Rapid process monitoring & control in mammalian cell culture using off-gas mass spectrometry analysis

In a concerted effort to investigate the implementation of a process analytical technology (PAT), we have evaluated the applicability of off-gas mass spectrometry (MS) analysis in mammalian cell culture based on the inlet and outlet gases from bioreactors. The limitations of the system & potential applications are also discussed. Compared to other invasive methods that require physical probes inserted directly into the culture (e.g. bio-capacitance probe, Raman spectrometry probe, etc.), the MS only requires gas going in and out of the cell culture which means it has minimal impact on the culture itself. With the Prima BT magnetic sector MS (Thermo Fisher Scientific), multiple gas streams can be analyzed (i.e. multiple bioreactors can be connected to the same Prima BT MS) which could reduce analytical equipment footprint (c.f. other equipment that may require 1 equipment per bioreactor). Distance between bioreactors and the MS is also not an issue given a high enough gas flow and a long enough gas tubing connection to the MS, i.e. the MS can be placed a distance away or even a different site from where all the bioreactors are situated. Our data showed that small changes in the gas traces (O2, CO2, N2 & Ar) on the MS, down to 0.001% mol, can be captured during the following events: 1) feed media addition (containing surfactant) 2) antifoam addition 3) any gas perturbation caused by external factors (non-culture related; gas supply leak or loss) 4) metabolic changes in the cell culture The MS data from two replicate 5L benchtop glass bioreactor runs are very comparable, as is the offline data between them; this indicates that the MS data can be used reliably to identify process deviations during bioreactor runs and be used as a convenient way to evaluate batch to batch variation, within predefined specifications, for robust manufacturing. We have also demonstrated that the same MS setup in the benchtop experiments, with minor modifications, can be applied in a 50L single-use bioreactor (SUB). We would also report that there are technical considerations with respect to the feasibility of integrating this system to the various cell culture platforms. The data so far suggests that the off-gas MS analysis can be: 1) incorporated as a feedback control to determine feed addition rates; 2) used to correlate the growth and perhaps viability of the culture with the MS gas traces; 3) identify cell culture contamination; 4) used to correlate cellular metabolic activity in the culture with the MS gas traces from the O2 & CO2; 5) used to elucidate more detailed metabolic states by analyzing volatile organic carbons (VOCs). In conclusion, we believe that our work presented here will be of significant relevance to the cell culture community who are keen adopters of PAT and its practical implementation

Applications of off-gas mass spectrometry in fed-batch mammalian cell culture

Off-gas analysis using a magnetic sector mass spectrometer was performed in mammalian cell cultures in the fed-batch mode at the 5 L bench and 50 L pilot scales. Factors affecting the MS gas traces were identified during the duration of the fed-batch cultures. Correlation between viable cell concentration (VCC) and oxygen concentration of the inlet gas into the bioreactor (O2-in) resulted in R2 ≈ 0.9; O2-in could be used as a proxy for VCC. Oxygen mass transfer (kLa) was also quantified throughout the culture period with antifoam addition at different time points which is shown to lower the kLa. Real-time specific oxygen consumption rate (qO2) of 2–20 pmol/cell/day throughout the bioreactor runs were within the range of values reported in literature for mammalian cell cultures. We also report, to our knowledge, the first instance of a distinct correlation between respiration quotient (RQ) and the metabolic state of the cell culture with regard to lactate production phase (average RQ > 1) and consumption phase (average RQ < 1)

Histidine‐functionalized diblock copolymer nanoparticles exhibit enhanced adsorption onto planar stainless steel

RAFT aqueous emulsion polymerization of isopropylideneglycerol monomethacrylate (IPGMA) is used to prepare a series of PGEO5MA46-PIPGMAy nanoparticles, where PGEO5MA is a hydrophilic methacrylic steric stabilizer block bearing pendent cis-diol groups. TEM studies confirm a spherical morphology while dynamic light scattering (DLS) analysis indicated that the z-average particle diameter can be adjusted by varying the target degree of polymerization for the core-forming PIPGMA block. Periodate oxidation is used to convert the cis-diol groups on PGEO5MA46-PIPGMA500 and PGEO5MA46-PIPGMA1000 nanoparticles into the analogous aldehyde-functionalized nanoparticles, which are then reacted with histidine via reductive amination. In each case, the extent of functionalization is more than 99% as determined by 1H NMR spectroscopy. Aqueous electrophoresis studies indicate that such derivatization converts initially neutral nanoparticles into zwitterionic nanoparticles with an isoelectric point at pH 7. DLS studies confirmed that such histidine-derivatized nanoparticles remain colloidally stable over a wide pH range. A quartz crystal microbalance is employed at 25°C to assess the adsorption of both the cis-diol- and histidine-functionalized nanoparticles onto planar stainless steel at pH 6. The histidine-bearing nanoparticles adsorb much more strongly than their cis-diol counterparts. For the highest adsorbed amount of 70.5 mg m–2, SEM indicates a fractional surface coverage of 0.23 for the adsorbed nanoparticles

Nurses' perceptions of using an evidence-based care bundle for initial emergency nursing management of patients with severe traumatic brain injury: A qualitative study.

Evidence to guide initial emergency nursing care of patients with severe traumatic brain injury (TBI) in Thailand is currently not available in a useable form. A care bundle was used to summarise an evidence-based approach to the initial emergency nursing management of patients with severe TBI and was implemented in one Thai emergency department. The aim of this study was to describe Thai emergency nurses' perceptions of care bundle use. A descriptive qualitative study was used to describe emergency nurses' perceptions of care bundle use during the implementation phase (Phase-One) and then post-implementation (Phase-Two). Ten emergency nurses participated in Phase-One, while 12 nurses participated in Phase-Two. In Phase-One, there were five important factors identified in relation to use of the care bundle including quality of care, competing priorities, inadequate equipment, agitated patients, and teamwork. In Phase Two, participants perceived that using the care bundle helped them to improve quality of care, increased nurses' knowledge, skills, and confidence. Care bundles are one strategy to increase integration of research evidence into clinical practice and facilitate healthcare providers to deliver optimal patient care in busy environments with limited resources

A laboratory-scale annular continuous flow reactor for UV photochemistry using excimer lamps for discrete wavelength excitation and its use in a wavelength study of a photodecarboxlyative cyclisation

This paper describes a new annular reactor for continuous UV photochemistry, which uses easily interchangeable excimer lamps of different wavelengths. The reactor has narrow clearance to form thin films of material for efficient irradiation of molecules. Its use is demonstrated by investigating the effect of discrete wavelength lamps (222, 282 and 308 nm) on the reaction of potassium N-phthalimidobutanoate 1. The ability of the reactor to be integrated into multistep processes is illustrated by combining it with an Amberlyst scavenger and a solid acid catalyst, NbOPO4, to access a second product 3 that is obtained in a single telescoped process. The tricyclic scaffold in 3 is a motif found in several biologically active compounds and has possibilities as a synthon for new pharmaceutical products

Renal supportive and palliative care: position statement.

Since the introduction of haemodialysis in the management of acute kidney injury in the 1940s and for chronic kidney disease (CKD) in the 1960s dialysis has become one of the most successful advances in medical technology, with almost 11 000 patients currently receiving dialysis in Australia and almost 2500 in New Zealand. Like all medical technologies, its place continues to evolve. For a time, dialysis was seen as a treatment best delivered only to younger patients without diabetes; today the greatest uptake of dialysis is in patients over age 65 and the most common cause of needing dialysis is diabetes. Along with these extended criteria for dialysis, that have evolved over many years, has come the recognition that the older dialysis patient often has considerable co-morbidity and frailty, that time spent on dialysis is not always beneficial to these patients and that their overall prognosis is considerably worse than their younger counterparts. CARI guidelines recommend that ‘an expectation of survival with an acceptable quality of life’ is a useful starting point for recommending dialysis

Application Note 38: α-photodecarboxylation of phthaloyl glycine

This application note illustrates the use of the Vapourtec UV-150 photochemical reactor for the conversion of phthaloyl glycine to N-methylphthalimide. The transformation has been systematically optimized in terms of reagent concentration and residence time. The optimized procedure was subsequently performed on large-scale, achieving a productivity of 1.15 g of product per hour (or 27.6 g per day). The reactor enabled continuous photochemistry in an easy-to-use, safe and efficient manner

A study of the electronic structure of some atoms and small molecules with high temperature photoelectron spectroscopy

This thesis describes the development of an intracavity frequency doubled dye laser from a commercial Spectra-Physics 380D single frequency ring dye laser, and the application of this system to anumber of spectroscopic studies in discharges. Evaluation of the stability of the laser cavity is carried out, by computer calculation, in order to optimise the optical component set used in the frequency doubled dye laser. Using an ADA (ammonium dihydrogen arsenate) crystal, 17 mW of continuous wave ultra-violet, tunable over the range 292-302 am, has been produced. The linewidth, when the laser is stabilised, is about 500 kHz in the UV. The CW UV output has been used to measure helium triplet (23S) metastable densities in a positive column discharge as a function of pressure (1-8 Torr) and current (2.5-80 mA). A maximum density of ioxlo12cm 3 is measured for a pressure of 2 Tort and current of 60mA. The measured metastable densities are used in a rate equation analysis to extract values of the rate coefficient for destruction (by collisions with electrons) of the 23S metastables (1.6x10®s 1 for p - 2 Torr). A study of the optogalvanic signal generated when irradiating a neon positive column discharge with laser light of wavelength 588.2 xc, corresponding to the 1s5 2p2 transition, is carried out. The signal is found to change sign as the dye laser power is increased, for some discharge conditions. Spatial studies of the optogalvanic signal in conjunction with absorption and emission studies are carried out to investigate the evolution of the population densities of levels connected to the 1s5 and 2p2 levels. A rate equation model is developed which demonstrates the importance of the 183 population density in explaining the observed sign reversal. A high resolution study of two transitions in the UV in the tungsten spectrum, is presented. The even isotope splittings and the hyperfine splitting between the two strongest hyperfine components on transitions at 294.4 in and 294.7 cm have been resolved by saturated absorption spectroscopy. From centre of gravity considerations the hyperfine splittings of the three levels involved in the two transitions have been predicted as 1630 MHz for the 7S3 level, -506 MHz for the 3692 level and 225 MHz for the 3683 level. Simple wavemeter using a corner-cube Michelson interferometer is described. Digital counting circuits to provide a direct reading of either the fundamental or second harmonic wavelength have been designed and constructed. Wavelength readings accurate to 1 part in 610 are achieved. The spectrometer, furnace assembly and induction heating system used for the evaporation of samples and the subsequent recording of their photoelectron spectra at temperatures up to 2600°K are described. The ab initioSCF and Bartree-Fock-Slater (HFS) computational schemes used in analysis of spectra are detailed. The photoelectron spectra of the vapours of tin, germanium and gallium are reported. Bands associated with ionisation from the outermost orbital have been observed from the ground as well as low-lying excited states of these elements, and relative band intensities have been used to estimate the excitation temperatures. For gallium evidence is presented to show that the Hel spectrum is perturbed by an autoionisation process. The Hei and Hell photoelectron spectra of ZnF2 and CUP have been recorded and interpreted with the aid of ab iniEio SCF calculations and a configuration interaction method. Results suggest that the 3d orbitals in CuF2 are valence in character, whereas in ZnF2 they are core-like. The Hell spectra of OF, ThF, UC14 and ThCl4 have been recorded and are campared with Eel and X-ray photoelectron spectra. The results show that the electronic structure of U and Th tetrahalides are similar, both in the solid and vapour phases, apart from the presence of two metal 5f electrons in uranium. The Eel spectrum of the first band of the dimethylsilaethylene molecule is reported and analysed by means of ab initio' SCF and BPS methods. Results are consistent with the presence of a C=Si double bond. Results of some unsuccessful but informative high temperature experiments are given. Details of a new multidetector photoelectron spectrometer are also presented.</p

Application note 56: photodecarboxylative addition of phenylacetic acid to N-methyl phthalimide

This application note illustrates the use of the Vapourtec UV-150 photochemical reactor for the photodecarboxylative benzylation of N-methylphthalimide. The transformation has been systematically optimized in terms of reagent concentration and residence time. The optimized procedure was subsequently performed on large-scale, achieving a productivity of 1.34 g (88%) of product per hour (or 32.16 g per day). The reactor enabled continuous photochemistry in an easy-to-use, safe and efficient manner