51 research outputs found

    Towards Protein Crystallization as a Process Step in Downstream Processing of Therapeutic Antibodies: Screening and Optimization at Microbatch Scale

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    Crystallization conditions of an intact monoclonal IgG4 (immunoglobulin G, subclass 4) antibody were established in vapor diffusion mode by sparse matrix screening and subsequent optimization. The procedure was transferred to microbatch conditions and a phase diagram was built showing surprisingly low solubility of the antibody at equilibrium. With up-scaling to process scale in mind, purification efficiency of the crystallization step was investigated. Added model protein contaminants were excluded from the crystals to more than 95%. No measurable loss of Fc-binding activity was observed in the crystallized and redissolved antibody. Conditions could be adapted to crystallize the antibody directly from concentrated and diafiltrated cell culture supernatant, showing purification efficiency similar to that of Protein A chromatography. We conclude that crystallization has the potential to be included in downstream processing as a low-cost purification or formulation step

    Adaptation and Validation of QUick, Easy, New, CHEap, and Reproducible (QUENCHER) Antioxidant Capacity Assays in Model Products Obtained from Residual Wine Pomace

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    Evaluation of the total antioxidant capacity of solid matrices without extraction steps is a very interesting alternative for food researchers and also for food industries. These methodologies have been denominated QUENCHER from QUick, Easy, New, CHEap, and Reproducible assays. To demonstrate and highlight the validity of QUENCHER (Q) methods, values of Q-method validation were showed for the first time, and they were tested with products of well-known different chemical properties. Furthermore, new QUENCHER assays to measure scavenging capacity against superoxide, hydroxyl, and lipid peroxyl radicals were developed. Calibration models showed good linearity (R2 > 0.995), proportionality and precision (CV < 6.5%), and acceptable detection limits (<20.4 nmol Trolox equiv). The presence of ethanol in the reaction medium gave antioxidant capacity values significantly different from those obtained with water. The dilution of samples with powdered cellulose was discouraged because possible interferences with some of the matrices analyzed may take place.The autonomous government of Castilla y León (Project BU268A11-2

    Bacteriocinogenic Effect of Lactobacillus sakei 2a on Microbiological Quality of Fermented Sardinella brasiliensis

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    Lactobacillus sakei 2a is a bacteriocin producer strain and, in this work, it’s effects as a starter culture in the fermentation process of sardine (Sardinella brasiliensis) fillets were observed at different concentrations of NaCl (2, 4 and 6%) and glucose (2 and 4%), to determine it’s ability to produce organic acids and consequent pH reduction. Experiments were carried out independently, with only one parameter (NaCl or glucose) varying at a time. After 21 days of fermentation the deteriorative bacteria concentration reached 9.7 Log10 CFU. g-1 corresponding to 6% NaCl and 4% glucose. Little differences were observed in lactic acid production when 2 and 4% glucose were added, since total acidity was 1.32 and 1.34% respectively, the experiments with 6% NaCl presented the best results. Initial pH of sardine fillets was 6 and after 21 days pH values were 3.8, 3.9 and 4 for the experiments with 2, 4 and 6% NaCl. This may have been due to the inhibitory properties of NaCl over the deteriorative bacteria. After 21 days of the fermentation process lactic acid bacteria concentrations were 14.5 Log10 CFU.g-1. The ratio protein nitrogen and total soluble nitrogen was typical of a cured fish

    Pathological crystallization of human immunoglobulins

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    Condensation of Igs has been observed in pharmaceutical formulations and in vivo in cases of cryoglobulinemia. We report a study of monoclonal IgG cryoglobulins overexpressed by two patients with multiple myeloma. These cryoglobulins form crystals, and we measured their solubility lines. Depending on the supersaturation, we observed a variety of condensate morphologies consistent with those reported in clinical investigations. Remarkably, the crystallization can occur at quite low concentrations. This suggests that, even within the regular immune response to infections, cryoprecipitation of Ig can be possible
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