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Validation of the Performance of the HORIBA Aqualog for Pharmaceutical and Industrial Analyses
A-TEEM for Biotech and Biopharma
Learn what A-TEEM Spectroscopy can do for pharmaceutical analysis.
A-TEEM is HORIBA’S proprietary fluorescence spectroscopy technology that simultaneously acquires, Absorbance, Transmission, and fluorescence Excitation and Emission Matrix measurements, correcting for the inner filter effect on the fly.
Fluorescence is insensitive to many common excipients, while being extremely sensitive to components containing conjugated rings. Proteins, small molecule APIs, aromatic amino acids, co-enzymes (NADH, Flavins) are easily characterized, whereas water, sugars, and other common excipients disappear.
A-TEEM can be used to characterize relevant components in complex matrices to sub-ppb levels. This makes it a natural fit for the characterization of many relevant biopharmaceutical products such as vaccines, insulin, viral vectors, anti-body drug conjugates, cell media, exosomes, CRISPr/CAS9, and more.
As a spectroscopic tool, A-TEEM can be deployed for process analytics, and used to monitor bioprocesses or sample purity.
Putting a sample in a cuvette is often the only preparation needed for measurement. For some quantitative measurements, sample dilution may be required. The Aqualog can be configured to work with an autosampler for unattended operation of multiple samples.
The Aqualog software is fully equipped to facilitate both calibration and validation of all instrumental absorbance and fluorescence functions following the specifications describe in the USP General Chapters for Fluorescence Spectroscopy <USP 853> and UV Spectroscopy <USP857>. IQ/OQ protocols ensure proper install and operation according to requirements.
A-TEEM is extremely sensitive to components containing conjugated rings. Molecules such as proteins, small molecule APIs, aromatic amino acids, co-enzymes (NADH, Flavins), vitamins, hydrolysates, by-products of bacterial contamination, and colored compounds are easily characterized.
By contrast, common excipients without conjugation, such as water, sugars, glycerin, polyethylene glycol, and others, are invisible to A-TEEM.
The high specificity and selectivity of A-TEEM fingerprints make it a natural fit for the characterization of biopharmaceutical products, enabling quantitative analysis at ppb levels of complex mixtures.
A-TEEM has be used to determine the quality of basal and feed media, to monitor bioprocesses, for reaction monitoring, and the characterization of vaccines, insulin, viral vectors, exosomes, anti-body drug conjugates, exosomes, CRISPr/CAS9 compounds, and more.
Samples are most often measured in a cuvette, which can be coupled with an auto-sampler for unattended measurements. As a spectroscopic tool, A-TEEM can be deployed for process analytics, used to monitor bioprocesses, or screen for batch-to-batch variance across a variety of relevant biomolecules.
Probe-based measurements in immersion or stand-off configurations are also possible, with many options for customization available.
The HORIBA Aqualog UV-800 facilitates both calibration and validation of all instrumental absorbance and fluorescence measurements, according to USP chapters <857> Ultraviolet-Visible Spectroscopy, and <853> Fluorescence Spectroscopy. The Calibration/Validation Toolbox is modular and intuitive, and along with HORIBA IQ/OQ documentation, guides users through the validation process. Individual Pass/Fail tests can be selected individually to minimize test redundance and facilitate more routine testing. The Aqualog is fully supported for pharmaceutical and industrial installations with a comprehensive IQ/OQ protocol.
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Benefit of Best-in-Class Molecular Sensitivity
Benefit of Quantification comparable to HPLC & GC
Benefit of high selectivity and concentration independent measurements:
The cost of cell culture media is a fraction of the value of the final product, therefore assessing its quality prior to the start of a bio-fermentation is a cost-effective way to ensure end-product quality and quantity. Standard approaches to perform this critical QC step (chromatography, mass spectrometry, test culture) are time consuming and expensive. A-TEEM provides a robust approach to assess the quality of cell culture media prior to the addition of cells, even for complex, non-chemically defined media using hydrolysate supplements.
A-TEEM for Cell Media characterization – boost upstream product yields
Standard separations-based approaches for vaccine characterization are too slow to keep up with the industry demand for rapid analysis. However, typical vaccine formulations (very low protein concentration, aqueous solutions, with confounding excipients) make these very challenging samples for the “standard” spectroscopic toolbox (Raman, FT-IR, NIR). The capabilities of A-TEEM make it highly suited for the analysis of vaccines. A-TEEM has been used to differentiate vaccine formulations, detect aggregation, identify amino acid substitutions and post-translational modifications, screen for batch-to-batch variation, and perform QC measurements for batch release.
A-TEEM for Vaccine Characterization – Differentiate similar formulations
Samples provided by Dr. Laura Sagle at the NIH Vaccine Research Center
A-TEEM for Vaccine QC – Detect batch-to-batch variation
The commercial deployment of biotherapeutics depends on advanced analytical tools for characterization, with a growing emphasis on speed and sustainability. A-TEEM has demonstrated the capacity to resolve AAV serotypes (AAV2 and AAV9), and to accurately quantify the payload filling percentage. A-TEEM, mostly owing to the high sensitivity of fluorescence, could be a suitable rapid alternative to other methods such as TEM for characterizing AAV materials including these certified reference materials.
A-TEEM for AAV characterization – differentiate serotypes, rapidly determine empty/full ratio
A-TEEM was evaluated for its potential to differentiate exosome types. Exosome standards (PC-3 and MCF7) and their mixtures were evaluated. The different exosomes were easily differentiated by A-TEEM, and a PLS model was readily able to predict the concentrations of the two components in samples comprised of the two exosomes mixed in known proportions.
From these initial results, it is expected that A-TEEM could be used as a rapid and cost-effective tool for manufacturing to find contaminants, detect batch-to-batch variation, and quantify payloads.
A-TEEM for Exosomes – Differentiate and quantify exosome types
Changes to the local solvent environment of the fluorescent residues monitored by A-TEEM fingerprints provides insights into aggregation and protein secondary structure. Short-acting and long-acting insulin have unique A-TEEM molecular fingerprints based on the different protein environments between formulations.
Source: “A-TEEM™, a new molecular fingerprinting technique: simultaneous absorbance-transmission and fluorescence excitation-emission matrix method,” published in Methods and Applications in Fluorescence, Volume 6, Number 2 https://doi.org/10.1088/2050-6120/aaa818. Acquired with Aqualog.
A-TEEM has also been used to study the ratio of intact vs damaged Lipid Nanoparticles (LNPs) delivered by different nebulizer devices. To quantify the integrity of the LNPs during the nebulization process, a fluorescent protein (calcein) was encapsulated in several LNP formulations. A-TEEM was used to quantify the relative amount of calcein released during this process. This measurement characterizes devices that are most effective in delivering intact LNPs to patients.
Read the publication:
Degradation of lipid based drug delivery formulations during nebulization
A-TEEM can be used to characterize relevant components in complex matrices to sub-ppb levels. This makes it a natural fit for the characterization of many relevant biopharmaceutical products such as vaccines, insulin, viral vectors, anti-body drug conjugates, cell media, exosomes, CRISPr/CAS9, and more. Use the filters below to find the application most suited for your needs.
A Simple, Fast, “Column Free” Molecular Fingerprinting Technology
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