In this mini-webinar and accompanying article, Craig Love, Senior Applications Scientist for Bioprocess Analytics, IDEX Health and Science shares his experience on how to gain process insights and disrupt process bottlenecks by enabling titer measurement on demand with the Tridex Protein Analyzer.
Increases in upstream operations have caused downstream bottlenecks
Mr. Love begins the webinar by explaining how advances in upstream bioprocess operations have yielded higher cell densities and increased titers. These increases have created several bottlenecks for downstream processes. These bottlenecks occur when one process is waiting on another to finish before production can move forward. For this reason, they are often compared with rate limiting steps in chemical reactions.
One such bottleneck is product titer. While viable cell density is increasingly measured on-line or at-line, product titer is typically performed by a QA/QC lab with turnaround times ranging from 24 to 72 hours. This creates a bottleneck for downstream capture processes, such as scaling Protein A capacity, column selection, or determining the number of columns and cycles for simulated moving bed systems.
This bottleneck also makes it difficult to gain process insights in real time. Ideally, titer levels could be monitored throughout the cell culture process to correlate titer changes with cell density or media composition. This would enable the creation of predictive models to better estimate product yields and ensure more consistent results.
Titer on demand
Accessing titer results on demand would disrupt these bottlenecks and ease the transition from upstream to downstream processes. Mr. Love shared how the Tridex protein analyzer puts the user in control of upstream titer measurements, by providing access to titer results in minutes, rather than days. Thus, enabling timely decisions for downstream processes, and providing an opportunity to maximize resources and improve process efficiencies. For example, having earlier access to product titers would improve scheduling of downstream personnel, and reduce the time that product is spent in holding tanks. This would free up valuable storage space and improve resource utilization.
Real-time titer results would also compliment existing on-line and at-line measurements to facilitate development of predictive models, gain valuable process insights, and improve process controls.
Fit for purpose
Next, Mr. Love compared existing titer measurement solutions with the capability of the Tridex Protein Analyzer. He explained that general purpose HPLC systems and bioprocess analyzers are highly configurable to support multiple assays. In contrast, the Tridex Protein Analyzer is a fit-for-purpose titer device, thus enabling the ability to provide such fast turnaround times. He shared this analogy that I thought was very useful, “HPLC systems and bioprocess analyzers are more like Swiss army knives, with an array of different tools, while the Tridex Protein Analyzer is more like a scalpel. The Swiss army knife offers greater versatility, while the scalpel offers better efficiency and ease of use”. When it comes to titer measurement, both types of instruments can produce excellent results, but the Tridex Protein Analyzer was designed for use in the manufacturing suite, where efficiency and ease of use are needed.
Designed to fit into current workflows
The Tridex Protein Analyzer was designed to compliment existing on-line and at-line instrumentation with real time titer measurement, Mr. Love explained. Titer measurement is conducted using a proprietary trap and elute analysis. The need for method development and reagent preparation have been eliminated by optimizing the analytical conditions and utilizing pre-formulated reagents. This not only saves valuable time, it also helps ensure consistent results.
Once the analysis is started, titer results are automatically displayed on the integrated touch screen. The Tridex analyzer is fully self-contained, so there is no tethered PC taking up valuable bench space and no additional software needed to obtain titer results.
The Tridex consumables simplify routine maintenance by providing up to 1000 analyses with 90-day on-board stability. The patented Analysis Module provides outstanding dynamic range by integrating the sample loop, sample trap and flowcell with an innovative fluidic manifold. The Reagent Pack features an ergonomic, leak-free design and is shelf-stable to free eliminate the need for valuable refrigerator space. Tridex is also easy to maintain with tool free consumable installation, and without the frequent service intervals of other titer measurement options.
Mr. Love closed the webinar by sharing data examples that illustrate the wide linear dynamic range, accuracy, and robustness of the Tridex Protein Analyzer. Please see the webinar for full data results.
Wide linear dynamic range of 0.1 to 10 g/L without dilution
In the first experiment, the wide linear dynamic range of the Tridex Protein Analyzer without sample dilution is demonstrated. For this experiment, cell culture media was spiked with a human IgG standard to prepare sample concentrations ranging from 0.1 to 10 g/L. The sample concentrations were then measured using triplicate injections below 1 g/L and duplicate injections at 1 g/L and above. Other than preparing the samples and IgG standards for a calibration curve, no sample dilution was required to obtain these results. Tridex measurements demonstrate a highly linear response with an R2 fit of 0.9998.
In the next experiment, the accuracy of the Tridex Protein analyzer was compared with Protein A HPLC using a simulated cell culture harvest sample. Protein A HPLC is generally considered to be the gold standard for antibody titer, so it provides a good basis for comparing titer accuracy of the Tridex results.
For this comparison, the cell culture media was enriched with up to 2.5% bovine serum albumin to simulate host cell proteins from a bioreactor. This mixture was then spiked with a human IgG standard to prepare titer concentrations ranging from 0.1 to 6.5 g/L. These samples were then measured using both the Tridex analyzer and a Protein A HPLC system. Only the creation of a calibration curve was required to begin acquiring sample data with the Tridex unit. Before sample data could be acquired with the HPLC system, the preparation of loading and elution buffers, methods for data acquisition and data processing, and the creation of a calibration curve were first required.
The Tridex measurements showed excellent agreement with the Protein A HPLC results and were not affected by cell culture media components or the addition of non-binding proteins.
Robustness and Versatility
This third example illustrates the robustness and versatility of the Tridex analyzer. For this experiment, samples were prepared by spiking a human IgG standard into PBS and three commercially available cell culture media formulations at concentrations ranging from 0.15 to 4.5 g/L. The titer values for all four sample preparations were measured in duplicate using a single calibration curve generated using human IgG standards prepared in PBS.
The Tridex analyzer yielded consistent titer results for all four samples across the sample concentration range. In terms of titer accuracy, all measurements were within the 10% upper and lower bounds. This data illustrates how a single calibration curve can be used for robust cell culture titer measurements with media compositions that are changing throughout the manufacturing run.
The preparation of calibration standards is also greatly simplified, because their composition does not need to match the sample composition. This makes standard dilutions much easier to prepare, because simple diluents, such as PBS can be utilized.
The associated technical notes or posters for these data examples can be found on the literature page of the Tridex website.
About our Presenter
Craig Love is a Senior Applications Scientist for Bioprocess Analytics with IDEX Health and Science. Craig has over 25 years of experience in the analytical instrumentation market and has been involved with the development of several commercial LC/MS instruments. He has overseen development of the Tridex Protein Analyzer from concept to commercial launch.
About IDEX Health and Science
IDEX Health and Science is a major OEM supplier for the healthcare, bioprocess and analytical markets with a broad portfolio of fluidic and imaging components in addition to the Tridex Protein Analyzer. Readers can connect with a Tridex expert via phone or email, or contact a sales agent to arrange a demo. Please contact IHSBioProcess@idexcorp.com for more information.
Additional product information and literature is also available on the Tridex website.
Samples may either be spun down or clarified using a 0.45 um syringe filter.
If the antibodies can be purified using Protein A chromatography, they should be compatible with the Tridex Protein Analyzer
No, Tridex only performs antibody titer measurements.
Tridex requires a sample volume of 100 µL and has a lower detection limit of 0.1 g/L.
Yes, Tridex has been tested with commercially available autosamplers from Flownamics and MAST.
No, Tridex is a fit-for-purpose antibody titer device.
Most Tridex users analyze 10 to 20 samples per day from multiple processes. Tridex consumables provide up to 1000 analyses over a 90 day period, or approximately 80 samples per week.
How complex is the training and what support is provided by the company? We have looked at a few other options that required a bit of training for techs.
The Tridex Protein Analyzer is simple to install and operate so IDEX Health & Science is able to offer remote installation support and introductory training. Users can also contact IDEX directly for technical support. Onsite installation and training services are available through Tridex manufacturer’s representatives. It typically takes new users just a few hours to install, calibrate and get ready to run actual titer samples.
Yes, Tridex has been tested with Flownamics and MAST automated sampling systems.
The run-to-run cycle time is approximately 4.5 minutes. No additional time is required for the next sample injection.
The Tridex analyzer does not require a warm-up period, but one or two blank injections are recommended prior to performing sample measurement.
I couldn’t find how data is saved. Can data be sent to a centralized management system. Is delivery to SCADA software available?
Data stored on the Tridex can be exported in .csv format via USB port on the front of the instrument. We are exploring alternatives for data delivery and would be interested in discussing requirements with anyone who needs that capability.
Yes, integrating Tridex with an online sampling system creates a closed system for sterile sampling directly from a bioreactor.
We are looking at a GMP process. Do you have a recommended method for qualifying the Tridex for GMP production?
The Tridex Protein Analyzer itself was designed for use in a production suite, however the software is not yet configured for GMP validation.
Tridex can be connected to an automated sampling system for sterile sampling from a bioreactor. Contamination risk during sampling for offline use will be no different than any other common analytical method such as HPLC.
Our lab would need to validate against our current HPLC method. Do you have a protocol for validation?
IDEX does not have a particular protocol for validation against HPLC because methods vary from site to site. We have demonstrated excellent correlation to HPLC and our customers have found similar results using their own validation protocols.
No scheduled maintenance is required because all the moving parts and critical flowpath components are life tested beyond the life cycle of the instrument.
Tridex has not yet been integrated with an automated sampling system sampling from a perfusion bioreactor. However, the analyzer is triggered by the sampling system so as long as the sample is cell free and the titer is within the range of detection (0.1 -10 g/L) Tridex would be a good fit for a perfusion process. If samples are injected manually then measuring titer with Tridex is the same for batch or perfusion processes.
The Tridex Protein Analyzer does not require any specialized cleaning procedures. The entire sample flowpath, from injection port to waste, is contained in the analysis module which should be replaced every 90 days.
If a PC isn’t connected, how is the data safeguarded against loss through equipment failure or when the system is offline.
The best way to safeguard data stored on the Tridex is to periodically export the data via USB port on the front panel and keep a back up copy.