An engineering study of key interactions within the process for antibody fragment production

This thesis illustrates the need for biologies manufacturing processes to be considered as a whole, as the operation of the upstream process can greatly alter the performance of downstream steps. It develops methods to allow this to be done at minimal cost and with the minimum requirement for process material. The industrial bioprocess for antibody fragment (Fab9) production in E. coli is used as the case study. Two variations of the antibody production process were examined. In the first lactose was used both to induce Fab' production and to provide the post-induction energy source. Cell growth was controlled by phosphate limitation. The second process used isopropyl p-D-l-thiogalactopyranoside (IPTG) for Fab' induction, with a glycerol feed providing energy post-induction. Both variations produced high cell density broths (ca. 20% solids w/v), and industrially relevant quantities of Fab' (ca. 1 g/L). However, the IPTG-induced fermentation was chosen for detailed study as it was the most controllable and reproducible of the two. The fermentation was investigated together with the initial unit operations of broth centrifugation, heat/chemical Fab9 extraction, and spheroplast centrifugation. Methods were developed to allow all aspects of the two centrifugation steps in the initial process to be mimicked at small-scale. Loss of cell integrity upon entry and exit to the industrial centrifuge was mimicked through the use of a rotating disc and capillary jet device respectively. Clarification was mimicked through the adaptation of an existing method to high cell density feeds (>10% w/v) by the addition of a dilution step and a mathematical correction based on the Richardson-Zaki equation. Dewatering of the solids cake was predicted to within 8% at small-scale using a novel method that maintains industrial spin speed, spin time, and height of the sediment cake at millilitre-scale. All small-scale methods developed were verified at pilot-scale. The heat/chemical Fab' extraction step was mimicked in microfuge tubes. Outputs, in terms of % total protein and Fab' release, were shown to be within 6% of pilot- scale results in all cases tested. Any differences between pilot- and small-scale performances were shown not to be statistically significant. Product quality, as assessed by SDS-PAGE, was shown to be very similar to pilot-scale. As the periplasmic extraction was identified as the step most detrimental to overall yield of the initial DSP the microfuge method was used in a 2-factor statistical design of experiments study to analyse the robustness of the extraction procedure. The process was then considered as a whole. Post-induction glycerol feed rate was varied and the impacts upon subsequent processing analysed using both the methods developed and pilot-scale data. This demonstrated the usefulness and accuracy of the small-scale methods developed, and also showed that optimizing the feed rate for maximum Fab' production during fermentation did not result in optimum Fab9 production after initial DSP. Instead a higher feed rate should be used to produce cells with weaker periplasmic membranes to facilitate more effective Fab9 release upon heat/chemical extraction. The thesis concludes with a discussion of the business relevance of the small-scale methods developed. Their general applicability for candidate screening, process development and process understanding is suggested, and the regulatory issues relevant to their use are explored

Osteonecrosis in children and young adults treated for acute lymphoblastic leukemia : a scoping review

Osteonecrosis (ON) is a common disabling complication of treatment for patients with acute lymphoblastic leukaemia (ALL). Reported incidence rates range from 1% to 61% and multiple possible risk factors have been identified. This review explored existing evidence to provide new perspectives and recommendations for future interdisciplinary research. PEDro, CINAHL, AMED, EMBSAE, OVID, EMCARE databases were systematically searched from their inception to March 2022. Published original research reporting the incidence rates of osteonecrosis in patients aged 10–25 with ALL were included. Study reporting quality was assessed against appropriate reporting guidelines (STROBE, CONSORT and CROSS). All relevant data reporting incidence rates and risk factors were extracted for narrative synthesis. 3146 report titles were screened, with 34 studies included (n = 12,056) (30 observational cohort studies, three randomised trials, and one questionnaire study). The median study quality reporting score was 68% (IQR 64–82%). Median overall incidence rate of ON was 51.8% (IQR 41.4–58.9%) and 15.65% (IQR 9.2–24.2%) for asymptomatic and symptomatic patient screening respectively. Five possible risk factor categories were identified: sex assigned at birth, age, ethnicity, steroid regimen, and genotype. The female sex and white ethnicity were consistently reported as risk factors independently associated with an increased risk of osteonecrosis in all studies. A heterogenous body of literature with moderate reporting quality identified a high incidence rate of osteonecrosis in patients with ALL. Future research investigating the efficacy of stratified treatments that focus on reducing the risk of osteonecrosis through modification of steroid regimen particularly in females of white ethnicity is needed. Obtaining multidisciplinary consensus with regards to screening methodologies and intervention outcomes may also help to improve evidence synthesis in this area. This may in turn facilitate early diagnosis and improve long term patient outcomes through treatment regimen modification and possible prevention of ON progression

Miniaturization in Biocatalysis

The use of biocatalysts for the production of both consumer goods and building blocks for chemical synthesis is consistently gaining relevance. A significant contribution for recent advances towards further implementation of enzymes and whole cells is related to the developments in miniature reactor technology and insights into flow behavior. Due to the high level of parallelization and reduced requirements of chemicals, intensive screening of biocatalysts and process variables has become more feasible and reproducibility of the bioconversion processes has been substantially improved. The present work aims to provide an overview of the applications of miniaturized reactors in bioconversion processes, considering multi-well plates and microfluidic devices, update information on the engineering characterization of the hardware used, and present perspective developments in this area of research

Bundles consisting of extended transmembrane segments of Vpu from HIV-1: computer simulations and conductance measurements.

Part of the genome of the human immunodeficiency virus type 1 (HIV-1) encodes for a short membrane protein Vpu, which has a length of 81 amino acids. It has two functional roles: (i) to downregulate CD4 and (ii) to support particle release. These roles are attributed to two distinct domains of the peptide, the cytoplasmic and transmembrane (TM) domains, respectively. It has been suggested that the enhanced particle release function is linked to the ion channel activity of Vpu, with a slight preference for cations over anions. To allow ion flux across the membrane Vpu would be required to assemble in homooligomers to form functional water-filled pores. In this study molecular dynamics simulations are used to address the role of particular amino acids in 4, 5, and 6 TM helix bundle structures. The helices (Vpu(6-33)) are extended to include hydrophilic residues such as Glu, Tyr, and Arg (EYR motif). Our simulations indicate that this motif destabilizes the bundles at their C-terminal ends. The arginines point into the pore to form a positive charged ring that could act as a putative selectivity filter. The helices of the bundles adopt slightly higher average tilt angles with decreasing number of helices. We also suggest that the helices are kinked. Conductance measurements on a peptide (Vpu(1-32)) reconstituted into lipid membranes show that the peptide forms ion channels with several conductance levels

Osteonecrosis in children and young adults treated for acute lymphoblastic leukemia: A scoping review

Osteonecrosis (ON) is a common disabling complication of treatment for patients with acute lymphoblastic leukaemia (ALL). Reported incidence rates range from 1% to 61% and multiple possible risk factors have been identified. This review explored existing evidence to provide new perspectives and recommendations for future interdisciplinary research. PEDro, CINAHL, AMED, EMBSAE, OVID, EMCARE databases were systematically searched from their inception to March 2022. Published original research reporting the incidence rates of osteonecrosis in patients aged 10–25 with ALL were included. Study reporting quality was assessed against appropriate reporting guidelines (STROBE, CONSORT and CROSS). All relevant data reporting incidence rates and risk factors were extracted for narrative synthesis. 3146 report titles were screened, with 34 studies included (n = 12,056) (30 observational cohort studies, three randomised trials, and one questionnaire study). The median study quality reporting score was 68% (IQR 64–82%). Median overall incidence rate of ON was 51.8% (IQR 41.4–58.9%) and 15.65% (IQR 9.2–24.2%) for asymptomatic and symptomatic patient screening respectively. Five possible risk factor categories were identified: sex assigned at birth, age, ethnicity, steroid regimen, and genotype. The female sex and white ethnicity were consistently reported as risk factors independently associated with an increased risk of osteonecrosis in all studies. A heterogenous body of literature with moderate reporting quality identified a high incidence rate of osteonecrosis in patients with ALL. Future research investigating the efficacy of stratified treatments that focus on reducing the risk of osteonecrosis through modification of steroid regimen particularly in females of white ethnicity is needed. Obtaining multidisciplinary consensus with regards to screening methodologies and intervention outcomes may also help to improve evidence synthesis in this area. This may in turn facilitate early diagnosis and improve long term patient outcomes through treatment regimen modification and possible prevention of ON progression