From lab coats to hard hats: Implementation of GMP continuous manufacturing on the road to commercial readiness

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An ultra-scale-down method to predict diafiltration performance during formulation of concentrated mAb solutions

Formulation of monoclonal antibody (mAb) solutions using membrane filtration processing is a critical unit operation in the preparation of antibody therapies. A key constraint in formulation process development, particularly in the early stages of development and when using high protein concentration solutions, is the availability of material for experimental studies. Ultra-scale down (USD) technologies use a combination of critical flow regime analysis, bioprocess modelling and experimentation at the milliliter scale to enable a more effective process development approach significantly reducing process material, cost and time requirements (Rayat et al, 2016). The ability to predict the performance of large-scale (LS) operations, e.g. flux profile characteristics and changes in protein structure, will help maximize the value of eventual high cost pilot-scale runs during process development. In this study a USD membrane device, comprising a sheared cell unit with a rotating disc and with an effective membrane area of 0.00021 m2 developed at University College London, is used to predict the performance of a LS cross-flow membrane cassette of area 0.11 m2. The USD set up was designed to mimic the LS in terms of processing volumes, membrane area and process times. Computational Fluid Dynamics (CFD) is implemented to characterize average shear rates as a function of suspension viscosities and disc speed of the USD membrane device. A series of trials at USD scale established the effect of average shear rate on flux and the rate of flux decline during a diafiltration operation reaching 7 diafiltration volumes. A series of LS runs were carried out at different cross flow rates covering a similar range of average shear rates as the USD trials. Good correlation was obtained between USD and LS performance using constant average shear rate over the membrane surface as the basis for scale translation between the two scales of operation. The predicted effect of change in shear rate on flux in USD matched that found in LS. This scale correlation on performance was additionally verified by studying the effect of type and concentration of mAb. The comparable process performance was achieved at USD with 520-fold reduction in effective membrane area, required process material and diafiltration buffer for the trial. Future studies will include membrane concentration operations and evaluating sensitivity to stress-related effects and the impact of operation at higher protein concentrations. Rayat, A.CME; Chatel, A; Hoare, M; Lye, G.J (2016). Ultra scale-down approaches to enhance the creation of bioprocesses at scale: impacts of process shear stress and early recovery stages. Current Opinion in Chemical Engineering 14:150-15

Design of a GMP-ready single-pass tangential flow filtration (SPTFF) and inline diafiltration (LILDF) system for continuous manufacturing operations

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Highly automated bioburden-free continuous manufacturing biologics GMP operations: How to get there?

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Ultra scale-down technologies for membrane processing using high monoclonal antibody concentrates

The availability of material is a key constraint in the development of full-scale bioprocesses. Using a combination of critical flow regime analysis, modelling and experimentation, ultra scale-down (USD) methods can yield bioprocess information using only millilitre quantities. A USD device comprising a stirred cell with 1.7 mL capacity is presented. This device resulted from a redesign of an existing device using computational fluid dynamics. The distance between the edge of the rotating disc and membrane surface is critical to reduce the presence of high shear regions. In addition, areas identified as being of low shear, ~ 40% of the membrane area, were blanked. The resulting design has an effective membrane area of 2.1 cm² with a rotating disc mounted 2.0 mm from the membrane surface leading to an approximately uniform shear rate (+/- 17% of average) in a defined layer above the membrane surface. The performance of the USD device was compared with tangential flow filtration (TFF) systems of the same ratio of feed volume to membrane area. The pilot-scale TFF system was characterised in terms of the shear rate using flow rate-pressure drop relationships for the cassette. It operates with ~500-fold larger feed volume and membrane area than the USD system. For a monoclonal antibody diafiltration stage, good agreement was attained in the performance between the two scales at equivalent average shear rates. This resulted in a methodology to predict flux using the USD device as a function of transmembrane pressure, concentration, flow conditions, and volume concentration factors. These predictions are compared to measured flux at pilot-scale. One difference between the systems is the processed sample turbidity. This is possibly due to high shear zones in the presence of membrane and metal surfaces. Mechanisms are described to help define the effect on the process material. A preliminary study of the USD methodology for the processing of nanobodies is used to explore the wider application of the thesis findings

Multistate coupled quantum dynamics of photoexcited cytosine in gas-phase: Nonadiabatic absorption spectrum and ultrafast internal conversions

International audienceQuantum dynamics simulations, with the Multiconfigurational Time Dependent Hartree method, are used to assign the very broad absorption spectrum of Cytosine in gas phase and study the relation between spectral features and the ultrafast internal conversions among its excited states. For each of the four populated tautomers of Cytosine we built a Linear Vibronic Coupling model, comprising all the low energy excited states up to ∼6.5 eV (7–9 states). We used Density Functional Theory and a general diabatization scheme based on the projection of the excited states on a set of reference ones. Vibronic progressions and inter-state couplings dominate the spectral shape, which is in nice agreement with experiment. Inter-state couplings contribute to the loss of vibronic resolution and to the spread of the absorption intensity along a large energy range. Their importance is different for each tautomer and is connected to the different decay times of the bright states

Solvent effect on the energetics of proton coupled electron transfer in guanine-cytosine pair in chloroform by mixed explicit and implicit solvation models

International audienceThe Watson-Crick hydrogen bonded pair formed by deoxyguanosine and deoxycytidine (GC) in chloroform has been analysed by classical Molecular Dynamics simulations, which shows the existence of several fairly stable solute/solvent hydrogen bonds. Time-Dependent Density Functional Theory (TD-DFT) calculations with M052X functional, including solvation effect by a mixed continuum/discrete model, show that solvent stabilizes G → C Charge Transfer excited states (CTGC). The Proton Transfer (PT) processes that can occur in CTGC have been mapped by TD-DFT combining State Specific and Linear Response implementations of the Polarizable Continuum Model. For the first time, the effect of explicit solute/solvent interactions on the PT is considered by studying models containing up to three CHCl3 molecules. Our study shows that PT from the N1 atom of G to the N3 of C is exoergonic also in solution but, at variance with what observed in gas-phase, a stable minimum is predicted for CTGC state in chloroform

The database of the PREDICTS (Projecting Responses of Ecological Diversity In Changing Terrestrial Systems) project

The PREDICTS project-Projecting Responses of Ecological Diversity In Changing Terrestrial Systems (www.predicts.org.uk)-has collated from published studies a large, reasonably representative database of comparable samples of biodiversity from multiple sites that differ in the nature or intensity of human impacts relating to land use. We have used this evidence base to develop global and regional statistical models of how local biodiversity responds to these measures. We describe and make freely available this 2016 release of the database, containing more than 3.2 million records sampled at over 26,000 locations and representing over 47,000 species. We outline how the database can help in answering a range of questions in ecology and conservation biology. To our knowledge, this is the largest and most geographically and taxonomically representative database of spatial comparisons of biodiversity that has been collated to date; it will be useful to researchers and international efforts wishing to model and understand the global status of biodiversity