Development of a high-throughput scale-down model in Ambr® 250 HT for plasmid DNA fermentation processes.

Recent advances in messenger ribonucleic acid (mRNA) vaccines and gene therapy vectors have increased the need for rapid plasmid DNA (pDNA) screening and production within the biopharmaceutical industry. High-throughput (HT) fermentor systems, such as the Ambr® 250 HT, can significantly accelerate process development timelines of pDNA upstream processes compared to traditional bench-scale glass fermentors or small-scale steam-in-place (SIP) fermentors. However, such scale-down models must be qualified to ensure that they are representative of the larger scale process similar to traditional small-scale models. In the current study, we developed a representative scale-down model of a Biostat® D-DCU 30 L pDNA fermentation process in Ambr® 250 HT fermentors using three cell lines producing three different constructs. The Ambr scale-down model provided comparable process performance and pDNA quality as the 30 L SIP fermentation process. In addition, we demonstrated the predictive value of the Ambr model by two-way qualification, first by accurately reproducing the prior trends observed in a 30 L process, followed by predicting new process trends that were then successfully reproduced in the 30 L process. The representative and predictive scale-down Ambr model developed in this study would enable a faster and more efficient approach to strain/clone/host-cell screening, pDNA process development and characterization studies, process scale-up studies, and manufacturing support.

Voltage dependent closure of PorB class II porin from Neisseria meningitidis investigated using impedance spectroscopy in a tethered bilayer lipid membrane interface.

Electrochemical impedance spectroscopy (EIS) was used to characterize voltage-dependent closure of PorB class II (PorBII) porin from Neisseria meningitidis incorporated in a tethered bilayer lipid membrane (tBLM). The tBLM's lower leaflet was fabricated by depositing a self assembled monolayer (SAM) of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) on a gold electrode, and the upper leaflet was formed by depositing1,2-dioleoyl-sn-glycero-3-phoshocholine (DOPC) liposomes. At 0mV bias DC potential, incorporation of PorBII decreased the membrane resistance (R(m)) from 2.5 MΩc m(2) to 0.6 MΩ cm(2), giving a ΔR(m) of 1.9 MΩ cm(2) and a normalized ΔR(m) (ΔR(m) divided by the R(m) of the tBLM without PorBII) of 76%. When the bias DC potential was increased to 200 mV, the normalized ΔR(m) value decreased to 20%. The effect of applied voltage on ΔR(m) was completely reversible, suggesting voltage-dependent closure of PorBII. The voltage dependence of PorBII was further studied in a planar bilayer lipid membrane made from 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhytPC). Following a single insertion event, PorBII exhibited multiple conductance states, with reversible, voltage-dependent closure of PorBII porin occurring at high transmembrane potentials. The trimetric porin closed in three discrete steps, each step corresponding to closure of one conducting monomer unit. The most probable single channel conductance was 4.2 nS. The agreement between results obtained with the tBLM and pBLM platforms demonstrates the utility of EIS to screen channel proteins immobilized in tBLM for voltage-gated behavior.

Functional characterization of PorB class II porin from Neisseria meningitidis using a tethered bilayer lipid membrane.

PorB class II from Neisseria meningitidis is a pore-forming, outer-membrane protein that can translocate to the host-cell membrane during Neisserial infections. This report describes development of tethered bilayer lipid membrane (tBLM) system to measure PorB conductance properties. The tBLM was fabricated by depositing a self-assembled monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) tethering lipid on a gold electrode and then using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes to deposit the upper tBLM leaflet. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to monitor tBLM formation and subsequent PorB incorporation. The highly insulating tBLM exhibited a membrane resistance and capacitance of 2.5MOmegacm(2) and 0.7 microF/cm(2), respectively. PorB was incorporated into the tBLM in an active conformation, as evidenced by its mediation of ion passage and the decrease in membrane impedance. After PorB incorporation, the membrane resistance decreased to 0.6MOmegacm(2). As expected, the PorB channel was found to be non-selective, allowing the transport of both cations and anions. Cyclic voltammetry indicated that ferricyanide ions can also pass through the pores. The PorB-containing biomimetic interface developed in this study could potentially be used to screen for compounds that modulate PorB activity.

Fabrication of highly insulating tethered bilayer lipid membrane using yeast cell membrane fractions for measuring ion channel activity.

A tethered bilayer lipid membrane (tBLM) was fabricated on a gold electrode using 1,2-dipalmitoyl-sn-glycero-phosphothioethanol as a tethering lipid and the membrane fractions of Saccharomyces pombe yeast cells to deposit the upper leaflet. The membrane fractions were characterized using transmission electron microscopy and dynamic light scattering and found to be similar in size to small unilamellar vesicles of synthetic lipids. The dynamics of membrane-fraction deposition and rupture on the tethering-lipid layer were measured using quartz crystal microgravimetry. The electrochemical properties of the resulting tBLM were characterized using electrical impedance spectroscopy and cyclic voltammetry. The tBLM's electrical resistance was greater than 1 MOmegacm(2), suggesting a defect-free membrane. The suitability of tBLM produced using membrane fractions for measuring ion-channel activities was shown by a decrease in membrane resistance from 1.6 to 0.43 MOmegacm(2) following addition of gramicidin. The use of membrane fractions to form high-quality tBLM on gold electrodes suggests a new approach to characterize membrane proteins, in which the upper leaflet of the tBLM is deposited, and overexpressed membrane proteins are incorporated, in a single step. This approach would be especially useful for proteins whose activity is lost or altered during extraction, purification, and reconstitution, or whose activities are strongly influenced by the lipid composition of the bilayer.