AFA™ Ultrasonic Technology is unique in its ability to control the amount of acoustic energy delivered to samples such as cells. By utilizing the control of the AFA to vary the energy that is delivered to a sample, it is possible to gently disrupt the cell membranes of mammalian cells (e.g., G-protein coupled receptor assays) or abruptly disrupt the cell walls of bacteria (e.g., total protein extraction).
Cell Lysis: Buffer is a Critical Component
Cell lysis differs greatly depending on the species the cells are derived from (e.g., plant cells are significantly harder to lyse than mammalian cells). Consequently, lysis buffer choice is a critical component in the cellular lysis process. Typically, a lysis buffer is chosen by acknowledging the stabilization characteristics of the target molecule, rather than for the lytic capabilities of the buffer or intended downstream analytical methods. While this improves the stability of the target molecules once extracted, it does not assure the most efficient extraction or compatibility with analytical methods.
Cell Lysis with AFA: Features and Benefits
The controlled mechanical energy provided by AFA improves the efficiency of cellular lysis independent of buffer constituents. Therefore, AFA provides the flexibility to homogenize cells and efficiently extract proteins, RNA, DNA, or other targets in buffers optimized for intended analytical methods.
Processing at controlled temperatures provides highest yields while preserving sample fidelity
Accurate and precise energy
Highly reproducible process
Non-contact, closed vessel
No cross-contamination, clean-up, or sample loss
AFA™ is a highly controllable and reproducible technology able to accurately and precisely introduce energy to samples to disrupt cell membranes and homogenize lysates. Combined with extraction buffers optimized for use with AFA provides the highest extraction yields while preserving labile metabolites.
Protein Biomarker Extraction
RNA Biomarker Extraction
DNA Biomarker Extraction
Bacterial cell lysis
Yeast Cell Lysis
Use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in tissue
Bindu Nanduri et al., Anal Biochem . 2016 December 15; 515: 9–13
This paper demonstrated the use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in bovine lung tissue. Focused ultrasonication retained the catalytic activity of the enzymes studied and reproducibility better than other methods.
Assessment of adaptive focused acoustics versus manual vortex/freeze-thaw for intracellular metabolite extraction from Streptomyces lividans producing recombinant proteins using GC-MS and multi-block principal component analysis
Kassama et al, The Royal Society of Chemistry 2010, Analyst, 2010, 135, 934–942
A study of the efficiency of intracellular metabolite extraction by an ultrasonic adaptive focused acoustics (AFA) technique compared to a manual vortex/freeze-thaw method. Relative peak response ratios to the internal standard of 10 metabolites were higher for the AFA extraction, suggesting a more efficient recovery of these metabolites than achieved with manual vortex/freeze-thaw methods.
A Microscale Yeast Cell Disruption Technique for Integrated Process Development Strategies
Wenger et al, The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, United Kingdom
Miniaturizing protein purification processes at the microliter scale (microscale) holds the promise of accelerating process development by enabling multi-parallel experimentation and automation. For intracellular proteins expressed in yeast, small-scale cell breakage methods capable of disrupting the rigid cell wall are needed that can match the protein release and contaminant profile of full-scale methods like homogenization, thereby enabling representative studies of subsequent downstream operations to be performed. In this study, a noncontact method known as adaptive focused acoustics (AFA) was optimized for the disruption of milligram quantities of yeast cells for the subsequent purification of recombinant human papillomavirus (HPV) virus-like particles (VLPs). AFA operates by delivering highly focused, computer-controlled acoustic radiation at frequencies significantly higher than those used in conventional sonication. With this method, the total soluble protein release was equivalent to that of laboratory-scale homogenization, and cell disruption was evident by light microscopy.
An ultra scale-down approach to study the interaction of fermentation, homogenisation and centrifugation for antibody fragment recovery from rec E. coli
Li et al, Biotechnology and Bioengineering Volume 110, Issue 8
One option for protein release is to use high-pressure homogenization followed by a centrifugation step to remove cell debris. While this does not give selective release of proteins in the periplasmic space, it does provide a robust process. An ultra scale-down (USD) approach based on focused acoustics is described to study rec E. coli cell disruption by high-pressure homogenization for recovery of an antibody fragment (Fab′) and the impact of fermentation harvest time.
Use of Focused Acoustics for Cell Disruption to Provide Ultra Scale-Down Insights of Microbial Homogenization and its Bioprocess Impact— Recovery of Antibody Fragments from rec E. coli
Li et al, Biotechnology and Bioengineering, Vol. 109, Issue 8
An ultra-scale-down (USD) device that provides insight of how industrial homogenization impacts bioprocess performance is desirable in the biopharmaceutical industry, especially at the early stage of process development where only a small quantity of material is available. This work studied the effectiveness of focused acoustics as the basis of an USD cell disruption method to mimic and study high-pressure, step-wise homogenization of rec Escherichia coli cells for the recovery of an intracellular protein, antibody fragment (Fab′). The release of both Fab′ and of overall protein follows first-order reaction kinetics with respect to time of exposure to focused acoustics.
Assessment of the Manufacturability of Escherichia coli High Cell Density Fermentations
Perez-Pardo et al, Biotechnology Progress, Volume 27, Issue 5
In this study, a combination of techniques, such as adaptive focus acoustics (AFA) and ultra scale-down (USD) centrifugation, that utilize milliliter quantities of sample were used to obtain an insight into the interaction between cells from the upstream process and initial downstream unit operations. 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).