Implications of feeds and supplements on the productivity and quality of recombinant proteins produced in CHO cells

Chinese Hamster Ovary (CHO) cells have become the preferred host for recombinant protein production due to their ability to secrete desired proteins with post-translational modifications similar to those observed in humans. Biopharmaceutical companies routinely utilize supplements and feed systems for maximizing the yield of recombinant proteins in processes involving CHO cells. Although high productivity is desirable for monoclonal antibodies (mAbs) production, utilizing supplements and feeds might affect the quality of recombinant proteins. Clinical efficacy and safety of recombinant proteins is dependent on key quality attributes including glycosylation pattern, aggregates, charge variants and low molecular weight species. Our work outlines the comparison of productivity and quality of recombinant proteins produced in fed-batch CHO-cell based processes using commercially available chemically defined (CD) medium for CHO cells, CD feeds and a variety of complex supplements and feed systems. The CD medium, feeds and supplements were tested in three CHO cell lines, each expressing a different IgG molecule. Each cell line was sequentially adapted to all the different CD medium being evaluated. Multiple fed-batch experiments were performed in shake-flasks with combinations of different commercially available supplements and feeds. At regular intervals, samples were assessed for viable cell density, cell viability, nutrient metabolism and IgG titers for the different conditions. The key IgG quality attributes including glycosylation pattern, aggregates, charge variants, low molecular weight species were also evaluated for the samples obtained at the end of the fed-batch process. The medium/feed combinations which demonstrated high protein productivity and high cell viability at the end of the culture process were further tested in bioreactors to evaluate scalability of the medium/feed combinations in the different cell lines. The use of CD medium with either CD feed or complex feed supplements resulted in higher cell viability at the end of the fed-batch process in addition to higher IgG titers. After evaluation of the product quality, the desired glycosylation pattern was obtained in certain combinations of medium, supplements and feeds. Lower amount of IgG aggregation was also observed. Due to the unique nutritional requirements of each cell line, different combinations of medium/supplement/feed were needed for optimal cell growth and productivity without affecting the product quality

New observations and critical assessments of incipient plasticity events and indentation size effect in nanoindentation of ceramic nanocomposites

The ceramic nanocomposites (CNCs) like zirconia toughened alumina (ZTA) ceramics are important futuristic materials for structural and functional applications in advanced strategic systems, structural components, biomedical prostheses and devices. In all structural materials including the ZTA CNCs, the very early stages of plastic deformation i.e., the incipient plasticity events (IPE) are most important to be understood so that the microstructure and mechanical properties can be tuned to suit a given end application. Here we report for the first time the mechanisms of IPE in the nanoindentation experiments conducted at 10-1000 mN loads in the 40 ZTA CNCs. Here 40 ZTA CNC stands for 40 vol% of 3 mol% Yttria partially stabilized zirconia toughened alumina (40ZTA) CNC. The role of load ranges in variations of the IPE related parameters in the 40 ZTA CNCs is also studied. Further, an attempt is made to assess how the amount of zirconia content in ZTA CNCs affects the variations of the IPE related parameters. Through the extensive usage of field emission scanning electron microscopy (FESEM) and theoretical estimations, efforts are also directed to check out the linkage, if any, between the localized shear deformation and/or microcracking with the IPE events that occur in the present CNCs. In addition, a new concept of damage resistance is introduced for the first time in the present work to explain the presence of a strong indentation size effect (ISE) in the 40 ZTA CNCs. Finally, an attempt is also directed to understand how the indentation load (P) controls the relative size of interaction zones of dislocation loops as well as the damage resistance and thereby, engineer the acuteness of the ISE in ZTA CNCs. The implication of these findings in futuristic design of especially the ZTA CNCs for various applications is also discussed

Modelling of nanoindentation behaviour in MgO doped alumina

Here we report the modelling of the nanoindentation behaviour of 1-5 wt% MgO doped alumina ceramics. It is well-known that the nanoindetation technique is highly efficient in digging out the mechanical properties of various materials including ceramics especially, at small length scale. The samples prepared by the pressureless sintering technique are characterized by the x-ray diffraction, field emission scanning electron microscopy and the nanoindentation techniques. The experimental results obtained from the nanoindentation experiments exhibit a unique nanoindentation size effect as well as the occurrences of localized plasticity events. This unique observation is what drives the inspiration for modelling such behaviours. The modelling of this nanoindentation size effect identifies that it could be linked to the load dependent spatial variations in both the dislocation loop interaction zone size and the deformation resistance. The localized plasticity events are found to happen due to the simultaneous contributions from dislocation nucleation, localized shear deformation bands formation and microcracking formations. The critical loads at which localized plasticity events are initiated increase with the amount of MgO. It happens due to the simultaneous enhancements in the relative density, spinel phase, relative amount of fine grains, and decrease in average size of the fine grains. The implications of these observations for microstructural design of structural ceramics like alumina with enhanced contact deformation resistance at the nanoscale are also discussed

Nanoscale plasticity in titania densified alumina ceramics

The present study explores the physics behind the loading rate (dP/dt or P asymptotic to 1 - 1000 mN s(-1)) dependent nanoscale plasticity (NSP) events observed during carefully controlled nanoindentation (NI) experiments on 1, 3, and 5 wt. % Titania Densified Alumina (TDA) ceramics. Characterizations of the TDA ceramics are carried out by x-ray diffraction, field emission scanning electron microscopy (FESEM), and NI techniques. A significant enhancement (similar to 30%) of the nanohardness of TDA ceramics occur with an enhancement in P . The results confirm that both the critical load (P-c) at which micro-pop-in or the NSP events initiate and the corresponding critical depth (h(c)) are sensitive functions of relative density, size of relatively finer grains, loading rate, and the amount of sintering aids. The experimentally observed empirical power law dependence of all the NSP related parameters on P is rationalized theoretically and qualitatively. It is suggested that the shear induced homogeneous dislocation nucleation underneath the nanoindenter may be the main factor contributing to the occurrence of the NSP events at relatively lower loading rates. However, especially at the relatively higher loading rates, the FESEM based evidence and the data obtained from the related NI experiments suggest that there is a more acute interconnection between the homogeneous dislocation nucleation induced profuse occurrence of the NSP events, shear band formations, and microcrack formation in the TDA ceramics. Finally, the design implications of the present results for the development of better alumina ceramics for load and strain tolerant applications are discussed.& nbsp;Published under an exclusive license by AIP Publishing

Nano to micrometer range particle size effect on the electrical and piezoelectric energy harvesting performances of hydroxide mediated crosslinked PVDF composites

The polar phase and piezoelectric response of filler-loaded PVDF-based composites very often depend on the interfacial interaction between the filler surface and PVDF dipoles. In this regard, hydrogen bonding interaction has shown a much stronger effect compared to other interactions. In order to induce hydrogen bonding interaction, the filler surface is commonly modified by various modifiers. In the present work, instead of filler surface modification, we introduce ZnSn(OH)6 filler (hydroxide filler having a high number of -OH groups) into the PVDF matrix in order to facilitate the hydrogen bonding interaction. Not only the hydroxide fillers but the effect of wide particle size variation (from nano to micrometer range) into PVDF has also been shown here for the very first time. ZnSn(OH)6 fillers with similar morphology but different sizes have been synthesized by using a variety of techniques and then incorporated into the PVDF matrix. The microstructural defects of the composite films have been found to be gradually increased with the increase in filler size which in turn caused to gradually increase their space charge polarization. Filler, with 915 nm size has shown the best polar phase formation (-84 %), dielectric permittivity (-10 at 1 kHz), and piezoelectric energy harvesting performance (output voltage -20 V) of the resulting PVDF-based composite and hence has been used for various real-life applications. All of these results have been suitably explained here on the basis of interfacial interaction, microstructural defect, and the mechanism of formation of space charge polarization

Air-plasma discharged PVDF based binary magnetoelectric composite for simultaneously enhanced energy storage and conversion efficiency

Different nanomaterials and their modified forms are very often added into a poly(vinylidene fluoride) (PVDF) matrix in order to improve the energy storage and conversion efficiency of the system. The improvement in energy storage density caused by this secondary nanomaterial addition is most often found to be accompanied by the reduction in energy storage efficiency due to increased amounts of space charges. Here, we show that both the capacitive energy storage density and efficiency can be simultaneously improved by air-plasma discharging on the PVDF based composite system. The energy storage density and efficiency of a 5 wt. % BiFeO3 loaded PVDF film (5BF) have been found to be increased to similar to 1.55 J/cm(3) and similar to 73%, respectively, from the values of similar to 1.36 J/cm(3) and 59% after air-plasma discharging. The dipole rotation caused by air-plasma discharging also helped in improving the mechanical to electrical energy conversion efficiency and magnetoelectric coupling of the studied composite system. Upon similar periodic applied stress, the pristine and air-plasma discharged 5BF film showed similar to 3 and 9.6 mu W/cm(2) of output electrical power density with similar to 13.5 and 19.2 V of open circuit output voltage, respectively. The air-plasma discharged 5BF film (5BFD) has also shown an excellent magnetoelectric coupling coefficient (alpha(33)) of similar to 35 mV cm(-1) Oe(-1) at 1 kHz frequency of fixed AC magnetic field (similar to 3 Oe) and 4 kOe of DC bias field. The simultaneous improvement of all of these parameters of the studied composite system caused by air-plasma discharging proves its multifunctional applicability in a variety of real life applications

Novel layered GO/Mg(OH)(2) nanocomposites for detection of Cd and Pb ions

Here we report the efficacy of the photoluminescence (PL) spectroscopy for novel detection of as low as 0.0001 to as high as 0.01 ppm of Cd2+ and Pb2+ ions in simulated wastewater solution. The solution comprises of 20 mg of layered graphene oxide (GO)-Mg(OH)(2) nanocomposite (LGOMHNC) powders in 100 ml of DI water. The LGOMHNC powders, synthesized by facile wet chemical route, are characterized by XRD, FESEM, TEM, FTIR, Raman spectroscopy, TGA-DTA, XPS and especially, the PL spectroscopy techniques. After adsorption of Cd2+ and Pb2+ ions, the novel LGOMHNC powders exhibit significant enhancement of the corresponding PL intensities as compared to those of the as-synthesized LGOMHNC powders. These results suggest that PL spectroscopy can indeed emerge as a very important tool for detection of even 0.0001 ppm of Cd2+ and Pb2+ ions in simulated wastewater. In addition, the novel LGOMHNC powders developed in the present work can have huge application potential in futuristic, optical sensor-based detection of the toxic, heavy metal ions like Cd2+ and Pb2+. (C) 2019 Elsevier B.V. All rights reserved

Studies on Multifunctional Properties of SILAR Synthesized CuO Thin Films for Enhanced Supercapacitor, Photocatalytic and Ethanol Sensing Applications

CuO thin films were successfully deposited using a simple, cost-effective and nearly room-temperature successive ionic layer adsorption and reaction routes. The material has been characterized using x-ray diffraction, field-emission scanning electron microscopy and transmission electron microscopy. Increase of grain size and decrease of microstrain were observed for a particular level of dipping (30 cycles) and further increase in dipping cycles shows a reverse tendency. The effect of the dipping cycle of the synthesized films on their supercapacitive, photocatalytic and ethanol-sensing performance were investigated. A 30-cycle dipped CuO thin film-based electrode provides a maximum specific capacitance of 585Fg(-1) at the voltage scan rate of 2 mVs(-1) from cyclic voltammetry measurement and 554Fg(-1) at a current density of 1 Ag-1 from the charging to discharging curve. This electrode exhibited long-term cycle stability with 92.3% capacitance retention after 4000 cycles. CuO films synthesized for 30 dipping cycles showed the highest photocatalytic activities with 91.1% degradation of methylene blue under exposure to visible light of 200-W energy in a time duration of 4h. Maximum sensitivity of 67% in the presence of 1500 ppm ethanol at the operating temperature 160 degrees C was obtained for 30 dipping cycles film. Such attractive properties of low cost and facile synthesized CuO thin films makes them a suitable candidate for different commercial applications

A critical note on nanoscale plasticity in 20 ZTA ceramics

The present work reports the very first observations on initiation of nanoscale plastic events in 20 ZTA (Zirconia Toughened Alumina) ceramics. The nanomechanical properties as well as the intrinsic contact deformation resistance of the present ZTA ceramic are studied here as a function of low loads (i.e., 10-1000 mN). Here we report for the very first time, the detailed mechanisms on the genesis of `micro pop-in' events that characterize the nanoscale plasticity initiation in the 20 ZTA ceramics. These new results along with field emission scanning electron microscopy (FESEM) based evidences confirm that the combined contributions from the maximum shear stress generated underneath the nanoindenter, the formations of shear bands and localized microcracking play significant roles in the initiation of nanoscale plastic events in the 20 ZTA ceramics

Indentation size effect and energy balance issues in nanomechanical behavior of ZTA ceramics

It is well known that physical, structural, mechanical as well as other functional properties change drastically at the nanoscale, thereby giving rise to size effects in all materials, especially ceramics. Therefore, it is extremely relevant today to understand the basic scientific issues involved in the development of size effects in materials, particularly ceramics which are characteristically brittle in nature. Hence, to be able to design better contact resistant ceramics; it is of significant importance to understand the genesis of indentation size effect (ISE) in the nanomechanical response of structural ceramics like zirconia toughened alumina (ZTA). Here we report the first ever systematic study on ISE in nanoindentation behavior of 10, 20 and 40 volume% (vol%) ZTA. The nanoindentation experiments were conducted at an ultra low load range of 1-1000 mN. As the experimental data showed the presence of strong ISE, the efficacies of existing models in explaining the same; were critically examined. Among existing models, the strain gradient plasticity model provided the real physical reason for the genesis of ISE in ZTA and hence, explained the data the best. Similarly, existing models were used to predict the variations in experimentally measured ratios of plastic to total energy spent in the nanoindentation process. The results showed that the Malzbender model predicted experimental data the best. This observation implied the best efficacy of the internally expanded cavity concept in explaining the nanoindentation response of the present ZTA ceramics. In addition, the other possible mechanisms of ISE in ZTA were discussed. Finally, the linkage of microstructural parameters to ISE in ZTA was explored