively. However, the proportions of these proteins varied considerably among genotypes, with the ratio of legumin:vicilin/convicilin ranging from 1:1 to 1:3. In addition, there was a significant (p < 0.01) negative correlation between the contents of these major fractions (r = -0.83). This study significantly extends the number of identified Vicia faba seed proteins and reveals new qualitative and quantitative variation in seed protein composition, filling a significant gap in the literature. Moreover, the germplasm and screening methods presented here are expected to contribute in selecting varieties with improved protein content and quality.

Solubility variation of wheat dough proteins: A practical way to track protein behaviors in dough processing.

To understand wheat dough protein behavior under dual mixing and thermal treatment, solubility of Mixolab-dough proteins were investigated using nine extraction buffers of different dissociation capacities. Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) demonstrated that overall changes of protein fractions and dynamic responses of specific proteins during dough processing were well reflected by their solubility variations. After starch pasting, the abundance of 0.5 M NaCl extractable proteins were decreased except for six protein groups including α-amylase inhibitors and superoxide dismutase (SOD). The solubility loss of glutenin proteins at C3 (32 min; 80 â„ƒ) was mainly ascribed to the un-extractable HMW-GSs, LMW-GSs, globulin and triticin, while the extract yield of α-, ß-, γ-gliadins and avenin-like proteins (ALPs) increased after starch pasting. Differential responses of dough proteins to extraction systems provides the basis for further exploring wheat protein dynamics in processing.

In vitro digestibility of proteins from historical and modern wheat cultivars.

BACKGROUND: Previous research has suggested that proteins and other quality parameters of wheats may have changed over a century of wheat breeding. These changes may affect protein digestibility. The in vitro protein digestibility of breads made with 21 cultivars of wheat introduced or released in the USA between 1870 and 2013 was therefore evaluated. RESULTS: Protein digestibility increased with release year, but was not normally distributed; three older cultivars had significantly lower digestibility than the other cultivars: 42.0 ± 0.3 mol% (primary amino N/total N) versus 34.7 ± 0.7 mol%; P < 0.001. High molecular weight (MW) protein fractions increased and low MW protein fractions decreased with release year, but these changes were not related to protein digestibility. Thus, other differences in protein composition or other flour components may contribute to diminished digestibility of the three older cultivars. CONCLUSIONS: This study identified differences in protein digestibility among wheat cultivars that may have important implications for human nutrition. Further investigation is required to determine the specific characteristics that differentiate high- and low-digestibility wheat cultivars. © 2020 Society of Chemical Industry.

Evaluation of the pattern and kinetics of degradation of adalimumab using a stability-indicating orthogonal testing protocol.

Forced degradation studies are crucial for the evaluation of the stability and biosimilarity. Here, adalimumab was subjected to oxidation, pH, temperature, agitation and repeated freeze-thaw in order to generate all possible degradation products. An orthogonal stability-indicating testing protocol comprising SE-HPLC, RP-HPLC, TapeStation gel electrophoresis, dynamic light scattering (DLS), and functional receptor binding assay was developed and validated. The assay protocol was used for the assessment of the pattern and kinetics of aggregation/degradation of adalimumab. SE-HPLC and DLS were used to show the formation of aggregates/fragments of adalimumab under nondenaturing conditions. TapeStation electrophoresis was performed under denaturing conditions to reveal the nature of aggregates. Results of the receptor binding assay agreed to those of SE-HPLC and DLS which indicated that it can be used as an activity-indicating assay for adalimumab. RP-HPLC demonstrated excellent selectivity for adalimumab in the presence of its oxidized forms. The kinetics of degradation was studied in each case and the results showed that it followed the first-order reaction kinetics. Correlation between the results supported the quality assessment of the tested product in industrial and clinical settings. This orthogonal protocol is a useful tool in stability assessment of monoclonal antibodies and a key criterion for the biosimilarity assessment.

Validation of a high performance liquid chromatography method for quantitation of foot-and-mouth disease virus antigen in vaccines and vaccine manufacturing.

Foot-and-mouth disease (FMD) is an infectious viral disease that affects the main meat and dairy production animals, including cattle, sheep, goats and swine. It is readily transmissible and countries where the disease is present suffer harsh international trade restrictions on livestock products and serious economic losses. Vaccines are important tools to contain outbreaks and maintain the status of free with or without vaccination, as defined by the World Organization for Animal Health (OIE). The efficacy of vaccines is reliant on the content and integrity of inactivated virus particles. The long-established method to quantify the viral content of vaccines along the manufacturing process and in the final product is the 140S sucrose density gradient analysis. This method has been a valuable tool for many decades. However, it requires gradient preparation for each sample, a lengthy ultracentrifugation and a manual UV reading of the gradient, rendering it highly operator dependent and almost impossible to automate. We present a method to quantify FMDV particles in vaccines and intermediate process samples that is based on separation of components by size exclusion high performance liquid chromatography (SE-HPLC) and measurement of virus by absorption at 254 nm. The method has been extensively validated; it is accurate, precise and linear. It is applicable to all FMDV strains and sample materials and has a good concordance with the 140S test. The proposed method uses off the shelf HPLC equipment and columns. It is easily automated for high throughput operation, affording a useful process analytical technology and a novel tool for control of final product by manufacturers and regulatory agencies.

A new size-exclusion chromatography method for fast rapeseed albumin and globulin quantification.

The method described in the article aims at the quantification of both main storage proteins, globulins and albumins, in aqueous extract from rapeseed, as an alternative to the current reference methods, Kjeldahl and SDS-PAGE electrophoresis. The new method lies on the analytical separation of extracted compounds by Size-Exclusion High Performance Liquid Chromatography (SE-HPLC) (Biosep-SEC-s2000, 5 µm). The elution of rapeseed extracts with water/acetonitrile/trifluoroacetic acid (45/55/0.1% v/v) during 30 min yields two distinct peaks for the main proteins of rapeseed. Based on the protein extinction coefficients, a calibrationless methodology was developed for their quantification on the basis of the UV signal. The SE-HPLC method was successfully compared to references: Kjeldahl and SDS-PAGE densitometry for the determination of the proportion of each protein. Then, it was successfully applied on two other oleoproteagineous plants, linseed and sunflower.

Formulation Stabilization and Disaggregation of Bevacizumab, Ranibizumab and Aflibercept in Dilute Solutions.

PURPOSE: Studies were conducted to investigate dilute solutions of the monoclonal antibody (mAb) bevacizumab, mAb fragment ranibizumab and fusion protein aflibercept, develop common procedures for formulation of low concentration mAbs and identify a stabilizing formulation for anti-VEGF mAbs for use in in vitro permeation studies. METHODS: Excipient substitutions were screened. The most stabilizing formulation was chosen. Standard dilutions of bevacizumab, ranibizumab and aflibercept were prepared in PBS, manufacturer's formulation, and the new formulation. Analysis was by SE-HPLC and ELISA. Stability, disaggregation and pre-exposure tests were studied. RESULTS: When Avastin, Lucentis and Eylea are diluted in PBS or manufacturer's formulation, there is a 40-50% loss of monomer concentration and drug activity. A formulation containing 0.3% NaCl, 7.5% trehalose, 10 mM arginine and 0.04% Tween 80 at a pH of 6.78 stabilized the mAbs and minimized the drug loss. The formulation also disaggregates mAb aggregation while preserving the activity. Degassing the formulation increases recovery. CONCLUSIONS: We developed a novel formulation that significantly stabilizes mAbs under unfavorable conditions such as low concentration or body temperature. The formulation allows for tissue permeation experimentation. The formulation also exhibits a disaggregating effect on mAbs, which can be applied to the manufacture/packaging of mAbs and bioassay reagents.

Anti-polymerization activity of tea and fruits extracts during rapeseed oil heating.

The aim of the study was to analyze the influence of natural antioxidants on polymerization of partially hydrogenated rapeseed oil heated in 170°C for 40h. In the research ethanolic extracts of green tea leaves (China Lung Ching), yellow tea leaves (China Kakecha), cranberry, blackberry, and lime were used. The yellow and green tea extracts were characterized by the highest content of total polyphenol and antioxidant activity. Polymers of triacylglycerols were found only in the polar fraction of heated oil. During heating, the increase of dimers, trimers, and oligomers was observed. However, it was dependent on the used additives and not directly related to the content of phenolic compounds and their antioxidant activity. The final content of polymers in oil samples increased in the fallowing order: green tea

Stability assessment of antibody-drug conjugate Trastuzumab emtansine in comparison to parent monoclonal antibody using orthogonal testing protocol.

Antibody-drug conjugates (ADC) represent an emerging, novel class of biopharmaceuticals. The heterogeneity originating from the sophisticated structure requires orthogonal analytical techniques for quality and stability assessment of ADC to ensure safety and efficacy. In this study, the stability of Trastuzumab (recombinant humanized IgG1 mAb, targeting HER2 receptor) and its ADC with DM1 (anti-tubulin anticancer drug), Trastuzumab emtansine (T-DM1) were studied. SE-HPLC was used to monitor formation of aggregates and/or fragments of the monoclonal antibodies (mAb). Correlation with the results of reducing and non-reducing sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering (DLS) were performed to interpret the obtained results. RP-HPLC was used for assessment of the stability of DM1 in ADC while spectrophotometry was employed to determine drug antibody ratio (DAR) . The studied drugs were subjected to several stress conditions including pH, temperature, mechanical agitation and repeated freeze and thaw to generate possible degradation products and ensure suitability of the assay protocol. The degradation pattern and extent were demonstrated under the indicated stress conditions. The correlation between the results of SE-HPLC and those of SDS-PAGE and DLS ensured the validity of the orthogonal assay protocol and indicated aggregates that were not detected using SE-HPLC. Results showed clearly that T-DM1 is relatively less stable than its parent mAb. This was attributed to the presence of the drug-linker part that is attached to the mAb. RP-HPLC showed that the cytotoxic drug moiety is liable for degradation under the studied conditions resulting in alteration of DAR as well as formation of degradation products. This confirmed the need for more robust coupling chemistries for production of safe and effective ADC and highlighted the importance of orthogonal testing protocols for quality assessment. The assay protocol should be applicable for quality and stability assessment of various ADC.

Protein interactions during flour mixing using wheat flour with altered starch.

Wheat grain proteins responses to mixing and thermal treatment were investigated using Mixolab-dough analysis systems with flour from two cultivars, Ventura-26 (normal amylose content) and Ventura-19 (reduced amylose content). Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) analysis revealed that, stress associated and metabolic proteins largely interacted with dough matrix of Ventura-26 after 26min (56°C); gliadins, avenin-like b proteins, LMW-GSs, and partial globulins showed stronger interactions within the dough matrix of Ventura-26 at 32min/C3 (80°C), thereafter, however, stronger protein interactions were observed within the dough matrix of Ventura-19 at 38min/C4 (85°C) and 43min (80°C). Thirty-seven proteins associated with changes in dough matrix due to reduced amylose content were identified by mass spectrometry and mainly annotated to the chromosome group 1, 4, and 6. The findings provide new entry points for modifying final product attributes.