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European Biopharmaceutical Review

Data Streaming

Market growth for biologics and biosimilar products is being driven by the success of monoclonal antibodies and the increasing use of other recombinant therapeutic proteins and vaccines. The market share of biologics is predicted to continue rising over the next five years, as big pharmaceutical companies increase the number of vaccines and protein therapeutics in their portfolios. This has resulted in an increasing need for better and faster analytical methods amenable to the monitoring of the whole cycle of bioprocess development and manufacturing.

For example, spent media analysis using nuclear magnetic resonance (NMR) technology has proven to be a rapid and cost-effective tool for the optimisation of cell feed strategy and upstream process development. NMR has been successfully applied to supporting clinical study supply and manufacturing scale, improving development efficiency and providing Quality by Design (QbD) compliance data (1). It may also be used in monitoring and establishing the quality of raw material and cell media, optimising media and feed conditions, and for troubleshooting bioprocess performance issues.

Faster, Better Characterisation


With the growth of the biologics and biosimilar products market expected to continue, there is an increased need for well-characterised and optimised biological batch processing at all scales to ensure fast development, quality, patient safety and manufacturers’ margins. While the market is expanding, development and production capacities remain expensive and, to some extent, limited. Biologics need to be optimally used, and this can be achieved in part by improving the monitoring of cell culture conditions, fermentation profiles and product quality attributes. Underperforming biomanufacturing or bioprocessing failures can indeed have dramatic impacts, hindering patient access to therapeutics. This risk is being particularly emphasised for biotech companies focusing on orphan diseases, for which production capacity is inherently limited.

Development and Manufacturing Efficiency

The Food and Drug Administration’s QbD initiative is a series of requirements that focus on ensuring product quality and consistency, and has been introduced in part to help lower development and manufacturing costs, while reducing the likelihood of production failures across both the small molecule and biological therapeutic areas. It entails “understanding the manufacturing process and identifying the key steps for obtaining and assuring a predefined product quality” (2).

A review of QbD within the industry stated that: “The QbD approach has been implemented in the biomanufacturing industry in two distinct but related ways. First, QbD increases up-front experimentation as part of process development to establish the operating boundaries between where quality is and isn’t affected. Second, QbD increases the quantity and fidelity of data collection and statistical analysis of manufacturing process parameters to detect changes in a process or in product quality. Understanding how quality relates to the manufacturing process is more difficult in biotech than in many other industries because of the sensitivity of biologic processes to seemingly small changes (both deliberate and unintentional) in process parameters” (3).

Monitoring a bioprocess requires a set of tools and methods that enable scientists and process engineers to monitor and collect process-relevant data in a robust and efficient way. For example, one might typically monitor pH, temperature, agitation, cell density/viability, O2/CO2, glucose, glutamine, and different metabolites such as lactate and ammonia. Considering the complexity of a cell culture, these are only a few parameters among hundreds that may impact the process at one level or another.

NMR-Based Techniques


NMR, or magnetic resonance imaging (MRI), is well-known by the public for its use in the field of clinical diagnostics. It is also a long-established analytical tool in chemistry. Nowadays, most chemistry work is supported by direct access to an NMR system. For instance, organic compounds generally contain protons (hydrogen atoms) which are differentially affected when submitted to a strong magnetic field – depending on their chemical environment and the structure of the molecule they are attached to. The protons will then behave differently, but predictably, when excited by specific radiowave frequencies, and will return a specific magnetic signature to the observer. These signatures can be interpreted and integrated to derive the chemical identity and concentration of the molecule(s) observed, either pure or in a complex mixture. These unique capabilities confer the unique analytical importance of this technique (see Figure 1).

Using custom-designed NMR databases, one can rapidly identify and quantitate the compounds present in a mixture as complex as fermentation media. Furthermore, NMR provides benefits over liquid chromatography-mass spectrometry (LC/MS) and tabletop profilers, including the number and classes of analytes that can be quantified (multiplex analysis), reproducibility and linearity of response across compound types and concentration ranges.

A further advantage of NMR is that – as it is a non-targeted analytical technique – unknown compounds or unexpected analytes can be detected and identified during standard analysis runs. Applying NMR to spent media also offers the advantage of being able to detect and quantify small weight molecules that other methods could miss in complex mixtures, such as formate, acetate or acetone.

The very detailed information content extracted from NMR data can further be used to establish critical markers related to the culture media parameters, which can explain performance variability observed between cell culture or fermentation batches. These markers can subsequently be identified from their NMR signature and integrated into predictive models, supporting raw material qualification customised to a specific culture process (see Figure 2).

Other key advantages of NMR include the minimal, rapid and straightforward sample preparation. Additionally, the data collection follows an automated and highly robust protocol. Finally, the accumulated spectra undergo data analysis through automated scripts to extract concentration data, providing rapid and cost-effective quantitative information on over 50 analytes per sample (see Figure 3).

Resolving Issues

When biologics reach production scale, all critical parameters have to be monitored and validated as part of the Good Manufacturing Practice qualification process, including the sterilisation of vessels or validation of the cell culture media and feed composition. This process must be completed by all production sites to ensure the safety and quality of products.

For this purpose, some companies run small-scale culture analysis to validate raw material, such as the media, and/or analyse specific components of the final media they receive from their suppliers to confirm that they are within certain specifications. Others rely strictly on the data delivered by their suppliers and analyse only a few parameters of their custom media preparations.

In line with QbD, the FDA requires some companies to apply a more extensive qualification of parameters of their raw materials, including final media preparation prior to manufacturing. Media analysis using NMR meets and exceeds this need as it typically covers many more parameters than usually required.

Furthermore, applying more extensive profiling of media prior to and during manufacturing, coupled with component analysis, partial least squares data analysis), would help to increase insight and understanding of the factors influencing the culture and thus help, over time, to minimise performance variability. By analysing spectra of multiple samples from a process, NMR gives information on the concentration of components present in each medium and provides quantitative data. Statistical analysis of each batch can help to rapidly identify outliers and components that are outside specifications, which would eventually lead to poor yield or modify the product quality attribute profile.

As this approach is by its nature ‘nontargeted’, one does not need a database or a hypothesis to start investigating. As the method can detect the outlier and support the identification of the variation, it can thus self-generate a hypothesis for why something is going wrong. Next to a production and/or quality assurance environment, this approach provides an increased level of safety, quality and productivity multivariate data analyses (principal in biologics production, as well as support for root-cause analysis and troubleshooting activities.

This has a direct impact on the margin made on a therapeutic by reducing failure and increasing productivity, without the need to change and re-qualify the process. This approach could foreseeably ease the process of transferring processes between sites, which would have a significant impact on costs.

Speeding Up

Because it can rapidly detect, identify and quantify a wide selection of feed components, metabolites and toxic compounds in culture media, NMR fingerprinting of cell culture can be used to benefit the fermentation process. This method can establish a detailed picture of the cell metabolism, which is specific to each individual cell line (1). Integrating the data collected over days of culture helps to establish, unambiguously, what cells consume and release during the culture process. The data collected can be used directly to rapidly fine-tune the media composition and feed strategy, improving and optimising cell viability, density and therapeutic production yield.

The Spedia-NMR method, which uses NMR-based profiling, has introduced an ensemble of technological advancements to significantly improve the throughput and robustness of the analysis, in order to match both scientists’ demands and quality requirements (see Figure 4). Because the analysis and access to data is fast, it can speed up the timeline between fermentation test batches, reducing the number of batches that are usually run for the design of experiment approach (DOE).

The key value here is that, by combining reduced DOE strategy and making optimum use of the response of the cell culture at a molecular level, it is possible to save weeks of development time and incubator capacity, which could mean significant development cost savings and faster access to products for clinical development and/or market release. In the short term, NMR can be utilised to facilitate and speed up cell culture development and upstream processing, allowing scientists to quickly and fully assess their cell culture.

Conclusion


The NMR profiling method has demonstrated its value in an upstream process development and production of biologics. It can be applied in a rapid and cost-effective manner, while also providing a source of QbDcompliant data. The use of NMR-based methods in biologics development and manufacturing application will continue rising concomitantly with the growing biologics and biosimilars market.

References
1. Bradley et al, Fermentanomics: monitoring mammalian cell cultures with NMR spectroscopy, J Am Chem Soc 132 (28): pp9,531-9,533, 2010
2. Food and Drug Administration,Advancing regulatory science at FDA, US Department of Health and Human Services, 2011. Visit: www.fda.gov/downloads/scienceresearch/specialtopics/regulatoryscience/ucm268225.pdf
3. Johnston R, Lambert J and Stump E, An industry perspective on quality by design, BioProcess International 10(3): pp26-35, 2010

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About the author

Frederic Girard
is Chief Executive Officer of Spinnovation Analytical BV, which he co-founded in 2004. He graduated in Organic Chemistry in 1997 and received his PhD cum laude in Biophysical Chemistry in 2000. Frederic spent three years on a post-doctoral study developing NMR-based techniques and investigating maturation mechanisms of hepatitis B genomic ribonucleic acid at Umea University, Sweden, and Radboud University Nijmegen, Netherlands. From his combined academic and industrial experiences, he has developed strong expertise in the application of advanced analytical techniques to support R&D and quality in pharma, biopharma and biotech.
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Frederic Girard
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