Chemical and biological characterisation of a sensor surface for bioprocess monitoring

Moore, Jonathan D.; Perez-Pardo, M. A.; Popplewell, Jonathan; Spencer, Steve J.; Ray, Santanu; Swann, Marcus J.; Shard, Alexander G.; Jones, Walis; Hills, Anna E.; Bracewell, Daniel G.

doi:10.1016/j.bios.2010.11.043

Cited by 23 publications

(12 citation statements)

References 28 publications

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“…Figure 2c shows the leakage profiles of the three prolonged fermentations, where Fab 0 leakage was 22.8 AE 3.2% of the total productivity at 35 h increasing to 36.1 AE 2.2% at 63 h of product formation. Similar levels of leakage shown here were also noted in the same cultivation by Moore et al 27 through the use of a biosensor for process monitoring. Because the product leakage is associated with the integrity of the cell wall, this fermentation process provides a good case study to verify the use of scale-down tools available, for predicting harvest time effects on downstream processing performance.…”

Section: Growth and Product Characterizationsupporting

confidence: 84%

Assessment of the manufacturability of Escherichia coli high cell density fermentations

Perez-Pardo

Ali

Balasundaram

et al. 2011

Biotechnology Progress

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The physical and biological conditions of the host cell obtained at the end of fermentation influences subsequent downstream processing unit operations. The ability to monitor these characteristics is central to the improvement of biopharmaceutical manufacture. In this study, we have used a combination of techniques such as adaptive focus acoustics (AFA) and ultra scale-down (USD) centrifugation that utilize milliliter quantities of sample to obtain an insight into the interaction between cells from the upstream process and initial downstream unit operations. This is achieved primarily through an assessment of cell strength and its impact on large-scale disc stack centrifugation performance, measuring critical attributes such as viscosity and particle size distribution. An Escherichia coli fed-batch fermentation expressing antibody fragments in the periplasm was used as a model system representative of current manufacturing challenges. The weakening of cell strength during cultivation time, detected through increased micronization and viscosity, resulted in a 2.6-fold increase in product release rates from the cell (as measured by AFA) and approximately fourfold decrease in clarification performance (as measured by USD centrifugation). The information obtained allows for informed harvest point decisions accounting for both product leakages during fermentation and potential losses during primary recovery. The clarification performance results were verified at pilot scale. The use of these technologies forms a route to the process understanding needed to tailor the host cell and upstream process to the product and downstream process, critical to the implementation of quality-by-design principles.

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Section: Growth and Product Characterizationsupporting

confidence: 84%

Assessment of the manufacturability of Escherichia coli high cell density fermentations

Perez-Pardo

Ali

Balasundaram

et al. 2011

Biotechnology Progress

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“…The product loss in the MSBR (453 ± 26 μg/mL) was also found to be similar to that in the 20‐L STR (450 ± 42 μg/mL) (Figure 8) showing that the environment in the MSBR did not affect product loss more than in the 20‐L STR. Product loss was also comparable to that found by Moore et al (2011) 41. Overall the growth and product characteristics compare well between both reactors suggesting successful scale down translation of this fermentation process.…”

Section: Resultssupporting

confidence: 86%

Characterization and feasibility of a miniaturized stirred tank bioreactor to perform E. coli high cell density fed‐batch fermentations

Ali

Perez-Pardo

Aucamp

et al. 2011

Biotechnology Progress

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The use of small scale bioreactors that are mechanically and functionally similar to large scale reactors is highly desirable to accelerate bioprocess development because they enable well-defined scale translations. In this study, a 25-mL miniaturized stirred tank bioreactor (MSBR) has been characterized in terms of its power input, hydrodynamics, and volumetric oxygen transfer coefficient (k(L)a) to assess its potential to grow high cell density (HCD) cultures using adequate scale-down criteria. Engineering characterization results show scale down, based on matched specific power input (P(G)/V), is feasible from a 20-L pilot scale stirred tank bioreactor. Results from fed-batch fermentations performed using Fab' producing E. coli W3110 at matched (P(G)/V) in the MSBR and 20-L STR demonstrated that the MSBR can accurately scale down the 20-L fermentation performance in terms of growth and Fab' production. Successful implementation of a fed-batch strategy in the MSBR resulted in maximum optical density of ca. 114 and total Fab' concentration of 940 μg/mL compared with ca. 118 and 990 μg/mL in 20-L STR. Furthermore, the use of the MSBR in conjunction with primary recovery scale-down tools to assess the harvest material of both reactors showed comparable shear sensitivity and centrifugation performance. The conjoint use of the MSBR with ultra scale-down (USD) centrifugation mimics can provide a cost-efficient manner in which to design and develop bioprocesses that account for good upstream performance as well as their manufacturability downstream.

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“…Previous systems have used BLI and MS techniques to evaluate protein binding of two and three component systems as well as integrity of receptor binding of expressed protein, but, in both instances, the methods were not developed to take advantage of the tandem EM/MS approach 8 9 . The only other interferometry system that combined MS analysis to help characterize interactions was a dual-polarization interferometry 10 . Unfortunately, this system is no longer available for general use.…”

Section: Discussionmentioning

confidence: 99%

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

Machen¹,

O’Neil²,

Pentelute³

et al. 2018

JoVE

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In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

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Chemical and biological characterisation of a sensor surface for bioprocess monitoring

Cited by 23 publications

References 28 publications

Assessment of the manufacturability of Escherichia coli high cell density fermentations

Assessment of the manufacturability of Escherichia coli high cell density fermentations

Characterization and feasibility of a miniaturized stirred tank bioreactor to perform E. coli high cell density fed‐batch fermentations

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

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