Development of functionally closed downstream operations for continuous biomanufacturing of recombinant therapeutic proteins

Traditionally, upstream processing in biologics manufacturing is performed in an aseptic and functionally closed manner while downstream processes are open operations designed to maintain a low-bioburden state. Consequently, the downstream process components such as skids, column hardware, and chromatography resins currently available have been designed for low bioburden rather than aseptic operations. One of the key design elements of Integrated Continuous Biomanufacturing (ICB) is the linkage of upstream and downstream operations and the capability of the system to run for prolonged durations at ambient temperature. Thus, a critical design criterion for ICB is the ability of the system to maintain a bioburden-free state. This study demonstrates the development of novel tools and methodologies to address the need for at-scale bioburden-free downstream operations for ICB. Both engineering and operational solutions were identified to achieve bioburden-free integration of a bioreactor with a periodic counter-current (PCC) skid. The engineering/operational solutions included the construction of a SIPable and functionally- closed manufacturing scale PCC skid, disposable and functionally-closed chromatography columns coupled with an innovative strategy to render them bioburden-free, as well as the use of tubing welders. To this end, we have successfully demonstrated at-scale operation for integrated capture of both a stable (monoclonal antibody) and an unstable molecule (recombinant enzyme) for over 40 days each. Samples from different points of the system showed that the operation was bioburden-free for the duration of the campaign. Additionally, no indication of time based performance decline was observed for either the mAb or enzyme capture step. Overall, our findings demonstrate successful scale-up of a functionally-closed, integrated and continuous capture step operated for weeks in a bioburden-free state. Such principles and solutions can be further extended to subsequent downstream unit operations, enabling the development of an end-to-end biomanufacturing platform. Environmental control requirements for a functionally closed end-to-end cGMP facility can potentially result in dramatic reductions for both OPEX and CAPEX

Behaviour of Quality Protein Maize (QPM) genotypes under well irrigated and water stress conditions in subtropical climate

Drought or water stress is one of the prime problems affecting production of maize at global level. A major objective of QPM breeding programs in semi arid tropics or subtropical climatic conditions is to increase genetic potential of QPM genotypes under water stress conditions. In order to identify drought tolerant single cross QPM hybrids an experiment with 85 genotypes was conducted under well irrigated and water stress conditions. Six drought tolerance indices viz, mean productivity (MP), geometric mean productivity (GMP), yield index (YI), tolerance index (TOL), stress susceptibility index (SSI), and superiority measures (SM) were used on the basis of grain yield in water stress (Ys) and well irrigated (Yp) conditions. Highest significant positive correlations were observed among MP, GMP and YI indices. The hybrids 75, 38, 27, and 50 were more drought tolerant based on drought tolerance indices. Three dimensional plot, bi-plot and cluster analysis confirmed these results. Principal component analysis reduced six indices down to two components with 90.71% proportional cumulative variance. Genotypes were grouped by two ways cluster analysis (using Ward’s method) based on Yp, Ys and drought tolerance indices. Also, the results of correlation, 3D graphs, bi-plot and cluster analysis reveals that the most suitable indices to screen QPM genotypes in drought stress conditions were MP, GMP and YI. These indices could be used in QPM breeding programs to introduce drought tolerance in single cross hybrids

Fluctuations of water near extended hydrophobic and hydrophilic surfaces

We use molecular dynamics simulations of the SPC-E model of liquid water to derive probability distributions for water density fluctuations in probe volumes of different shapes and sizes, both in the bulk as well as near hydrophobic and hydrophilic surfaces. To obtain our results, we introduce a biased sampling of coarse-grained densities, which in turn biases the actual solvent density. The technique is easily combined with molecular dynamics integration algorithms. Our principal result is that the probability for density fluctuations of water near a hydrophobic surface, with or without surface-water attractions, is akin to density fluctuations at the water-vapor interface. Specifically, the probability of density depletion near the surface is significantly larger than that in bulk. In contrast, we find that the statistics of water density fluctuations near a model hydrophilic surface are similar to that in the bulk

Improving Thermal Conduction Across Cathode/Electrolyte Interfaces in Solid-State Lithium-Ion Batteries by Hierarchical Hydrogen-Bond Network

Effective thermal management is an important issue to ensure safety and performance of lithium-ion batteries. Fast heat removal is highly desired but has been obstructed by the high thermal resistance across cathode/electrolyte interface. In this study, self-assembled monolayers (SAMs) are used as the vibrational mediator to tune interfacial thermal conductance between an electrode, lithium cobalt oxide (LCO), and a solid state electrolyte, polyethylene oxide (PEO). Embedded at the LCO/PEO interface, SAMs are specially designed to form hierarchical hydrogen-bond (H-bond) network with PEO. Molecular dynamics simulations demonstrate that all SAM-decorated interfaces show enhanced thermal conductance and dominated by H-bonds types. The incorporation of poly(acrylic acid) (PAA) SAM drastically enhances interfacial thermal conductance by approximately 211.69%, largely due to the formation of a strong H-bond, -COOH···:O, between PAA and PEO. Even with weaker H-bonds such as -OH···:O, it still outperforms the pristine interface as well as interfaces decorated with non-H-bonded SAMs, e.g. PE. Such improvement is attributed to the unique hierarchical H-bond network at the interface, which removes discontinuities in temperature field, straighten SAM chains, make materials strongly adhere, and couple the vibrational modes of materials. The study is expected to guide surface engineering for more effective thermal management in lithium-ion batteries

Instantaneous Liquid Interfaces

We describe and illustrate a simple procedure for identifying a liquid interface from atomic coordinates. In particular, a coarse grained density field is constructed, and the interface is defined as a constant density surface for this coarse grained field. In applications to a molecular dynamics simulation of liquid water, it is shown that this procedure provides instructive and useful pictures of liquid-vapor interfaces and of liquid-protein interfaces.Comment: 15 pages, 4 figure

Molecular dynamics simulations of the growth of poly(chloro-para-xylylene) films

Parylene C, poly(chloro-para-xylylene) is the most widely used member of the parylene family due to its excellent chemical and physical properties. In this work we analyzed the formation of the parylene C film using molecular mechanics and molecular dynamics methods. A five unit chain is necessary to create a stable hydrophobic cluster and to adhere to a covered surface. Two scenarios were deemed to take place. The obtained results are consistent with a polymer film scaling growth mechanism and contribute to the description of the dynamic growth of the parylene C polymer

Sitting at the edge: How biomolecules use hydrophobicity to tune their interactions and function

Water near hydrophobic surfaces is like that at a liquid-vapor interface, where fluctuations in water density are substantially enhanced compared to that in bulk water. Here we use molecular simulations with specialized sampling techniques to show that water density fluctuations are similarly enhanced, even near hydrophobic surfaces of complex biomolecules, situating them at the edge of a dewetting transition. Consequently, water near these surfaces is sensitive to subtle changes in surface conformation, topology, and chemistry, any of which can tip the balance towards or away from the wet state, and thus significantly alter biomolecular interactions and function. Our work also resolves the long-standing puzzle of why some biological surfaces dewet and other seemingly similar surfaces do not.Comment: 12 pages, 4 figure

Disaccharide topology induces slow down in local water dynamics

Molecular level insight into water structure and structural dynamics near proteins, lipids and nucleic acids is critical to the quantitative understanding of many biophysical processes. Un- fortunately, understanding hydration and hydration dynamics around such large molecules is challenging because of the necessity of deconvoluting the effects of topography and chemical heterogeneity. Here we study, via classical all atom simulation, water structure and structural dynamics around two biologically relevant solutes large enough to have significant chemical and topological heterogeneity but small enough to be computationally tractable: the disaccharides Kojibiose and Trehalose. We find both molecules to be strongly amphiphilic (as quantified from normalized local density fluctuations) and to induce nonuniform local slowdown in water translational and rotational motion. Detailed analysis of the rotational slowdown shows that while the rotational mechanism is similar to that previously identified in other aqueous systems by Laage, Hynes and coworkers, two novel characteristics are observed: broadening of the transition state during hydrogen bond exchange (water rotation) and a subpopulation of water for which rotation is slowed because of hindered access of the new accepting water molecule to the transition state. Both of these characteristics are expected to be generic features of water rotation around larger biomolecules and, taken together, emphasize the difficulty in transferring insight into water rotation around small molecules to much larger amphiphilic solutes.This work is part of the research program of the “Stichting voor Fundamenteel Onderzoek der Materie (FOM)” which is financially supported by the “Nederlandse organisatie voor Wetenschap- pelijk Onderzoek (NWO)”. Further financial support was provided by a Marie Curie Incoming International Fellowship (RKC). We gratefully acknowledge SARA, the Dutch center for high- performance computing, for computational time and Huib Bakker and Daan Frenkel for useful critical reviews on an earlier version of this work. We thank two anonymous reviewers for their excellent work, especially for bringing to our attention calculations done on the transition state geometry of dimers and the overstructuring of the O-O radial distribution function of SPC/E water