Exclusion High Performance Liquid Chromatography Research Articles

Machine Learning and Accelerated Stress Approaches to Differentiate Potential Causes of Aggregation in Polyclonal Antibody Formulations During Shipping.

Therapeutic proteins are among the most widely prescribed medications, with wide distribution and complex supply chains. Shipping exposes protein formulations to stresses that can trigger aggregation, although the exact mechanism(s) responsible for aggregation are unknown. To better understand how shipping causes aggregation, we compared populations of aggregates that were formed in a polyclonal antibody formulation during live shipping studies to populations observed in accelerated stability studies designed to mimic both the sporadic high g-force and continuous low g-force stresses encountered during shipping. Additionally, we compared the effects on aggregation levels generated in two types of secondary packaging, one of which was designed to mitigate the effects of large g-force stresses. Aggregation was quantified using fluorescence intensity of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) dye, size exclusion high performance liquid chromatography (SECHPLC), and flow imaging microscopy (FIM). FIM was also combined with machine learning methods to analyze particle morphology distributions. These comparisons revealed that the morphology distributions of aggregates formed during live shipping resemble distributions that result from low g-force events, but not those observed following high g-force events, suggesting that low g-force stresses play a predominant role in shipping-induced aggregation.

(-)-Epicatechin-An Important Contributor to the Antioxidant Activity of Japanese Knotweed Rhizome Bark Extract as Determined by Antioxidant Activity-Guided Fractionation.

The antioxidant activities of Japanese knotweed rhizome bark extracts, prepared with eight different solvents or solvent mixtures (water, methanol, 80% methanol(aq), acetone, 70% acetone(aq), ethanol, 70% ethanol(aq), and 90% ethyl acetate(aq)), were determined using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging assay. Low half maximal inhibitory concentration (IC50) values (2.632–3.720 µg mL−1) for all the extracts were in the range of the IC50 value of the known antioxidant ascorbic acid at t0 (3.115 µg mL−1). Due to the highest extraction yield (~44%), 70% ethanol(aq) was selected for the preparation of the extract for further investigations. The IC50 value calculated for its antioxidant activity remained stable for at least 14 days, while the IC50 of ascorbic acid increased over time. The stability study showed that the container material was of great importance for the light-protected storage of the ascorbic acid(aq) solution in a refrigerator. Size exclusion–high-performance liquid chromatography (SEC-HPLC)–UV and reversed phase (RP)-HPLC-UV coupled with multistage mass spectrometry (MSn) were developed for fractionation of the 70% ethanol(aq) extract and for further compound identification, respectively. In the most potent antioxidant SEC fraction, determined using an on-line post-column SEC-HPLC-DPPH assay, epicatechin, resveratrol malonyl hexoside, and its in-source fragments (resveratrol and resveratrol acetyl hexoside) were tentatively identified by RP-HPLC-MSn. Moreover, epicatechin was additionally confirmed by two orthogonal methods, SEC-HPLC-UV and high-performance thin-layer chromatography (HPTLC) coupled with densitometry. Finally, the latter technique enabled the identification of (−)-epicatechin. (−)-Epicatechin demonstrated potent and stable time-dependent antioxidant activity (IC50 value ~1.5 µg mL−1) for at least 14 days.

Moment of Bentonite Addition, Co-Addition of Tannins, and Bentonite Type Affect the Differential Affinity of Pathogenesis-Related Grape Proteins towards Bentonite during Fermentation.

To test the effect of the moment of bentonite addition, co-addition of tannins, and bentonite type on the differential affinity of pathogenesis-related (PR) proteins towards bentonite during grape must fermentation, three separate experiments were set up. PR proteins in the obtained wines were analyzed by reverse phase and size exclusion high-performance liquid chromatography (HPLC). The most significant reduction of bentonite dose and PR protein concentration was achieved by applying bentonite in the last third of fermentation. Particular thaumatin-like proteins (TLP) and proteins with lower molecular mass in general were more affected than others, while TLPs were more affected than chitinases. Exogenous enological tannins interacted with particular PR proteins, mostly TLPs, and lowered the total bentonite dose required. The combined application of tannins and bentonite in fermentation removed more PR proteins than bentonite alone, but did not achieve a synergistic effect in reducing the bentonite dose. Various bentonite types, including two Na-activated bentonites, an activated Na bentonite with specifically adsorbed silica, and an active Na-Ca bentonite, exhibited differential affinity towards different PR proteins. The results obtained could be used in developing wine fining protocols which combine treatments with complementary affinity for adsorption and removal of PR proteins, and in this way achieve greater efficiency of bentonite fining by reducing its total dose, which is of significant interest to the wine industry.

Analytical comparability study of anti-CD20 monoclonal antibodies rituximab and obinutuzumab using a stability-indicating orthogonal testing protocol: Effect of structural optimization and glycoengineering

Glycoengineering and biosimilarity are the key factors for growing, promising and progressive approaches in monoclonal antibodies development. In this study, the physicochemical stability of anti-CD20 rituximab (RTX); originator and biosimilar was compared to its glycoengineered humanized version; obinutuzumab (OBZ). An orthogonal stability-indicating protocol using a set of validated bioanalytical techniques; size exclusion high performance liquid chromatography (SE-HPLC), reversed phase liquid chromatography (RP-HPLC), quantitative gel electrophoresis by TapeStation, receptor binding assay and dynamic light scattering (DLS) was used to investigate the effect of different stress factors on the pattern and kinetics of degradation. SE-HPLC results supported with spectral purity showed similar degradation extent with a different pattern of degradation between RTX and OBZ. A lower tendency to form degraded fragments and a relatively higher favorability for degradation through aggregate formation has been revealed in case of OBZ. Results were in agreement with those of DLS and receptor binding assay which showed specificity to the intact antibodies in the presence of their degradation products. Furthermore, results were additionally confirmed through denaturing quantitative gel electrophoresis which suggested reducible covalent bonds as the mechanism for aggregates formation. RP-HPLC results showed two oxidized forms via excessive oxidation of RTX and OBZ with nearly the same degradation percent. Comparability data of RTX and OBZ using the applied methodologies showed that although glycoengineering; carried out to enhance the therapeutic and biological activity of OBZ altered the pattern of degradation but did not significantly affect the overall stability. Results showed also consistent stability profile between the biosimilar and its originator RTX products.

Food protein network formation and gelation induced by conductive or microwave heating: A focus on hen egg white

Heat-induced protein polymerization, gelation and the associated molecular scale changes were investigated during conductive and microwave heating (CH and MH, respectively). Given identical temperature-time profiles, polymerization of bovine serum albumin, ovalbumin, egg white (EW) and wheat gluten protein occurred faster and to a greater extent with CH than with MH. Moreover, lanthionine cross-links were present in some MH but in none of the CH samples. Gelation of EW protein by CH or MH was studied in more detail. Differential scanning calorimetry and fluorescence spectroscopy indicated that EW proteins initially denatured to a larger extent with CH than with MH. Size exclusion high performance liquid chromatography demonstrated that CH resulted in more heavily cross-linked EW protein networks than MH. Homogeneous EW gels were obtained by 15 min CH at 80 °C while 120 min MH at this temperature still resulted in inhomogeneous (with liquid and solid zones) gels. This was affirmed by low resolution 1H nuclear magnetic resonance measurements which indicated more mobile water in MH than in CH samples. However, MH gels had higher firmness than the corresponding CH gels. Thus, MH here led to less covalent cross-linking than CH, but still resulted in firmer gels most likely because its protein network relied heavily on non-covalent interactions. Industrial relevanceThis study highlighted differences between microwave and conventional, conductive, heating of different protein materials. While literature is divided about the origin of differences noted between these different heating modes, it is clear from our data that heating mode, given identical temperature-time profiles, impacts the changes that proteins undergo during heating. The knowledge that microwave heating favors protein unfolding, aggregation and cross-linking to a lower extent than conductive heating can likely be exploited in industrial applications.

Purification of Lumbricus terrestris Mega-Hemoglobin for Diverse Oxygen Therapeutic Applications.

Oxygen therapeutics are being developed for a variety of applications in transfusion medicine. In order to reduce the side-effects (vasoconstriction, systemic hypertension, and oxidative tissue injury) associated with previous generations of oxygen therapeutics, new strategies are focused on increasing the molecular diameter of hemoglobin obtained from mammalian sources via polymerization and encapsulation. Another approach towards oxygen therapeutic design has centered on using naturally occurring large molecular diameter hemoglobins (i.e. erythrocruorins) derived from annelid sources. Therefore, the goal of this study was to purify erythrocruorin from the terrestrial worm Lumbricus terrestris for diverse oxygen therapeutic applications. Tangential flow filtration (TFF) was used as a scalable protein purification platform to obtain a >99% pure LtEc product, which was confirmed by size exclusion high performance liquid chromatography and SDS-PAGE analysis. In vitro characterization concluded that the ultra-pure LtEc product had oxygen equilibrium properties similar to human red blood cells, and a lower rate of auto-oxidation compared to human hemoglobin, both of which should enable efficient oxygen transport under physiological conditions. In vivo evaluation concluded that the ultra-pure product had positive effects on the microcirculation sustaining functional capillary density compared to a less pure product (~86% purity). In summary, we purified an LtEc product with favorable biophysical properties that performed well in an animal model using a reliable and scalable purification platform to eliminate undesirable proteins.

Immunomodulatory effect of a purified peptide fraction isolated from Mesobuthus eupeus scorpion venom on astrocytoma cell line (1321N1)

Objective(s): Multiple Sclerosis is a central nervous system disease which belongs to the category of autoimmune diseases. The prevalence of this disease in Iran is approaching the European level. Astrocyte cells are nerve tissues that regulate the immune system activity by secreting various cytokines such as IL- 17. The aim of this study was partial purification of toxin from M.eupeus scorpion venom that has immunomodulatory effect on astrocyte cell line (1321N1) Materials and Methods: In the present study, purified crude venom of M.eupeus scorpion. Size exclusion and reverse-phase high-performance liquid chromatography was used for fractionation. The fractional molecular weight was determined by Using SDS and Tricine electrophoresis, Astrocyte cells (1321N1) were selected as functional cells in testing the immunomodulatory effect of venom. The viability of cells were determined by MTT and LDH assays. Astrocyte cells were activated by lipopolysaccharide and the release of interleukin-17 in activated cells was estimated using ELISA kit. Results: fraction 331 (F331) from RP-HPLC contain the purified peptide with molecular weight of about 4500 Dalton. When activated cells exposed to purified peptide the rate of interleukin-17 release was found to be 85 pg/ml which is almost similar to un-activated cells (78 pg/ml). However in activated cells by LPS without treatment with purified peptide the rate of IL-17 release was found to be 147 pg/ml which was significantly (p <0.05) higher than control group. Conclusion: The purified peptide (F331) from venom of Mesobouthus eupeus can inactivate the astrocyte 1321N1 cells activated by LPS as indicated by decreased secretion of IL-17 from the cells.

Effect of thermosonication pre-treatment on mung bean (Vigna radiata) and white kidney bean (Phaseolus vulgaris) proteins: Enzymatic hydrolysis, cholesterol lowering activity and structural characterization.

Protein hydrolysates have attained great attention due to a good nutritive food ingredient and higher biological activities. In this study, thermosonication, ultrasound and heat were used as a pre-treatment to obtain (<3KDa) hydrolysate from mung bean and white kidney bean to understand the mechanism of cholesterol absorption into micelle and inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) activity. Size exclusion high performance liquid chromatography (SE-HPLC) results of mung bean showed that the concentration of peptides (0.5KDa-1KDa and 1-3KDa) in the hydrolysate were significantly (p<0.05) increased after thermosonication while, the peptides concentration (1-3KDa) in white kidney bean was significantly (p<0.05) decreased. Thermosonication of mung bean hydrolysate exhibited higher inhibition of cholesterol solubilization, hydrophobicity and antioxidant activities. In addition, there was no difference observed in HMG-CoA activity and hydrophobicity between ultrasound alone and ultrasound combined with heat i.e. thermosonication treated hydrolysate of white kidney bean. Changes in secondary and tertiary structures were also analyzed under different processing conditions with maximum change due to thermosonication. Results indicated that mung bean hydrolysate had a great potential for inhibition of cholesterol synthesis and its solubility in the micelle, antioxidant activity and also convinced for its application in food and nutraceutical industries.

Nitrogen and sulfur effects on hard winter wheat quality and asparagine concentration

Grain protein concentration and composition are key factors affecting winter wheat quality and are influenced by wheat genotype, available fertility, and growing conditions. These same parameters can affect free asparagine concentration in grain, and elevated asparagine can lead to acrylamide production in baked food products, which can be a health concern. The objectives of this study were to determine the effect of genotype, nitrogen (N), and sulfur (S) fertility on protein concentration, protein quality, dough rheology, and asparagine concentration in winter wheat grown on S-deficient soils. Treatments were arranged in a 3 × 2 × 4 factorial design in 2017 and 3 × 2 × 5 factorial design in 2018. There were three levels of N (56, 101 and 146 kg ha−1), two levels of S (0 and 22 kg ha−1), four levels of genotype in 2017, and five levels of genotype in 2018. Protein composition was evaluated as the percent polymeric protein using size exclusion high performance liquid chromatography. In both years, the ratio of polymeric to monomeric protein was increased by sulfur fertilization. Solvent retention capacity (SRC) was evaluated using the whole grain lactic acid-sodium dodecyl sulfate test. In 2018, S application increased the SRC by 217%–308%. However, in 2017, SRC improvement was limited to two genotypes and was modest, likely a consequence of the reduced protein concentration in S-treated plots. Free asparagine concentration averaged 9.8 μmol/g and 20.9 μmol/g in 2017 and 2018, respectively. Asparagine concentration in grain was affected by N, S, genotype, and their interactions. Sulfur application substantially reduced asparagine concentrations in both years. Dough rheology was evaluated in the 2018 trial using the farinograph test. Sulfur application increased average farinograph stability from 9.2 min to 14.6 min. Farinograph stability was effectively predicted by the SRC test (R2 = 0.78). These results demonstrate the importance of ensuring adequate S fertility in winter wheat production.

Osborne extractability and chromatographic separation of protein from quinoa (Chenopodium quinoa Willd.) wholemeal

The composition of quinoa proteins has hardly been studied. We here applied an Osborne type extraction procedure. High protein yield and little protein denaturation was obtained with extraction (2x) with water followed by extraction (2x) with 0.4 mol/l sodium chloride. Albumins, globulins, prolamins and glutelins accounted for 41–50, 7–11, 4–6 and 14–16% of the total protein of four quinoa cultivars, respectively. Protein extracts were separated by size exclusion high performance liquid chromatography (SE-HPLC) with a sodium dodecyl sulfate containing elution medium. Electrophoretic separation followed by mass spectrometry allowed identifying the proteins in the Osborne type extracts. Aqueous extracts contained different chloroplastic and cytoplasmic enzymes as well as legumin-like and vicilin-like proteins. Dilute salt extracts mainly contained legumin-like proteins. Aqueous alcohol extracts contained small proteins while dilute base extracts proteins were similar to those present in the aqueous and dilute salt extracts. In one cultivar, only the monomer of the 11S globulin seed storage protein 2-like was observed while the other cultivars studied also contained its acidic and basic subunits separately. This SE-HPLC method allows analyzing food processing induced changes in quinoa protein extractability and size distribution.

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 ℃) was mainly ascribed to the un-extractable HMW-GSs, LMW-GSs, globulin and triticin, while the extract yield of α-, β-, 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.

Physicochemical Characterization of Altebrel™, a Proposed Etanercept Biosimilar.

Background: Etanercept is prescribed for the rapid and effective treatment of chronic immune-mediated inflammatory disorders. Due to the expiration of etanercept patents and worldwide demand for comparable and more affordable substitutes, several biosimilars of etanercept have been approved in different countries and new ones are in the process of approval. Objectives: In the present study, Altebrel™ as an etanercept proposed biosimilar was investigated in a side by side comparison using various orthogonal analytical methods. Materials and Methods: Three batches of the Altebrel™ and Enbrel® samples were used for the study. Several physicochemical properties of samples were compared according to international guidelines, incliding; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Capillary electrophoresis sodium dodecyl sulfate (CE-SDS), size exclusion high performance liquid chromatography (SE-HPLC), hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC) and its biological activity was evaluated using surface plasmon resonance affinity analysis and tumor necrosis factor alpha (TNFα) neutralization biological assay. Amino acid analysis was applied to check the primary sequence and far-UV circular dichroism (CD) spectroscopy investigated the secondary structure. Results: The obtained results indicated a high degree of similarity between Altebrel™ and Enbrel®. Results of SDS-PAGE, CE-SDS, HIC-HPLC and SE-HPLC implied a comparable pattern of size variants for all samples. Based on the data achieved via in vitro bioactivity assays and SPR analysis, the functional property of Altebrel™ was proved comparable to that of the reference product. Moreover, amino acid analysis indicated similar primary structure and circular dichroism study implied a similar secondary structure for Altebrel™ and Enbrel®. Conclusion: Overall, our data provide analytical evidence for structural and in vitro functional similarity between Altebrel™ and Enbrel®.

Antioxidant and antibacterial activity and in vitro digestion stability of cottonseed protein hydrolysates

In this study, five fractions (F–I to F–V) were successfully isolated from cottonseed protein hydrolysates (CPH) by using ion exchange resin system, and proved to be mainly composed of peptides with molecular weights less than 1 kDa by size exclusion high performance liquid chromatography (SEC-HPLC). All fractions possessed the DPPH radical and ABTS radical cation scavenging abilities. However, the antibacterial experiment revealed F–I and CPH had no inhibition against E. coli. By analyzing the composition of amino acids, the antioxidant and antibacterial activities were found to be positively correlated with basic amino acid contents (Arg and Lys), but negatively correlated with acidic residual (Glu and Asp). Furthermore, the in vitro digestion had slightly effects on antibacterial activity of the fractions (CPH, Fraction I-Ⅴ), while it showed great impacts on the antioxidant capacity. The work here provided basic theory and a new strategy for preparing bifunctional peptides from cottonseed protein, which could be used as an alternative in the functional food formulations.

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.

Extended Stability of Reconstituted and Diluted SB3 (Trastuzumab Biosimilar) Assessed by Physicochemical and Biological Properties.

IntroductionStability information for the trastuzumab biosimilar SB3 is limited to 48 h at 2–8 °C for the reconstituted solution and 24 h at up to 30 °C for diluted solutions. Extended physicochemical stability and biological activity were assessed to evaluate the advanced preparation of reconstituted and diluted SB3.MethodsUnder controlled and aseptic conditions, the stability of reconstituted and diluted SB3 was evaluated using several assessments and according to the UK’s National Health Service guidance. Reconstituted SB3 was stored at 25 ± 2 °C with 60 ± 5% relative humidity for 3 days, and subsequently diluted SB3 (0.32–4 mg/mL) was stored in an infusion bag in the absence of light at 25 ± 2 °C with 60 ± 5% relative humidity for 28 days and 5 ± 3 °C for 28 days, respectively. Physicochemical stability (appearance, pH, protein concentration, size exclusion high-performance liquid chromatography, non-reducing capillary electrophoresis–sodium dodecyl sulfate, imaged capillary isoelectric focusing), biological activity (competitive inhibition binding assay to human epidermal growth factor receptor 2 by fluorescence resonance energy transfer, anti-proliferation assay), and properties with a potential safety impact (subvisible particulates, submicronic aggregation by dynamic light scattering) were determined.ResultsNo physicochemical instability signs or biological activity changes were observed for either reconstituted or diluted SB3 up to 28 days; all stability acceptance criteria were met. No major change was noted in the proportion of molecular weight variants (high molecular weight impurity, total purity) or relative percentages of acidic, main, and basic charge isoforms of the protein. No increases in particulates or aggregates in terms of a potential safety impact were noted.ConclusionThe physicochemical stability and biological activity of reconstituted and diluted SB3 are maintained for extended time periods beyond those denoted in the product labeling, which allows for advanced SB3 preparation and may reduce drug wastage and preparation time.FundingSamsung Bioepis Co., Ltd.

Protein aggregation in cooked pork products: New details on the supramolecular organization

The molecular weight distribution of protein aggregates from raw meat and cooked pork products was assessed by size exclusion–high performance liquid chromatography (SE–HPLC). Electrophoretic analysis under reducing conditions showed that the high molecular weight SE–HPLC peak (peak 1) of the cooked products contained protein aggregates in addition to high molecular weight muscle proteins, while the second peak (peak 2) still contained aggregates and <50 kDa proteins. The protein aggregates composition was investigated by HPLC–tandem mass spectrometry. Different classes of proteins were identified and the cooked products showed a more complex composition and organization, according to the muscle structure and the technological procedures, respectively. The key role of actin in the building of the protein networks was also confirmed. The different multi-protein systems found in the cooked products suggest protein re-organization in heat-induced supramolecular structures, which might be responsible for the texture and the structural properties of the final products.

Molecular Aggregation of Marketed Recombinant FVIII Products: Biochemical Evidence and Functional Effects.

Background Recombinant (rec-) coagulation factor VIII concentrates available for hemophilia A (HA) treatment differ in cell line production and structure, which could affect their pharmacodynamics and immunogenicity. Clinical trials showed that previously untreated patients with severe HA present higher rates of inhibitor development if treated with rec-FVIII products and that differences do exist as to inhibitor's formation among different rec-FVIII products. This finding could arise from several causes, such as absence of von Willebrand factor, different glycosylation profiles, or processes of molecular aggregation of the recombinant FVIII molecules. Objectives/Methods In this study, using size exclusion high-performance liquid chromatography (SE-HPLC), dynamic light scattering (DLS) spectroscopy, and functional biochemical assays, we investigated the purity grade, FX activating ability, and aggregation status of three recombinant marketed products (Advate [Baxalta], Refacto AF [Pfizer], and Kogenate [Bayer]). Results The overall analysis of the results obtained with SE-HPLC and DLS spectroscopy showed that the three recombinant FVIII concentrates contain low but significant amounts of molecular aggregates. This phenomenon was less evident for the Advate product. Molecular aggregation negatively affects the in vitro pharmacodynamics of the concentrates with higher aggregates' content. Conclusions This study shows that the three pharmaceutical formulations of recombinant FVIII contain variable amounts of molecular aggregates after their reconstitution at therapeutic concentrations. This phenomenon negatively affects the in vitro potency of the products with higher aggregates' content and might be invoked as a contributing cause of their increased risk to induce the formation of FVIII inhibitors.

Quantitation of the monoclonal antibody Denosumab by bioassay and validated LC methods

The monoclonal antibody Denosumab (DmAb) is clinically used to treat osteoporosis and bone loss. We developed a bioassay based on the ability of DmAb to inhibit the effect of human receptor activator of nuclear factor-κB ligand (RANKL) to stimulate the formation of osteoclasts derived from RAW 264.7 cells. This bioassay was applied in conjunction with size exclusion high-performance liquid chromatography (SE–HPLC) and reversed-phase high-performance liquid chromatography (RP–HPLC) methods, with diode array detection (DAD), validated for the quantitation of this biotechnology-derived medicine. The SE–HPLC(DAD) method was carried out on a TSKGel G2000SWXL column and the mobile phase consisted of potassium phosphate buffer with sodium chloride, pH 7.4. The gradient RP–HPLC(DAD) method was carried out on a Vydac 214TP C4 column at 60 °C. The mobile phases consisted of 0.1% v/v trifluoroacetic acid (TFA) in water and 0.1% v/v TFA in acetonitrile. Calibration curves were linear over the concentration ranges 6–200 μg mL−1 and 6–300 μg mL−1 for the SE–HPLC(DAD) and RP–HPLC(DAD) methods respectively. The bioassay results correlated with the LC methods results, indicating the capabilities of these methods to quantitate DmAb, which will contribute to ensure the batch-to-batch consistency and efficacy of this biotherapeutic.

Optimizing yield and quality in malting barley by the governance of field cultivation conditions

This study evaluates how cultivation location, year, soil conditions and starter fertilizer interact and determine yield and quality of malting barley. Size exclusion high performance liquid chromatography (SE-HPLC) was applied to determine protein composition in mature and malted barley.The effects of starter fertilizer treatments on yield and quality of malting barley was largely hidden by the impact of growing location and cultivation year on these parameters. Here, the highest yield and the lowest grain protein concentration was found in samples cultivated on sandy soil with adequate precipitation and a relatively low soil temperature (below 15–17 °C) during grain filling. In malted grains, the general change in protein composition as related to mature grains, was a decrease in the polymerized protein fractions and an increase in the more easily extractable and smaller proteins. However, cultivation conditions impacted the changes of the protein fractions; the highest break-down rates of polymerized proteins was found for samples cultivated on sandy soil with enough precipitation and a relatively low soil temperature (below 15–17 °C) during grain filling. Thus, the sandy soil, enough precipitation and relatively low soil temperature were the most beneficial both for grain yield and for protein based quality of the malting barley.

Cytosolic distributions of highly toxic metals Cd and Tl and several essential elements in the liver of brown trout (Salmo trutta L.) analyzed by size exclusion chromatography and inductively coupled plasma mass spectrometry

Cytosolic distributions of nonessential metals Cd and Tl and seven essential elements among compounds of different molecular masses were studied in the liver of brown trout (Salmo trutta) from the karstic Krka River in Croatia. Analyses were done by size exclusion high performance liquid chromatography and high resolution inductively coupled plasma mass spectrometry. Common feature of Cd and Tl, as highly toxic elements, was their distribution within only two narrow peaks. The increase of cytosolic Cd concentrations was reflected in marked increase of Cd elution within low molecular mass peak (maximum at ∼15 kDa), presumably containing metallothioneins (MTs), which indicated successful Cd detoxification in brown trout liver under studied exposure conditions. Contrary, the increase of cytosolic Tl concentrations was reflected in marked increase of Tl elution within high molecular mass peak (maximum at 140 kDa), which probably indicated incomplete Tl detoxification. Common feature of the majority of studied essential elements was their distribution within more peaks, often broad and not well resolved, which is consistent with their numerous physiological functions. Among observed associations of essential metals/nonmetal to proteins, the following could be singled out: Cu and Zn association to MTs, Fe association to storage protein ferritin, and Se association to compounds of very low molecular masses (<5 kDa). The obtained results present the first step towards identification of metal-binding compounds in hepatic cytosol of brown trout, and thus a significant contribution to better understanding of metal fate in the liver of that important bioindicator species.