Combination of the 2A/furin technology with an animal component free cell line development platform process

The recently described 2A/furin technology combines both chains of the antibody in a single open reading frame. Upon translation and secretion, the peptide is processed by the cell to generate native fully functional IgG antibodies. Here, we describe the results of an evaluation study of this technology for an industrial CHO cell line development process. The 2A/furin expression cassette setup was combined with a Novartis vector system. A transfection, selection, and cloning procedure in chemically defined media was established at Novartis and applied for a monoclonal test antibody. The productivity of 2A/furin-vector-derived clones in non-optimized generic shake flask fed-batch models was in a comparable range with clones derived from the reference control vector. Higher clonal production stability was seen for the majority of clones generated with the 2A/furin technology compared to the clones generated with the reference control vector. Product quality was analyzed by SDS-PAGE and no significant difference was detected between the two systems. Thus, it was shown that the 2A/furin technology can be successfully combined with a Novartis CHO expression system and platform. Due to the single ORF setup, the 2A/furin technology may therefore offer a suitable approach to reduce vector size and complexity.     

First-time urea breath tests performed at home by 36,629 patients: a study of Helicobacter pylori prevalence in primary care

Background: The aim of the current study was (1) to describe the use of a ¹³C‐urea breath test (UBT) that was performed by patients at their homes as a part of a test‐and‐treat strategy in primary care and (2) to investigate the prevalence of Helicobacter pylori in patients taking a first‐time UBT. Material and Methods: The patients performed UBTs at home based on the discretion of the general practitioner and mailed the breath bags to a central laboratory for analysis. Each patient was identified by a unique civil registration number. The study was population‐based, and the background population was approximately 700,000 people. Results: From 2003 to 2009, 44,487 UBTs were performed. Of these, 36,629 were first‐time UBTs. In total, 726 of 45,213 breath bags received (1.6%) were unable to be analyzed because of errors with the bags. For both women and men who were ≤45 years of age, positive H. pylori declined over the time course of the study (women: 19.6% in 2003 to 17.6% in 2009, p < .01; men: 20.7% in 2003 to 16.9% in 2009, p < .001). Patients who were older than 45 years had significantly higher positive H. pylori results than younger patients. Conclusions: A test‐and‐treat system was possible to implement that allowed patients to perform UBTs at their homes. The results of the first‐time UBTs demonstrated that approximately one of five patients who presented with dyspepsia in the clinical setting of Danish primary care was infected with H. pylori.     

Effects of preincubation temperature on the detection of fastidious organisms in delayed-entry samples in the BacT/ALERT 3D blood culture system

This study evaluates the effect of preincubation on delayed-entry samples for fastidious organisms including the HACEK group, Streptococcus species, Neisseria meningitidis, Haemophilus species and Corynebacterium species for the BacT/ALERT 3D System (bioMérieux) using the FA (aerobic) medium.Bottles were inoculated with two different concentrations (0.5 McFarland and a 1:100,000 dilution) of each organism and either loaded into the system immediately or stored at 4 °C, room temperature (RT) or 37 °C for 24 hours (h) prior to loading.The detection rate (DR) was 92.5% for bottles loaded immediately for both concentrations with a mean time to detection (TTD) of 26.7 h (standard deviation (SD): 14.7 h) for the low concentration and 9.21 h (SD: 5.3 h) for the high concentration. Preincubation at 4 °C did not affect the DR for either of the two concentrations in comparison to no preincubation. The DR at RT was 90.0% for the low concentration and 83.6% for the high concentration. At 37 °C the DR was 76.3% and 66.3% for the low and the high concentrations respectively. The average TTD was inversely correlated with the preincubation temperature. An incubation of four days was sufficient, with the exception of Eikenella corrodens and Gemella sanguinis. The serotype of Streptococcus pneumoniae or Neisseria meningitidis did not influence the TTD. Kingella kingae remained undetected.For the retrieval of the above mentioned bacteria we recommend storage of bottles at room temperature. In case of erroneous storage at 37 °C subcultivation is advisable.All cases with a negative result on day four should be reevaluated and eventually new material for alternative diagnostic procedures should be retrieved.     

Reorganization of cortical microtubules during cell differentiation in tobacco explants

Summary Using indirect immunofluorescence on polyethylene glycol embedded material, the organization of cortical microtubules (MTs) has been studied in explants ofNicotiana tabacum. Within 6 hours after explantation the orientation of the cortical MTs shifts from transverse to longitudinal to the long axis of the cell in all cells. This change of direction is followed by further shifts that occur only locally and predict the orientation of future cell divisions. These reorientations are independent of the formation of protrusions and buds that will develop in the explants (after 4–7 days) and they represent a stage of de-differentiation of the explants. After two days of culturing clusters of cells can be recognized, at the proximal side of the explants, with randomly oriented cortical MTs. These regions represent the origin of the protrusions from which floral buds will develop. The formation of these clusters represent the first signs of re-differentiation and formation of new polar axes in the explants. The cells thus show a very early commitment (within 2 days) as to their differentiation.Abbreviations BAP benzyl-amino-purine - DMSO dimethylsulfoxid - EGTA ethylene glycol bis(2-aminoethylether)-N,N,N,N-tetraacetic acid - GA glutaraldehyde - MTs microtubules - MTOCs microtubule organizing centres - NAA -naphthalene acetic acid - PEG polyethylene glycol - PFA paraformaldehyde - PPBs preprophase bands     

Nonlinear state estimation as tool for online monitoring and adaptive feed in high throughput cultivations

Robotic facilities that can perform advanced cultivations (e.g., fed-batch or continuous) in high throughput have drastically increased the speed and reliability of the bioprocess development pipeline. Still, developing reliable analytical technologies, that can cope with the throughput of the cultivation system, has proven to be very challenging. On the one hand, the analytical accuracy suffers from the low sampling volumes, and on the other hand, the number of samples that must be treated rapidly is very large. These issues have been a major limitation for the implementation of feedback control methods in miniaturized bioreactor systems, where observations of the process states are typically obtained after the experiment has finished. In this work, we implement a Sigma-Point Kalman Filter in a high throughput platform with 24 parallel experiments at the mL-scale to demonstrate its viability and added value in high throughput experiments. The filter exploits the information generated by the ammonia-based pH control to enable the continuous estimation of the biomass concentration, a critical state to monitor the specific rates of production and consumption in the process. The objective in the selected case study is to ensure that the selected specific substrate consumption rate is tightly controlled throughout the complete Escherichia coli cultivations for recombinant production of an antibody fragment.     

Design of NK‐2‐derived peptides with improved activity against equine sarcoid cells

Equine sarcoid is a topically accessible model for the evaluation of anticancer peptides acting by physical membrane disruption avoiding the complexity of a systemic application. We aim at evaluating and improving natural peptides for host defence as lead structures, where we focus on the cationic and amphipathic peptide NK‐2. Cytotoxicity tests, fluorescence microscopy and a chip‐based biosensor, which enabled real‐time monitoring of cell metabolism, were applied. Cancer cell killing was dynamic with an initial phase of increased cellular respiration, followed by membrane destruction. NK‐2 was substantially improved and shortened. Novel peptides exhibited a fivefold improved activity against sarcoid cells, while haemolysis remained almost unaltered. Similar Zeta potential and similar amount of surface phosphatidylserine of sarcoid and normal skin cells are responsible for a lack of selectivity between these two cell types. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.