Acceleration of vaccine development by improvement of process understanding - Analysis of the host cell proteome

While regulatory agencies require stringent product quality and safety to be upheld in biopharmaceutical products, today’s competitive biopharmaceutical market requires short process development times. The demand to accelerate especially the development of vaccines became obvious with the COVID-19 pandemic. By expanding process understanding with the use of process design tools the development time of the purification could be significantly shortened. High throughput experimentation (HTE) provides an automated experimentation platform, which minimizes the amount of used samples and saves experimental time. In this approach, HTE is used to acquire experimental data to regress parameters used as inputs for a chromatographic mechanistic model with the objective to establish an E. coli vaccine purification process development platform for a recombinant subunit vaccine. To provide a generic process development strategy that can be applied to novel antigens, the focus lies on the description of the adsorption behavior of the impurities such as host cell proteins (HCPs) during the capture step. Therefore our approach focuses on the present impurities, in specific the HCPs (Figure 1). When using the same E.coli strain the knowledge regarding the host cell proteins could be transferred to a new product. The first step is the identification of HCPs. Over a thousand HCPs are identified in the E.coli harvest sample investigated by means of mass spectrometry based proteomics. A database containing the properties of these proteins can provide assistance in the decision on chromatography resins suited for the purification process of a new developed antigen. Please click Download on the upper right corner to see the full abstract

Model-based process development for complex vaccine mixtures

The regulations, safety and purity demands are extremely high for vaccine processes and likewise reflected in process development time and cost. Reducing time-to-market is key for pharmaceutical companies, hence saving lives and money, and therefore the need raised for systematic, general and efficient process development strategies (Hanke & Ottens, 2014). Despite the tremendous variation between vaccine purification processes, platform processes for similar types of vaccines could aid to generally accelerate the process development and would be beneficial in terms of knowledge, resources, costs and regulatory aspect. High throughput process development (HTPD) approaches can be used to establish platform processes. HTPD combines high throughput technologies and statistical or mechanistic modeling in an efficient manner. In particular mechanistic models, that aim to describe the real process based upon physical processes occurring, can be of great merit to extend the level of process understanding and thereby support in making decision regarding the process design (Pirrung et al., 2019). Please click Download on the upper right corner to see the full abstract

Continuous adsorption in food industry: The recovery of sinapic acid from rapeseed meal extract

Efficient recovery and utilization of valuable components from industrial food side streams is a main driver towards a circular economy. Among different available purification techniques, adsorption can effectively recover these components. However, the conventional batch mode of operation can limit its applicability in food processes due to limited efficiency. This work compares conventional batch packed bed adsorption with semi-continuous adsorption (so-called CaptureSMB) for the recovery of sinapic acid at industrial scale, using a food grade resin AmberliteTM FPX66. A mathematical mechanistic model able to describe semi-continuous operation is successfully validated and used to identify optimum operating parameters to maximize productivity and resin capacity utilization in batch and semi-continuous operating modes. The results indicate that CaptureSMB outperforms batch operation, increasing productivity from 5.18 g/L/h to 10.3 g/L/h for a given yield (>97%). A resin capacity utilization (RU) of around 70% is observed in both operating modes when productivity is maximized. A 92% RU can be accomplished for a given yield using the CaptureSMB process at a productivity of 7.0 g/L/h, higher than for conventional batch operation. The use of semi-continuous adsorption operation in food industry contributes to more efficient processes at reduced purification costs

A Hybrid Text Classification and Language Generation Model for Automated Summarization of Dutch Breast Cancer Radiology Reports

Breast cancer diagnosis is based on radiology reports describing observations made from medical imagery, such as X-rays obtained during mammography. The reports are written by radiologists and contain a conclusion summarizing the observations. Manually summarizing the reports is time-consuming and leads to high text variability. This paper investigates the automated summarization of Dutch radiology reports. We propose a hybrid model consisting of a language model (encoder-decoder with attention) and a separate BI-RADS score classifier. The summarization model achieved a ROUGE-L F1 score of 51.5% on the Dutch reports, which is comparable to results in other languages and other domains. For the BI-RADS classification, the language model (accuracy 79.1 %) was outperformed by a separate classifier (accuracy 83.3 %), leading us to propose a hybrid approach for radiology report summarization. Our qualitative evaluation with experts found the generated conclusions to be comprehensible and to cover mostly relevant content, and the main focus for improvement should be their factual correctness. While the current model is not accurate enough to be employed in clinical practice, our results indicate that hybrid models might be a worthwhile direction for future research

Arteriogenesis requires toll-like receptor 2 and 4 expression in bone-marrow derived cells

Adaptive collateral growth (arteriogenesis) is an important protective mechanism against ischemic injury in patients with cardiovascular disease. Arteriogenesis involves enlargement of pre-existent arterial anastomoses and shares many mechanistic similarities with inflammatory processes. Although infusion of the Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS) has shown to result in a significant stimulation of arteriogenesis and both Toll-like receptor 2 and 4 are involved in structural arterial adaptations, the requirement for TLRs in arteriogenesis has not yet been established. We therefore subjected TLR 2 null and TLR 4 defective mice to unilateral femoral artery occlusion. At 7 days, both TLR 2 null and TLR 4 defective mice showed a significant reduction (similar to 35%) of collateral perfusion. Histological staining showed that TLR 2 and TLR 4 expression during arteriogenesis is mostly restricted to infiltrating leukocytes. To distinguish between the functional importance of vascular and leukocytic TLRs in arteriogenesis, cross-over bone marrow transplantation was performed 6 weeks before femoral artery occlusion. Perfusion measurements showed that transplantation of wild-type bone marrow into TLR 2 null and TLR 4 defective mice rescued the impaired arteriogenesis, while injection of TLR 2 null and TLR 4 defective bone marrow into wild-type mice significantly reduced collateral vessel growth to levels of TLR null/defective mice. RT-PCR analysis demonstrated a significant upregulation of two endogenous TLR ligands EDA and Hsp60 (91.7 fold and 1.9 fold respectively) in regions of collateral vessel formation. This study illustrates the involvement of TLR 2 and TLR 4 in adaptive collateral artery growth and shows the importance of TLR 2 and 4 expression by bone-marrow derived cells for this process. (C) 2010 Elsevier Ltd. All rights reserve

Bridging a diagnostic Kawasaki disease classifier from a microarray platform to a qRT-PCR assay

BackgroundKawasaki disease (KD) is a systemic vasculitis that mainly affects children under 5 years of age. Up to 30% of patients develop coronary artery abnormalities, which are reduced with early treatment. Timely diagnosis of KD is challenging but may become more straightforward with the recent discovery of a whole-blood host response classifier that discriminates KD patients from patients with other febrile conditions. Here, we bridged this microarray-based classifier to a clinically applicable quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay: the Kawasaki Disease Gene Expression Profiling (KiDs-GEP) classifier.MethodsWe designed and optimized a qRT-PCR assay and applied it to a subset of samples previously used for the classifier discovery to reweight the original classifier.ResultsThe performance of the KiDs-GEP classifier was comparable to the original classifier with a cross-validated area under the ROC curve of 0.964 [95% CI: 0.924-1.00] vs 0.992 [95% CI: 0.978-1.00], respectively. Both classifiers demonstrated similar trends over various disease conditions, with the clearest distinction between individuals diagnosed with KD vs viral infections.ConclusionWe successfully bridged the microarray-based classifier into the KiDs-GEP classifier, a more rapid and more cost-efficient qRT-PCR assay, bringing a diagnostic test for KD closer to the hospital clinical laboratory.ImpactA diagnostic test is needed for Kawasaki disease and is currently not available. We describe the development of a One-Step multiplex qRT-PCR assay and the subsequent modification (i.e., bridging) of the microarray-based host response classifier previously described by Wright et al. The bridged KiDs-GEP classifier performs well in discriminating Kawasaki disease patients from febrile controls. This host response clinical test for Kawasaki disease can be adapted to the hospital clinical laboratory