An ultra scale‐down approach to study the interaction of fermentation,homogenization, and centrifugation for antibody fragment recovery from rec E. coli

Escherichia coli is frequently used as a microbial host to express recombinant proteins but it lacks the ability to secrete proteins into medium. One option for protein release is to use high‐pressure homogenization followed by a centrifugation step to remove cell debris. While this does not give selective release of proteins in the periplasmic space, it does provide a robust process. An ultra scale‐down (USD) approach based on focused acoustics is described to study rec E. coli cell disruption by high‐pressure homogenization for recovery of an antibody fragment (Fab′) and the impact of fermentation harvest time. This approach is followed by microwell‐based USD centrifugation to study the removal of the resultant cell debris. Successful verification of this USD approach is achieved using pilot scale high‐pressure homogenization and pilot scale, continuous flow, disc stack centrifugation comparing performance parameters such as the fraction of Fab′ release, cell debris size distribution and the carryover of cell debris fine particles in the supernatant. The integration of fermentation and primary recovery stages is examined using USD monitoring of different phases of cell growth. Increasing susceptibility of the cells to disruption is observed with time following induction. For a given recovery process this results in a higher fraction of product release and a greater proportion of fine cell debris particles that are difficult to remove by centrifugation. Such observations are confirmed at pilot scale. Biotechnol. Bioeng. 2013 9999:XX–XX. © 2013 Wiley Periodicals, Inc. Biotechnol. Bioeng. 2013; 110: 2150–2160. © 2013 Wiley Periodicals, Inc.     

Effects of energy dissipation rate on islets of Langerhans: Implications for isolation and transplantation

Acute physical stresses can occur in the procurement and isolation process and potentially can contribute to islet death or malfunction upon transplantation. A contractional flow device, previously used to subject suspended cells to well‐defined hydrodynamic forces, has been modified and used to assess the vulnerability of porcine islets of Langerhans to hydrodynamic forces. The flow profiles and velocity gradients in this modified device were modeled using commercial CFD software and characterized, as in previous studies, with the scalar parameter, energy dissipation rate (EDR). Porcine islets were stressed in a single pass at various stress levels (i.e., values of EDR). Membrane integrity, oxygen uptake rate, caspase 3/7 activity, and insulin release were not affected by the levels of fluid stress tested up to an EDR of 2 × 10³ W/m³. Visual observation of the stressed islets suggested that cells at the islet exterior were peeled away at EDR greater than 10,000 W/m³, however, this observation could not be confirmed using image analysis software, which determined the ratio of surface perimeter to total area. The result of this study suggests an upper limit in fluid stress to which islets can be subjected. Such upper limits assist in the design and operation of future islet processing equipment and processes. Biotechnol. Bioeng. 2009;103: 413–423. © 2008 Wiley Periodicals, Inc.     

Use of focused acoustics for cell disruption to provide ultra scale-down insights of microbial homogenization and its bioprocess impact--recovery of antibody fragments from rec E. coli

An ultra scale-down (USD) device that provides insight of how industrial homogenization impacts bioprocess performance is desirable in the biopharmaceutical industry, especially at the early stage of process development where only a small quantity of material is available. In this work, we assess the effectiveness of focused acoustics as the basis of an USD cell disruption method to mimic and study high-pressure, step-wise homogenization of rec Escherichia coli cells for the recovery of an intracellular protein, antibody fragment (Fab'). The release of both Fab' and of overall protein follows first-order reaction kinetics with respect to time of exposure to focused acoustics. The rate constant is directly proportional to applied electrical power input per unit volume. For nearly total protein or Fab' release (>99%), the key physical properties of the disruptate produced by focused acoustics, such as cell debris particle size distribution and apparent viscosity show good agreement with those for homogenates produced by high-pressure homogenization operated to give the same fractional release. The only key difference is observed for partial disruption of cells where focused acoustics yields a disruptate of lower viscosity than homogenization, evidently due to a greater extent of polynucleic acids degradation. Verification of this USD approach to cell disruption by high-pressure homogenization is achieved using USD centrifugation to demonstrate the same sedimentation characteristics of disruptates prepared using both the scaled-down focused acoustic and the pilot-scale homogenization methods for the same fraction of protein release.     

Integration of host strain bioengineering and bioprocess development using ultra‐scale down studies to select the optimum combination: An antibody fragment primary recovery case study

An ultra scale‐down primary recovery sequence was established for a platform E. coli Fab production process. It was used to evaluate the process robustness of various bioengineered strains. Centrifugal discharge in the initial dewatering stage was determined to be the major cause of cell breakage. The ability of cells to resist breakage was dependant on a combination of factors including host strain, vector, and fermentation strategy. Periplasmic extraction studies were conducted in shake flasks and it was demonstrated that key performance parameters such as Fab titre and nucleic acid concentrations were mimicked. The shake flask system also captured particle aggregation effects seen in a large scale stirred vessel, reproducing the fine particle size distribution that impacts the final centrifugal clarification stage. The use of scale‐down primary recovery process sequences can be used to screen a larger number of engineered strains. This can lead to closer integration with and better feedback between strain development, fermentation development, and primary recovery studies. Biotechnol. Bioeng. 2014;111: 1971–1981. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Evaluation of a contraction flow field on hydrodynamic damage to entomopathogenic nematodes-A biological pest control agent

Mechanized production and delivery of biological pesticides presents challenges because the biological agents must remain viable during these processes. This study evaluates the effect of flow through an abrupt contraction, where flow characteristics similar to that found within bioprocesses and spray equipment are developed, on damage to a benchmark biological pest control agent, entomopathogenic nematodes (EPNs). An opposed-pistons, contraction flow device generated volumetric flow rates ranging between 8.26 cm(3)/s and 41.3 cm(3)/s. Four EPN species were evaluated: Heterorhabditis bacteriophora, Heterorhabditis megidis, Steinernema carpocapsae, and Steinernema glaseri. Damage was quantified by counting living and dead EPNs. Optical and cold field emission scanning electron microscope (CFE-SEM) images provided qualitative information to describe how the damage occurred. The experimental flow field was completely described using FLUENT, a computational fluid dynamics program. Local flow parameters computed in FLUENT were compared to EPN damage. The type and extent of damage varied between EPN species. Damaged Heterorhabditis spp. generally remained whole with an internal rupture located near the center of the body, while Steinernema spp. most often broke into several pieces. The fast-transient stress field generated at the entrance to the contraction caused a momentary tensile loading and then relaxation that damaged the EPNs. At high flow rates, the tensile stresses became large enough to cause failure of the EPN structural membrane. The relative elasticity of the EPN structural membrane may explain the differences in damage observed between the species. It is speculated that the internal rupture of the Heterorhabditis spp. occurred during the processes of stretching and relaxing at the contraction entrance. Appreciable damage was observed at lower average energy dissipation rates for H. bacteriophora (1.23E + 8 W/m(3)), H. megidis (1.72E + 8 W/m(3)), and S. glaseri (2.89E + 8 W/m(3)) compared to S. carpocapsae (3.70E + 8 W/m(3)). Energy dissipation rates within an equipment component should be kept below 1E + 8 W/m(3) to avoid hydrodynamic damage to EPNs. The relationship between average energy dissipation and EPN damage provides important information for future simulation efforts of actual spray equipment components.     

An approach to quantitative assessment of hemodynamic differences between unruptured and ruptured ophthalmic artery aneurysms

Background and purpose: Hemodynamic parameters are important in the pathogenesis, evolution and rupture of intracranial aneurysm. Energy loss (EL) has been applied for the rupture risk prediction of artery aneurysms recently. We proposed a new EL and further investigate its effects on the rupture of aneurysms. Materials and methods: Sixty-four patient-specific ophthalmic aneurysm datasets were divided into ruptured and unruptured groups based on their clinical history. Based on patient-specific 3D-DSA data, realistic models were retrospectively reconstructed and then analyzed by using computational fluid dynamic method. Results: The flow field feature EL in ruptured cases was significantly higher than that in unruptured cases. The average wall shear stress (WSS) and the maximum WSS in ruptured cases were higher than those in unruptured cases. Modified pressure loss coefficient (PLC_M) in ruptured cases was slight higher than that in unruptured cases but the difference has no statistical significance. Multivariate logistic regression analysis demonstrated flow field feature EL (p < 0.05) and the maximum WSS (p < 0.05) were the only independently significant variables to predict rupture of ophthalmic aneurysm. There were no differences in PLC_M, the maximum oscillatory shear index (OSI), the average OSI and AR between the two groups. Conclusion: Flow field feature EL may be a reliable factor to predict the rupture risk of aneurysms.     

Free energy simulations and MM-PBSA analyses on the affinity and specificity of steroid binding to antiestradiol antibody

Antiestradiol antibody 57-2 binds 17beta-estradiol (E2) with moderately high affinity (K(a) = 5 x 10(8) M(-1)). The structurally related natural estrogens estrone and estriol as well synthetic 17-deoxy-estradiol and 17alpha-estradiol are bound to the antibody with 3.7-4.9 kcal mol(-1) lower binding free energies than E2. Free energy perturbation (FEP) simulations and the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method were applied to investigate the factors responsible for the relatively low cross-reactivity of the antibody with these four steroids, differing from E2 by the substituents of the steroid D-ring. In addition, computational alanine scanning of the binding site residues was carried out with the MM-PBSA method. Both the FEP and MM-PBSA methods reproduced the experimental relative affinities of the five steroids in good agreement with experiment. On the basis of FEP simulations, the number of hydrogen bonds formed between the antibody and steroids, which varied from 0 to 3 in the steroids studied, determined directly the magnitude of the steroid-antibody interaction free energies. One hydrogen bond was calculated to contribute about 3 kcal mol(-1) to the interaction energy. Because the relative binding free energies of estrone (two antibody-steroid hydrogen bonds), estriol (three hydrogen bonds), 17-deoxy-estradiol (no hydrogen bonds), and 17alpha-estradiol (two hydrogen bonds) are close to each other and clearly lower than that of E2 (three hydrogen bonds), the water-steroid interactions lost upon binding to the antibody make an important contribution to the binding free energies. The MM-PBSA calculations showed that the binding of steroids to the antiestradiol antibody is driven by van der Waals interactions, whereas specificity is solely due to electrostatic interactions. In addition, binding of steroids to the antiestradiol antibody 57-2 was compared to the binding to the antiprogesterone antibody DB3 and antitestosterone antibody 3-C4F5, studied earlier with the MM-PBSA method.     

Production of bifunctional proteins by Aspergillus awamori: llama variable heavy chain antibody fragment (V(HH)) R9 coupled to Arthromyces ramosus peroxidase (ARP)

The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5′- or 3′-terminal ends of the gene encoding llama variable heavy chain antibody fragment V_HH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which produced up to 30 mg l⁻¹ fusion protein in the culture medium. Both fusion proteins showed peroxidase activity in an ABTS activity test. Considerable amounts of fusion protein were detected intracellularly, suggesting that the fungus encounters problems in secreting these kind of proteins. ELISA experiments showed that ARP-R9 was less able to bind its antigen, the azo-dye RR6, as compared to R9-ARP. Furthermore, in contrast to R9-ARP, ARP-R9 bound to RR6 did not show peroxidase activity anymore. These results indicate that fusion of ARP to the C-terminus of the antibody fragment V_HH R9 (R9-ARP) is the preferred orientation.