An integrated and continuous downstream process for microbial virus-like particle vaccine biomanufacture

In this study, we present the first integrated and continuous downstream process for the production of microbial virus‐like particle vaccines. Modular murine polyomavirus major capsid VP1 with integrated J8 antigen was used as a model virus‐like particle vaccine. The integrated continuous downstream process starts with crude cell lysate and consists of a flow‐through chromatography step followed by periodic counter‐current chromatography (PCC) (bind‐elute) using salt‐tolerant mixed‐mode resin and subsequent in‐line assembly. The automated process showed a robust behavior over different inlet feed concentrations ranging from 1.0 to 3.2 mg ml−1 with only minimal adjustments needed, and produced continuously high‐quality virus‐like particles, free of nucleic acids, with constant purity over extended periods of time. The average size remained constant between 44.8 ± 2.3 and 47.2 ± 2.9 nm comparable to literature. The process had an overall product recovery of 88.6% and a process productivity up to 2.56 mg h−1 mlresin−1 in the PCC step, depending on the inlet concentration. Integrating a flow through step with a subsequent PCC step allowed streamlined processing, showing a possible continuous pathway for a wide range of products of interest.Lukas Gerstweiler, Jagan Billakanti, Jingxiu Bi, Anton P. J. Middelber

Nanoparticle elasticity regulates phagocytosis and cancer cell uptake

The ability of cells to sense external mechanical cues is essential for their adaptation to the surrounding microenvironment. However, how nanoparticle mechanical properties affect cell-nanoparticle interactions remains largely unknown. Here, we synthesized a library of silica nanocapsules (SNCs) with a wide range of elasticity (Young’s modulus ranging from 560 kPa to 1.18 GPa), demonstrating the impact of SNC elasticity on SNC interactions with cells. Transmission electron microscopy revealed that the stiff SNCs remained spherical during cellular uptake. The soft SNCs, however, were deformed by forces originating from the specific ligand-receptor interaction and membrane wrapping, which reduced their cellular binding and endocytosis rate. This work demonstrates the crucial role of the elasticity of nanoparticles in modulating their macrophage uptake and receptor-mediated cancer cell uptake, which may shed light on the design of drug delivery vectors with higher efficiency.Yue Hui1, Xin Yi2, David Wibowo1*, Guangze Yang1, Anton P. J. Middelberg3, Huajian Gao4,5†, Chun-Xia Zha

Chimeric virus-like particles and capsomeres induce similar CD8+ T cell responses but differ in capacity to induce CD4+ T cell responses and antibody responses

Despite extensive research, the development of an effective malaria vaccine remains elusive. The induction of robust and sustained T cell and antibody response by vaccination is an urgent unmet need. Chimeric virus-like particles (VLPs) are a promising vaccine platform. VLPs are composed of multiple subunit capsomeres which can be rapidly produced in a cost-effective manner, but the ability of capsomeres to induce antigen-specific cellular immune responses has not been thoroughly investigated. Accordingly, we have compared chimeric VLPs and their sub-unit capsomeres for capacity to induce CD8+ and CD4+ T cell and antibody responses. We produced chimeric murine polyomavirus VLPs and capsomeres each incorporating defined CD8+ T cell, CD4+ T cell or B cell repeat epitopes derived from Plasmodium yoelii CSP. VLPs and capsomeres were evaluated using both homologous or heterologous DNA prime/boost immunization regimens for T cell and antibody immunogenicity. Chimeric VLP and capsomere vaccine platforms induced robust CD8+ T cell responses at similar levels which was enhanced by a heterologous DNA prime. The capsomere platform was, however, more efficient at inducing CD4+ T cell responses and less efficient at inducing antigen-specific antibody responses. Our data suggest that capsomeres, which have significant manufacturing advantages over VLPs, should be considered for diseases where a T cell response is the desired outcome.David J. Pattinson, Simon H. Apte, Nani Wibowo, Tania Rivera-Hernandez, Penny L. Groves, Anton P. J. Middelberg, and Denise L. Doola

Engineering materials from the bottom up - Overview

Self-assembly is a ubiquitous phenomenon in nature. Proteins, polynucleotides, polysaccharides, and lipids may assemble themselves to form multitudes of structures from the molecular level up to the macroscopic world of everyday experience. This phenomenon presents materials with versatile functionality and highly specialized, sometimes unique performance. In particular, proteins or peptides, informational polymers composed of permutations of a set of 20+ natural (and synthetic) amino acids, offer an attractive engineering challenge to design tailored structures and materials with novel properties and functionalities. Nature, through an evolutionary process, has demonstrated the versatility and multiplicity of applications to be addressed by peptides and proteins. An historical overview of materials research sets the scene for a series of papers that reflect upon the current challenges and successes of research in the field of self-assembling peptides and proteins. It leads the reader from understanding the underlying physics of self-assembly; both from a theoretical as well as an experimental perspective to the challenges of making large quantities of peptides that enable industrial exploration and commercialization in a variety of applications. To date, the self-assembly of peptide and proteins patent literature indicates a major application focus on the medical and pharmaceutical market. However, over the last few years, the general literature on peptide and protein self-assembly research shows a rapidly growing opportunity for advanced material engineering in many other areas

The effect of homogeniser impact distance on the disruption of Escherichia coli

The effect of homogeniser impact distance on the disruption efficiency of stationary and growth-phase Escherichia coli is examined. Disruption efficiency decreases as impact distance increases as seen previously for yeasts. However, the dependence on impact distance is considerably less than previously reported for yeast. Results suggest that homogeniser performance can be improved by decreasing impact distance. This effect is modelled.Andrew R. Kleinig, Brian K. O'Neill, Anton P. J. Middelber

Rapid screening of surfactant and biosurfactant surface cleaning performance

Surface Plasmon Resonance (SPR) and rubisco protein stain were used as tools to screen the effectiveness of detergent formulations in cleaning a protein stain from solid surfaces. Surfactant and biosurfactant-based formulations, with and without added protease, were screened for cleaning performance. Enzyme-free detergent formulations at 1500 ppm total surfactant were insufficient to cause complete surface cleaning, despite the high concentration of surfactant. The cleaning performance of a "home-made" formulation containing 2 ppm subtilisin A (SA) and 2 ppm sodium dodecyl benzyl sulphonate (SDOBS) was as efficient as the best amongst the three enzyme-free 1500 ppm formulations. The cleaning performance of 2 ppm SA in the absence of SDOBS was less effective than the combined formulation, even though 2 ppm SDOBS alone did not cause any protein removal. The observed synergistic performance was attributed to the cooperative mechanisms (chemical and physical attack) by which these two agents act on a rubisco stain. Replacing SDOBS in the enzyme-surfactant formulation with the same amount of surfactin biosurfactant (2 ppm) gave the best rubisco removal of all formulations examined in this study, irrespective of the surface chemistry underlying the protein film. It was found that 75% and 80% of immobilised rubisco stain could be removed from hydrophobic and hydrophilic surfaces, respectively, by the biosurfactant-SA formulation (compared with 60% and 65%, respectively, using the SDOBS-SA formulation). Our results suggest that it may be possible to generate fully renewable biochemical-based cleaning formulations that have superior cleaning performance to existing technologies. In developing optimised formulations, there is a pressing need for chip-based tools similar to that developed in this research

Continuous downstream bioprocessing for intensified manufacture of biopharmaceuticals and antibodies

Continuous production delivers higher productivity and better quality than traditional batch-wise approaches. It intensifies production lowering capital cost and enables better control. Despite obvious advantages, continuous processing has not yet guided biomanufacturing to 21st Century Advanced Manufacturing status. Production relies primarily on batch-wise methods that have served the industry through its infancy. While great improvements have been achieved on the upstream side, downstream processing lacks development in continuous processing and is now the handbrake on modernisation. Nevertheless, there are hopeful advances. Research on continuous chromatographic purification for antibodies is maturing, and work has commenced on other unit operations and on process system integration. This exciting field of process intensification research is at a turning point, though considerably more research is needed. This review aims to summarize the latest developments and capabilities of continuous downstream processing applied in biopharmaceutical research and gives an overview of recent developments.Lukas Gerstweiler, Jingxiu Bi, Anton P.J. Middelber

Control strategy for multi-column continuous periodic counter current chromatography subject to fluctuating inlet stream concentration

Fluctuations of the inlet feed stream concentration are a challenge in controlling continuous multicolumn counter current chromatography systems with standard methods. We propose a new control strategy based on calculated product column breakthrough from UV sensor signals by neglecting an impurity baseline and instead using the impurity to product ratio. This calculation is independent of the inlet feed concentration. In-silico simulation showed that the proposed method can calculate the product column breakthrough perfectly even with fluctuating and highly unstable inlet feed concentration during a loading cycle. Applying the proposed method to control a three column periodic counter current chromatography process with fluctuating inlet feed concentration resulted in constant column loading in each cycle, while using the standard method failed to do so. Unavoidable band broadening caused by diffusion and dispersion has been identified as an inherent limiting factor for accurate calculation of column breakthrough comparing inlet and outlet UV signals. The proposed advanced calculations increase the robustness of periodic counter current chromatography and extend the capability to process unstable inlet streams.Lukas Gerstweiler, Jagan Billakanti, Jingxiu Bi, Anton P.J. Middelber

Affinity purification of viral protein having heterogeneous quaternary structure: Modeling the impact of soluble aggregates on chromatographic performance

Prokaryote-expressed polyomavirus structural protein VP1 with an N-terminal glutathione-S-transferase tag (GST-VP1) self-assembles into pentamer structures that further organize into soluble aggregates of variable size (3.4 x 10(2)-1.8 x 10(4) kDa) [D.I. Lipin, L.H.L. Lua, A.P.J. Middelberg, J. Chromatogr. A 1190 (2008) 204]. The adsorption mechanism for the full range of GST-VPI soluble aggregates was described assuming a dual-component model [T.Y. Gu, G.J. Tsai, G.T. Tsao, AICHE J. 37 (1991) 1333], with components differentiated by size, and hence pore accessibility, rather than by protein identity. GST-VPI protein was separated into two component groups: aggregates small enough to access resin pores (LMW: 3.4. 10(2)-1.4 x 10(3) kDa) and aggregates excluded from the resin pores (HMW: 9.0 x 10(2)-1.8 x 10(4) kDa). LMW aggregates bound to resin at a higher saturation concentration (29.7 g L(-1)) than HMW aggregates (13.3 g L-1), while the rate of adsorption of HMW aggregates was an order of magnitude higher than for LMW aggregates. The model was used to predict both batch and packed bed adsorption of GST-VPI protein in solutions with known concentrations of HMW and LMW aggregates to Glutathione Sepharose HP resin. Asymmetrical flow field flow fractionation with UV absorbance was utilized in conjunction with adsorption experimentation to show that binding of HMW aggregates to the resin was strong enough to withstand model-predicted displacement by LMW aggregates. High pore concentrations of LMW aggregates were also found to significantly inhibit the diffusion rate of further protein in the resin pores. Additional downstream processing experimentation showed that enzymatic cleavage of LMW aggregates to remove GST tags yields more un-aggregated VP1 pentamers than enzymatic cleavage of HMW aggregates. This model can be used to enhance the chromatographic capture of GST-VP1, and suggests an approach for modeling chromatographic purification of proteins that have a range of quaternary structures, including soluble aggregates. (C) 2009 Elsevier B.V. All rights reserved