Patrick Bennett is Strategic Marketing Director - Pharma for Thermo Fisher Scientific.

Patrick Bennett

The authors discuss the development of a simple, sensitive, and universal platform for identification and quantification of trace-level protein impurities in biotherapeutics. IgG monoclonal antibody spiked with low abundant proteins was used as the model to test the assay sensitivity and dynamic range. The simultaneous qualitative and quantitative analysis of low abundance proteins was achieved with single injection on a hybrid quadrupole-Orbitrap mass spectrometer.

	This LC–MS based approach can be applied for host-cell proteins generated during drug process development by recombinant DNA technology.

Articles

The authors discuss the development of a simple, sensitive, and universal platform for identification and quantification of trace-level protein impurities in biotherapeutics.

Identification and Quantification of Trace-Level Protein Impurities

Bioanalysis has migrated from a very general vocation to one that requires more specialisation, particularly in GLP applications. Previously, a bioanalytical scientist was as familiar with the design, electronics and operation of their instruments, software and LIMS system as they were with their developed methods. However, the overall performance of commercial instrumentation has become more sensitive, rugged and simpler to operate, allowing a wider user base. Also, the increase in commercially available software applications, including electronic laboratory notebooks and laboratory information management systems (LIMS), have made it easier for scientists be more effective from a compliance standpoint as well as throughput. As more experienced users gained access to mass spectrometers, there was a drive towards more innovation in the drug discovery arena and this has resulted in a wide variety of technologies and applications that reduce the time to perform routine and non-routine analysis. These include the introduction of 96-well plates, significantly improved sample extraction products, improved HPLC column performance and UPLC.

Bioanalysis falls between the most rigid GMP processes and more fluid R&D decision-making processes. Because of the combination of complex biological matrices containing the analyte and metabolites being quantified and the rigid GLP regulatory guidelines required for their analysis, bioanalysis requires a wide range of technologies and skills. These include the typical, basic laboratory instrumentation and equipment, as well as advanced software, hardware and separation technologies. These must be flexible enough for a wide range of users, experimental designs and reporting requirements, while maintaining GLP compliance. 


The practice of bioanalytical sample preparation and analysis has changed markedly in recent years, and the main challenges currently facing bioanalytical scientists performing sample preparation and analysis today are different to those in prior years. These challenges are not always analytical in nature and result from multiple sources, including organisational changes, limitations in analytical instrumentation and separations technology, more complex chemistry of the analytes or their formulations and maintaining GLP compliance while adapting to the above challenges. 


Organisational changes are occurring throughout the pharmaceutical industry irrespective of company size and affect not only internal operations, but bioanalytical CROs too. These changes have resulted in problems with skilled personnel availability, reduced information flow and throughput of results, and challenges of communicating analytical requirements. There is now less emphasis on high throughput for bioanalytical sample preparation for the pure sake of throughput, and an increased emphasis on throughput effectiveness. 


From an instrumentation and supply technology perspective, analyte carryover and matrix effects persist as issues in the bioanalytical sample preparation and analysis sector. Because of the volume of matrix sample available, sensitivity can still be challenging even as we continue to see gains in the sensitivity of the mass spectrometers we use. Incurred sample reanalysis is an important driver in the need for sensitivity and selectivity in situations where there is limited sample volume. 


From a chemistry perspective, endogenous materials (e.g., phospholipids), increasingly complex formulations (e.g., PEGs, lipids, protein-linked molecules), degradation products or metabolites of these formulations, unstable molecule and prodrugs all present bioanalytical scientists with an array of challenges. 


In GLP laboratories, challenges relate to the ability to track critical processes related to sample, reagent and sample storage, stability, use and handling, and to linking various software programs e.g., temperature monitoring, ELN, LIMS, data acquisition and processing software, and reporting software. The need to comply with these requirements presents an ongoing challenge. 


The application of dried blood spot (DBS) technology to sample collection is not new. DBS can greatly simplify sample collection, transportation, storage and, in some cases, can improve analyte storage stability. However, whilst it presents a means of overcoming certain challenges, until automation for DBS technology is developed, it presents an additional challenge for bioanalytical scientists in the near term. 

Reducing the time from sample collection to reporting results is paramount. Continued development of effective chromatographic separation technology combined with faster and higher mass resolution data acquisition by the mass spectrometers can also help overcome bioanalytical challenges. 

As analytical capacity increases, the rate-limiting step will once again be software and data handling. Simple user interfaces and reduced time to bring less experienced users up to speed is important. Because the majority of laboratories will have a mixture of software applications to perform a variety of tasks, software that can seamlessly link these applications will be extremely useful. These “connecting” packages will allow data to flow from unassociated applications to improve the reporting process. 


In the future, an increased focus on technologies that incorporate sample collection with the bioanalytical analysis procedure is very likely. The technology is likely to evolve to the point where samples will be collected on media that is then used for extraction and introduction into the mass spectrometer. A variety of methods ranging from simple liquid-liquid extraction of a paper-based media through to individual cartridge-based systems that can be formatted in 96 or more wells will be available. A smaller but significant driver for this type of technology is the increasing need to analyse prodrugs and drugs or metabolites that are unstable in blood or plasma. The addition of reagents to inhibit this instability is challenging in both drug discovery and development settings where samples are collected. By removing the need to treat samples immediately following sample collection, these unstable compounds can be rapidly collected, processed and stored in a simple and standardised format. 

Continued advancement of high-resolution mass spectrometers will drive shorter analytical cycle times and is likely to allow denser formats than 96 wells. These advances must be combined with higher throughput sample processing hardware, including multiplexing and both on-line and off-line extractions. A powerful commercial system would perform multiplexed injections of extracts (or whole plasma) onto a chromatographic system. Peaks would be collected into a plate format according to a pre-validated method. These individual peaks should be free of potential interfering material that could result in matrix effects, imprecision or ion suppression and can be injected with little or no chromatography into a mass spectrometer. This would shift expensive mass spectrometer utilisation time to less costly chromatographic systems. 

Fast and simple introduction ionization techniques such as Desorption Electrospray Ionization (DESI), Direct Analysis in Real Time (DART) and Laser Diode Thermal Desorption (LDTD) will continue to be desired, which will result in continued research in the application of these technologies. In addition, the introduction of similar products should occur. 

However, among the most significant gains would come from technologies that reduce or eliminate matrix effects in the ion source, such as the above off-line chromatographic separation and collection of individual peaks followed by introduction to a mass spectrometer, or alternative ionization techniques will be powerful. 

Bioanalysis has migrated from a very general vocation to one that requires more specialisation, particularly in GLP applications.

Bioanalysis: hurdles and hopes

The full version of this analytical technologies feature can be read in the May issue of our digital magazine: http://www.pharmtech.com/ptedigital0510

Bioanalysis has migrated from a very general vocation to one that requires more specialisation, particularly in GLP applications. Previously, a bioanalytical scientist was as familiar with the design, electronics and operation of their instruments, software and LIMS system as they were with their developed methods. However, the overall performance of commercial instrumentation has become more sensitive, rugged and simpler to operate, allowing a wider user base. Also, the increase in commercially available software applications, including electronic laboratory notebooks and laboratory information management systems (LIMS), have made it easier for scientists be more effective from a compliance standpoint as well as throughput. As more experienced users gained access to mass spectrometers, there was a drive towards more innovation in the drug discovery arena and this has resulted in a wide variety of technologies and applications that reduce the time to perform routine and non-routine analysis. These include the introduction of 96-well plates, significantly improved sample extraction products, improved HPLC column performance and UPLC. 

The practice of bioanalytical sample preparation and analysis has changed markedly in recent years, and the main challenges currently facing bioanalytical scientists performing sample preparation and analysis today are different to those in prior years. These challenges are not always analytical in nature and result from multiple sources, including organisational changes, limitations in analytical instrumentation and separations technology, more complex chemistry of the analytes or their formulations and maintaining GLP compliance while adapting to the above challenges.  

Organisational changes are occurring throughout the pharmaceutical industry irrespective of company size and affect not only internal operations, but bioanalytical CROs too. These changes have resulted in problems with skilled personnel availability, reduced information flow and throughput of results, and challenges of communicating analytical requirements. There is now less emphasis on high throughput for bioanalytical sample preparation for the pure sake of throughput, and an increased emphasis on throughput effectiveness.  

From an instrumentation and supply technology perspective, analyte carryover and matrix effects persist as issues in the bioanalytical sample preparation and analysis sector. Because of the volume of matrix sample available, sensitivity can still be challenging even as we continue to see gains in the sensitivity of the mass spectrometers we use. Incurred sample reanalysis is an important driver in the need for sensitivity and selectivity in situations where there is limited sample volume.  

From a chemistry perspective, endogenous materials (e.g., phospholipids), increasingly complex formulations (e.g., PEGs, lipids, protein-linked molecules), degradation products or metabolites of these formulations, unstable molecule and prodrugs all present bioanalytical scientists with an array of challenges.   

In GLP laboratories, challenges relate to the ability to track critical processes related to sample, reagent and sample storage, stability, use and handling, and to linking various software programs e.g., temperature monitoring, ELN, LIMS, data acquisition and processing software, and reporting software. The need to comply with these requirements presents an ongoing challenge. 

The application of dried blood spot (DBS) technology to sample collection is not new. DBS can greatly simplify sample collection, transportation, storage and, in some cases, can improve analyte storage stability. However, whilst it presents a means of overcoming certain challenges, until automation for DBS technology is developed, it presents an additional challenge for bioanalytical scientists in the near term.  

Reducing the time from sample collection to reporting results is paramount. Continued development of effective chromatographic separation technology combined with faster and higher mass resolution data acquisition by the mass spectrometers can also help overcome bioanalytical challenges.  

As analytical capacity increases, the rate-limiting step will once again be software and data handling. Simple user interfaces and reduced time to bring less experienced users up to speed is important. Because the majority of laboratories will have a mixture of software applications to perform a variety of tasks, software that can seamlessly link these applications will be extremely useful. These "connecting" packages will allow data to flow from unassociated applications to improve the reporting process.  

In the future, an increased focus on technologies that incorporate sample collection with the bioanalytical analysis procedure is very likely. The technology is likely to evolve to the point where samples will be collected on media that is then used for extraction and introduction into the mass spectrometer. A variety of methods ranging from simple liquid-liquid extraction of a paper-based media through to individual cartridge-based systems that can be formatted in 96 or more wells will be available. A smaller but significant driver for this type of technology is the increasing need to analyse prodrugs and drugs or metabolites that are unstable in blood or plasma. The addition of reagents to inhibit this instability is challenging in both drug discovery and development settings where samples are collected. By removing the need to treat samples immediately following sample collection, these unstable compounds can be rapidly collected, processed and stored in a simple and standardised format. 

However, among the most significant gains would come from technologies that reduce or eliminate matrix effects in the ion source, such as the above off-line chromatographic separation and collection of individual peaks followed by introduction to a mass spectrometer, or alternative ionization techniques will be powerful.  

Bioanalysis has migrated from a very general vocation to one that requires more specialization, particularly in GLP applications.