Process related impurity breakthrough from depth filtration during monoclonal antibody purification

Upstream advances have led to increased mAb titers above 5g/L in 14-day fed-batch cultures. This is accompanied by higher cell densities and process-related impurities such as DNA and Host Cell Protein (HCP), which have caused challenges for downstream operations. Depth filtration remains a popular choice for harvesting CHO cell culture. However, manufacturers are looking to move away from natural materials such as cellulose and Diatomaceous Earth (DE) for better filter consistency and security of supply. This thesis investigates the impact of high cell density (30-40 million cells/ml) feed material on traditional cellulose and DE filters compared to synthetic polyacrylate + silica depth filters. The study focuses on the retention of process-related impurities such as DNA and HCP through breakthrough studies and a novel confocal microscopy method for imaging foulant in-situ. Further investigation is carried out to understand the effects on Protein A chromatography. In a 2:1 primary: secondary depth filter scale-down model, it was found that the primary filter was the limiting step in terms of pressure and that soluble impurities were mostly removed by the secondary filters. A direct comparison of secondary synthetic and cellulose/DE filters was performed by scaling up the primary depth filter. The viability of the cell culture, and hence DNA concentration at input, was an important factor for DNA retention. HCP was not significantly removed by the depth filtration train. The confocal imaging of the secondary filter showed that cell debris and DNA foulant were distributed differently based on viability and filter type, leading to differences in the pressure profile and impurity retention. In order to gain a deeper understanding of process interaction, three different depth filtration trains were compared where the variables were filter materials and loading. The filtrate was used in a scale-down Protein A chromatography lifetime study and a low pH hold with 0.2μm filtration. Results show that a 36% loading increase in the primary synthetic filter negatively. affected DNA retention in the secondary filter. Confocal imaging of the depth filters showed that the foulant was pushed down through the filter with higher loading. The additional two layers in the primary synthetic filter led to better pressure profiles in both primary and secondary filters but did not help to retain HCP or DNA. Increased solids in the filtrate were associated with the synthetic filter trains, as was precipitation in the Protein A column feed. Confocal imaging of resin after 100 cycles showed that DNA build-up around the outside of the bead was associated with synthetic filter trains, leading to potential mass transfer problems

Depth filter material process interaction in the harvest of mammalian cells

Upstream advances have led to increased mAb titers above 5 g/L in 14-day fed-batch cultures. This is accompanied by higher cell densities and process-related impurities such as DNA and Host Cell Protein (HCP), which have caused challenges for downstream operations. Depth filtration remains a popular choice for harvesting CHO cell culture, and there is interest in utilising these to remove process-related impurities at the harvest stage. Operation of the harvest stage has also been shown to affect the performance of the Protein A chromatography step. In addition, manufacturers are looking to move away from natural materials such as cellulose and Diatomaceous Earth (DE) for better filter consistency and security of supply. Therefore, there is an increased need for further understanding and knowledge of depth filtration. This study investigates the effect of depth filter material and loading on the Protein A resin lifetime with an industrially relevant high cell density feed material (40 million cells/mL). It focuses on the retention of process-related impurities such as DNA and HCP through breakthrough studies and a novel confocal microscopy method for imaging foulant in-situ. An increase in loading of the primary-synthetic filter by a third, led to earlier DNA breakthrough in the secondary filter, with DNA concentration at a throughput of 50 L/m2 being more than double. Confocal imaging of the depth filters showed that the foulant was pushed forward into the filter structure with higher loading. The additional two layers in the primary-synthetic filter led to better pressure profiles in both primary and secondary filters but did not help to retain HCP or DNA. Reduced filtrate clarity, as measured by OD600, was 1.6 fold lower in the final filtrate where a synthetic filter train was used. This was also associated with precipitation in the Protein A column feed. Confocal imaging of resin after 100 cycles showed that DNA build-up around the outside of the bead was associated with synthetic filter trains, leading to potential mass transfer problems

Effect of Doping Element and Electrolyte’s pH on the Properties of Hydroxyapatite Coatings Obtained by Pulsed Galvanostatic Technique

Hydroxyapatite (HAp) is the most widely used calcium phosphate as a coating on metal implants due to its biocompatibility and bioactivity. The aim of this research is to evaluate the effect of the pH’s electrolyte and doping element on the morphology, roughness, chemical, and phasic composition of hydroxyapatite-based coatings obtained by pulsed galvanostatic electrochemical deposition. As doping elements, both Sr and Ag were selected due to their good osseoinductive character and antibacterial effect, respectively. The electrolytes were prepared at pH 4 and 5, in which specific concentrations of Sr, Ag, and Sr + Ag were added. In terms of morphology, all coatings consist in ribbon-like crystals, which at pH 5 appear to be a little larger. Addition of Sr did not affect the morphology of HAp, while Ag addition has led to the formation of flower-like crystals agglomeration. When both doping elements were added, the flowers like agglomerations caused by the Ag have diminished, indicating the competition between Sr and Ag. X-Ray Diffraction analysis has highlighted that Sr and/or Ag have successfully substituted the Ca in the HAp structure. Moreover, at higher pH, the crystallinity of all HAp coatings was enhanced. Thus, it can be said that the electrolyte’s pH enhances to some extent the properties of HAp-based coatings, while the addition of Sr and/or Ag does not negatively impact the obtained features of HAp, indicating that by using pulsed galvanostatic electrochemical deposition, materials with tunable features dictated by the function of the coated medical device can be designed

Structural, mechanical, wear and anticorrosive properties of CrSiCN coatings used for industrial woodworking applications

The woodworking applications are a fast-growing field that aims to create advanced coatings with superior wear resistance, reduced friction, and robust corrosion protection. Chromium silicon carbonitride (CrSiCN) coatings have emerged as a promising solution that offers a unique combination of properties ideal for various industrial applications. The C/N ratio significantly influences the coatings' mechanical and tribological properties. By optimizing the C/N ratio, this research aims to reveal new insights for CrSiCN coatings, enhancing their application in environments that require durability, efficiency, and longevity. In this paper, the effect of the C/N ratio on the structural, mechanical, and corrosion resistance of CrSiCN coatings deposited by cathodic arc evaporation on different steel substrates was studied. The main purpose was to enhance the mechanical and anticorrosion properties of the CrSiCN coatings and to select the optimum parameters for the deposition of layers with superior properties. The results showed that the final properties can be tailored by choosing specific deposition conditions. In this case, the C/N ratio proved to be critical since coatings with higher carbon content presented enhanced corrosion resistance, being able to withstand operating conditions similar to real-life

Electrochemical Surface Biofunctionalization of Titanium through Growth of TiO2 Nanotubes and Deposition of Zn Doped Hydroxyapatite

The current research aim is to biofunctionalize pure titanium (Ti, grade IV) substrate with titania nanotubes and Zn doped hydroxyapatite-based coatings by applying a duplex electrochemical treatment, and to evaluate the influence of Zn content on the physico-chemical properties of hydroxyapatite (HAp). The obtained nanostructured surfaces were covered with HAp-based coatings doped with Zn in different concentrations by electrochemical deposition in pulsed galvanostatic mode. The obtained surfaces were characterized in terms of morphology, elemental and phasic composition, chemical bonds, roughness, and adhesion. The nanostructured surface consisted of titania nanotubes (NT), aligned, vertically oriented, and hollow, with an inner diameter of ~70 nm. X-ray Diffraction (XRD) analysis showed that the nanostructured surface consists of an anatase phase and some rutile peaks as a secondary phase. The morphology of all coatings consisted of ribbon like-crystals, and by increasing the Zn content the coating became denser due to the decrement of the crystals’ dimensions. The elemental and phase compositions evidenced that HAp was successfully doped with Zn through the pulsed galvanostatic method on the Ti nanostructured surfaces. Fourier Transform Infrared spectroscopy (FTIR) and XRD analysis confirmed the presence of HAp in all coatings, while the adhesion test showed that the addition of a high quantity leads to some delamination. Based on the obtained results, it can be said that the addition of Zn enhances the properties of HAp, and through proper experimental design, the concentration of Zn can be modulated to achieve coatings with tunable features

Investigation of cast and annealed Ti25Nb10Zr alloy as material for orthopedic devices

In the present work, we report the preparation of a novel titanium-based alloy, namely Ti25Nb10Zr, by cold crucible levitation melting technique. The cast alloy consists of a complex microstructure with large Beta phase grains (54%, 50-150 μm) with a regularly connected net of Alpha′ (orthorhombic, 46%) phase running along boundaries and across the grains and keeping a regular misorientation with respect to the Beta phase. An intermeshed 51% Alpha and 49% Beta phases with lamellar microstructure were found by annealing. The electrochemical tests showed that both alloys were affected by the corrosion process. A good corrosion resistance in SBF at 37 °C was found for the cast form. The cast alloy is more resistant when immersed into solutions with pH2 and pH7, while the annealed one is resistant in pH5 solution. Surface potential of both alloys is negative, with the annealing process leading to a slight decrease of that property. Collectively, the biological results indicate a more favorable viability on cast form as compared to annealed one, suggesting that the cast alloy is promising for biomedical applications.Fil: Bolmaro, Raul Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Física de Rosario. Universidad Nacional de Rosario. Instituto de Física de Rosario; ArgentinaFil: Parau, Anca C.. National Institute of Research and Development for Optoelectronics; RumaniaFil: Pruna, Vasile. Romanian Academy. Institute of Cellular Biology and Pathology Nicolae Simionescu; RumaniaFil: Surmeneva, Maria A.. National Research Tomsk Polytechnic University; RusiaFil: Constantin, Lidia R.. National Institute of Research and Development for Optoelectronics; RumaniaFil: Avalos, Martina Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Física de Rosario. Universidad Nacional de Rosario. Instituto de Física de Rosario; ArgentinaFil: Cotrut, Cosmin M.. National Research Tomsk Polytechnic University; Rusia. University Politehnica of Bucharest; RumaniaFil: Tutuianu, Raluca. Romanian Academy. Institute of Cellular Biology and Pathology Nicolae Simionescu; RumaniaFil: Braic, Mariana. National Institute of Research and Development for Optoelectronics; RumaniaFil: Cojocaru, Danut V.. National Research Tomsk Polytechnic University; RusiaFil: Dan, Ioan. SC R&D Consulting and Services; RumaniaFil: Croitoru, Sorin. Tehnomed Impex Co; RumaniaFil: Surmenev, Roman A.. Romanian Academy. Institute of Cellular Biology and Pathology Nicolae Simionescu; RumaniaFil: Vladescu, Alina. National Institute of Research and Development for Optoelectronics; Rumania. Romanian Academy. Institute of Cellular Biology and Pathology Nicolae Simionescu; Rumani