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Apr 15, 2007 (Vol. 27, No. 8)

Techniques for Characterizing Biologics

As Novel Therapeutic Platforms Are Discovered, New Analytical Tools Are Necessary

  • The development of protein-based pharmaceuticals is increasing, and the need for complete characterization of these molecules requires sophisticated techniques. Fortunately, significant advances in analytical technology have provided powerful tools for the biotechnology and pharmaceutical industry to assess the batch-to-batch quality of biologics and divergence in manufactured products in fine detail, as well as detecting minute differences in structure and composition within a short time. The California Separation Science Society and the FDA sponsored the recent “Well-Characterized Biological Pharmaceuticals” conference, which show cased some of these new technologies and the biologics they have been used to create.

  • Chromatography Technology

    Waters was one of the first companies to market HPLC instruments, and it continues to advance the technology in the marketplace. In 2004 Waters introduced its ultra performance liquid chromatography (UPLC) system.

    The Acquity UPLC system is the result of technological advances in particle chemistry, column packing, higher-pressure fluidic modules, minimized system volume, detector design, and data processing and control, reports Jeffrey R Mazzeo, Ph.D., biopharmaceutical business director. The high resolution combined with high sensitivity of the UPLC system allows scientists to analyze modified peptides, for instance deamidation, oxidation, and glycopeptides, with much detail.

    Glycopeptides are large heterogeneous molecules that tend to elute at relatively broader peaks in traditional HPLC, making interpretation of results a challenge. “With the UPLC, you get better resolution and peak shape,” adds Dr. Mazzeo.

    Waters developed a whole family of UPLC chemistries to serve the biopharmaceutical industries in two key applications for well-characterized biologics. The first application is amino acid analysis with the introduction of the AccQ Tag Ultra Chemistry. This product consists of reagents, columns, and methodologies for derivatizing amino acids for the purpose of characterization, analysis, and cell culture monitoring. The second application is the peptide separation technology columns. These columns are specifically designed and tested for conducting peptide analysis and peptide mapping.

    Waters recently introduced an innovation to mass spectrometry called high definition mass spectrometry. This technology takes advantage of the mobility characteristics of ions and couples it with mass spectrometry, thus allowing scientists to not only determine the molecular weight of a protein, but also gain information about protein size.

    “We are seeing a lot of interest in using this technology in the biopharmaceutical arena because it allows customers to look at their proteins in a new way. They are able to detect small differences in size and shape with IMS (ion mobility spectrometry), which allows more comprehensive characterization and provides a tool for comparing different batches of therapeutic proteins,” states Dr. Mazzeo.

  • Macromolecule Characterization

    Capillary electrophoresis (CE) has been an emerging technology in the biotechnology industry for the characterization and analysis of macromolecules such as proteins, carbohydrates, and nucleic acids. In its relatively short history, CE has found particular applicability in bioanalysis. The simplified but robust instrumentation that is required is making CE into a routine laboratory assay. Beckman Coulter’s ProteomeLab™ PA 800 is a protein characterization system based on CE; it is used for the determination of purity, heterogeneity, and identity of macromolecules. “Our platform technology supports multiple test analyses, using simplified, reproducible, and supported methods. This feature is important when it comes time for technology transfer and method harmonization,” reports Mark Lies, Ph.D., strategic marketing manager. This characterization system is capable of determining heterogeneity of protein molecules. For instance, it allows for the characterization of microheterogeneity due to glycosylation and therefore the determination of the presence of impurities or any differentially modified antibody species.

    This same instrument platform can use other techniques to run different assays. Capillary gel electrophoresis can be used as an alternative to SDS-PAGE for determining the molecular weight and purity of a protein. Carbohydrate separation and analysis is also used for the analysis of complex carbohydrates to determine molar ratio, degree of polymerization of oligosaccharides, and to detect changes in the extent or nature of oligosaccharide distribution.

    Capillary isoelectric focusing can determine the heterogeneity of the molecule based on its charge. Capillary zone electrophoresis is used in routine analysis of proteins, nucleic acids, and derivatives, including bases, nucleosides, nucleotides, and oligonucleotides, to study characterization properties like purity and identity. Other benefits of CE include high-resolution, efficient separations, short analysis times, and low volume of samples within a controlled environment. Wyatt Technology claims it was the first company to develop and commercialize the laser light-scattering instrumentation that, in recent years, has become an important analytical tool in the pharmaceutical industry. During this period, it became apparent that, under certain circumstances, protein-based products had the potential to induce immunogenicity in the patient.

    “Biological products are complex and, therefore, difficult to develop and manufacture. Because of the potential for immunogenic activity, it is important to closely monitor product formulation and associated structural changes. One of the major sources of immunogenicity is the formation of large aggregates that may frequently occur in such products,” says Philip J. Wyatt, Ph.D., CEO.

  • Irreversible Aggregates

    Wyatt has developed two basic light-scattering instruments with particular emphasis on their ability to monitor products that have the potential to form large irreversible aggregates. In both instruments, the sample, often as small as 0.4 mL, is illuminated by a fine laser beam. The classical multiangle light scattering (MALS) instrument determines the average molar mass of the suspended molecules, whereas the dynamic light scattering (DLS) instrument need only make measurements at a single angle to determine their average size. The newest DLS instrumentation, developed for operation in a high-throughput mode, is capable of measuring, unattended, up to 1,500 samples prepared in a microtiter plate, with less than 10 seconds per sample, reports Dr. Wyatt.

    Whereas the MALS measurement determines the average molar mass by measuring the intensity of the scattered light relative to the incident light intensity, the DLS determination is based on measuring the fluctuations of the scattered light relative to its average value. From these fluctuations, the diffusion coefficient of the scattering molecules is calculated, and from that, their hydrodynamic radius.

    “The DLS instrumentation is capable of measuring size differences between particles as small as 0.1 nm, whereas MALS instrumentation provides a particularly powerful method for precisely measuring oligomeric mass changes,” notes Dr. Wyatt. “Light-scattering techniques represent a direct, fast, and absolute method to confirm the composition, consistency, and quality of a product, and they provide a rapid and a powerful means to monitor the presence and formation of both large and small aggregates.”

  • Protein Quantification

    Gyros offers scientists involved in drug development a diverse range of applications all using a single system. Gyrolab Workstation is an open platform for protein quantification in an immunoassay format based on microfluidic principles. The process is miniaturized into a CD micro-laboratory. On each CD, there are 112 microstructures with the capacity to handle nanoliter volumes. Each microstructure contains prepacked columns, which can be used for specific applications under the control of Gyrolab Workstation LIF.

    By spinning the CD microlaboratory at precisely controlled speeds, defined volumes of samples and reagents can be drawn into the microstructure by capillary action and centrifugal force. This feature eliminates any volume transferring errors and enhances reproducibility and reliability. Furthermore, working at the nanoliter scale provides the additional benefit of significantly reducing reaction times.

    “You can process as many as 112 datapoints in less than an hour and up to 560 datapoints with unattended operation. It represents a huge difference compared to HPLC or classical immunoassay formats for scientists working with biopharmaceutical process development,” says Frida Sandegren, product manager. The fact that this technology is an open platform gives it an extra advantage; scientists can develop or transfer their own immunoassays to the CD microlaboratory.

    Experimental set-up can be designed to suit analytical needs, minimizing the need for sample dilution. Gyros’ platform provides a broad dynamic range, making it suitable for many different applications in the drug development process such as quantification of monoclonal IgG and contaminants in process development and pharmacokinetic studies, reports Sandegren.

  • Gene Delivery Vectors

    Targeted Genetics discovers, develops, and delivers molecular medicine to treat inflammatory arthritis, HIV/AIDS, congestive heart failure, and Huntington’s disease. Its platform technology is based on a gene delivery technology in which DNA sequences are delivered to cells with the goal of preventing or treating disease.

    Adeno associated virus (AAV) is a nano particle of 25 nm; it is not known to be associated with any human disease. Recombinant AAV (rAAV) vectors have a protein coat that encloses a piece of DNA which, when introduced into a cell, will result in expression of the gene of interest encoded by the DNA fragment. The AAV capsid forms the protein coat, which has affinity with certain cell-surface receptors.

    “When the particle is injected, it is recognized by the receptor and is transported into the cell, whereupon the gene is expressed,” explains Pervin Anklesaria, Ph.D., vp of therapeutic development.

    Targeted Genetics intra-articularly delivers a gene that expresses a TNF-a antagonist for local treatment of rheumatoid arthritis. In another application, AAV is used to deliver DNA-encoding HIV antigens that can be used as a prophylactic vaccine to induce immune response. “The AAV vector genomes are more stable and persist in cells for a longer time than traditional plasmids or adenovirus vectors, so they require less frequent dosing. The AAV is also safe because it does not integrate into chromosomal DNA,” adds Dr. Anklesaria.

    Targeted Genetics has developed a manufacturing process for AAV based on the same technology used to produce mAbs and therapeutic proteins. The company’s strengths reside in an understanding of the different attributes of the AAV products and the manufacturing and purification process, according to Dr. Anklesaria. It has a scalable manufacturing process for its AAV vector products and is able to quantify and evaluate the products with appropriate analytical methods, thus resulting in a well-characterized process and products.

    “Our product is a biological product. Even though it is different from traditional protein products, it is important that it is tested, analyzed, and characterized in the same way as the other biologics. We make sure we have the right analytical tools and that the process that we use to produce them is state-of-the-art,” concludes Dr. Anklesaria.

    Progress in the development of new, more powerful analytical instrumentation for biologics-based materials is ongoing. As scientists continue to look at their molecules in greater detail and resolution they will be able to make quicker research decisions. The efficiency of new drug development will increase and push science forward.

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