LYTAG-driven purification strategies as a key to integrate and intensify the downstream processing of monoclonal antibodies

Monoclonal antibodies (mAbs) are currently the most important class of recombinant protein therapeutics in the biotechnological and biopharmaceutical industry with more than 250 mAbs currently undergoing clinical trials. High titer producing cultures and complex mixtures containing high cell densities, together with an increasing growing demand for highly pure mAbs is making recovery and purification processes hot targets for improvement and opens important technological challenges in mAbs manufacturing platforms. This work explores the use of an affinity dual ligand based on a choline binding polypeptide tag (LYTAG) fused with the synthetic antibody Z domain (LYTAG-Z) as a tool to integrate and optimized the downstream processing of mAbs. Upon addition of this ligand to an animal cell culture broth, antibody-LYTAG-Z complexes are formed which can be easily captured and separated from host cell impurities by affinity partitioning in aqueous two-phase systems (ATPS) composed of polyethylene glycol –PEG, as PEG molecules have the ability to binding to the choline binding sites of LYTAG. Integration of clarification and primary mAbs recovery was successfully accomplished using a system composed of 6% PEG 3350 Da and 7% dextran 500,000 Da in which an extraction yield of 89% and a clarification higher than 95% were achieved. IgG-rich phases were further processed by chromatography, using three different strong anion exchange matrices charged with quaternary methyl amines (a choline analogue) – CIMmultus QA, HiTrap Q FF and gPore NW Q. A two-elution method was developed for the separation of the antibody-LYTAG-Z complexe, allowing simultaneous purification of the antibody and recovery of the ligand. The process was successfully scale-up 10000 times allowing a global antibody recovery of 70% with a purity of 89% and enabling 100% cell removal

Three-Dimensional Study of Natural Convection in a Horizontal Channel With Discrete Heaters on One of Its Vertical Walls

Three-dimensional natural convection in a horizontal channel with an array of discrete flush-mounted heaters on one of its vertical walls is numerically studied. Effects of thermal conductivities of substrate and heaters and convection on outer sides of the channel walls on heat transfer are examined. The substrate affects heat transfer in a wider range of thermal conductivities than do the heaters. At lower heater thermal conductivities a higher heat portion is transferred by direct convection from the heaters to the adjacent coolant. However, higher substrate conductivity is associated with higher heat portion transferred through the substrate. The innermost heater column is found to become the hottest heater column due to the lower coolant accessibility. The heat transfer in the channel is strongly influenced by convection on the outer sides of the channel walls. Correlations are presented for dimensionless temperature maximum and average Nusselt number

Preparation, characterization, and process performance of composite fibrous adsorbents as cation exchangers for high throughput and high capacity bioseparations

Fibrous materials are proposed as novel chromatographic supports depicting high throughput and high product capacity. In this work, a composite fiber harboring strong cation-exchange moieties has been investigated. Such materials were characterized by a plethora of physical methods including degree of swelling (DS), scanning electron microscope (SEM), confocal laser scanning microscopy (CLSM), and Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR). The composite showed a high degree of grafting (∼30%) and exhibited a high swelling ratio (∼300%). Moreover, homogenous grafting and the development of an internal (functional) hydrogel were observed. The fibrous adsorbent was packed utilizing a designed " double roll" supported-structure and subsequently tested for packing efficiency and chromatography performance. The mentioned system showed similar packing efficiency of height equivalent to a theoretical plate (HETP) value and higher permeability coefficient (0.92×10 -7cm 2) than commercial resins. Experimentally determined Peclet number (Pe) values were within the range 60-90, suggesting a close-to-plug-flow condition. Total ionic capacity of the fibrous adsorbent was determined by the transition pH method. A capacity of 6.5mequiv./g was obtained. Moreover, a high dynamic binding capacity for lysozyme was found to be 283mg/g. On the other hand, a bed of randomly packed fiber also demonstrated high-resolution ability when a mixture of model protein was utilized to that end. Resolution was maintained at high flow rates (up to 900cm/h) and utilizing shorter gradient development routines. Direct sequestration of a model protein (lysozyme) was also possible from an artificial mixture containing 1.5% yeast homogenate. Summarizing, the composite fibrous adsorbents exhibited superior performance during early protein capture and intermediate-resolution applications.Fil: Gavara, Poondi Rajesh. Universitat Bremen; AlemaniaFil: Cabrera, Rosa. Universitat Bremen; AlemaniaFil: Vennapusa, Rami Reddy. Universitat Bremen; AlemaniaFil: Grasselli, Mariano. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Fernandez-Lahore, Marcelo. Universitat Bremen; Alemani

Chromatographic Characterization and Process Performance of Column-Packed Anion Exchange Fibrous Adsorbents for High Throughput and High Capacity Bioseparations

Fibrous materials are prominent among novel chromatographic supports for the separation and purification of biomolecules. In this work, strong anion exchange, quaternary ammonium (Q) functional fibrous adsorbents were evaluated with regards to their physical and functional characteristics. A column packed with Q fibrous adsorbent illustrated the good column packing efficiency of theoretical plate height (H) values and higher permeability coefficients (>0.9 × 10−7 cm2) than commercial adsorbents. For pulse experiments with acetone and lactoferrin as tracers under nonbinding conditions, the total porosity (for acetone) and the interstitial porosity (for lactoferrin) measured 0.97 and 0.47, respectively. The total ionic capacity of the chemically-functionalized Q fiber was 0.51 mmol/mL. The results indicated that the Q fiber had a static binding capacity of 140 mg/mL and a dynamic binding capacity (DBC) of 76 mg/mL for bovine serum albumin (BSA) and showed a linearly-scalable factor (~110 mL) for a column volume with high capacity and high throughput. Furthermore, this adsorptive material had the ability to bind the high molecular weight protein, thyroglobulin, with a capacity of 6 mg/mL. This work demonstrated the column-packed Q fibrous adsorption system as a potential chromatography support that exhibits high capacity at higher flow rates

Safety Assessment of Ubiquinol Acetate: Subchronic Toxicity and Genotoxicity Studies

Coenzyme Q10 (CoQ10) is a lipid soluble, endogenous antioxidant present at highest levels in the heart followed by the kidney and liver. The reduced CoQ10 ubiquinol is well known for its chemical instability and low bioavailability. The present study was designed to synthesize ubiquinol acetate, which is more stable and biologically active, and further evaluate its safety and genotoxic potential. Synthesized ubiquinol acetate showed better stability than that of ubiquinol at the end of 3 months. In vitro genotoxicity studies (AMES test, in vitro micronucleus and chromosomal aberration) showed ubiquinol acetate as nongenotoxic with no clastogenic or aneugenic effects at high dose of 5000 and 62.5 μg/mL, respectively. In subchronic toxicity study, ubiquinol acetate was administered orally to Sprague Dawley rats at 150, 300, and 600 mg/kg/day for 90 days. No treatment related adverse effects were observed in males at 600 mg/kg/day; however, females showed treatment related increase in AST and ALT with small focal irregular white-yellow spots in liver on gross necropsy examination. Histopathological evaluation revealed hepatocellular necrosis in high dose females which was considered as adverse. Based on the results, the No-Observed-Adverse-Effect Level (NOAEL) of ubiquinol acetate in males and females was determined as 600 and 300 mg/kg/day, respectively