Biologic drugs continue to be one of the fastest-growing categories of the pharmaceutical market. US sales of biologics grew 9.7% annually from 2010 to 2012 to $65.5 billion, or 47% of the global market, according to EvaluatePharma. According to the Pharmaceutical Research and Manufacturers of America, 907 biologic medicines and vaccines are in development at US firms. EvaluatePharma forecasts biologics’ share of the pharmaceutical market to increase from 22% last year to 37% by 2020, when they will account for 52% of the 100 best-selling drugs.

Furthering the development of biologics have been advancements in analytical technology. Compared to small-molecule drugs, biologic drugs are more complex. Therapeutic proteins weigh 5–300 kDa, are structurally heterogeneous and manufacturing can affect product attributes. Analysis requires a range of techniques as well as orthogonal approaches. Biologics characterization includes testing to determine structural characteristics, purity, physicochemical properties, and process- and product-related impurities. Analytical tools for biologics development and manufacturing is a focus of several instrument providers, including AB SCIEX, Agilent Technologies and Malvern Instruments. The demands of biologics analysis is advancing technology development at these companies as they seek to make workflows more accessible, collaborate with customers and accelerate development of newer drug types.

Malvern Instruments, a Spectris business, is a supplier of materials-characterization techniques, such as particle-sizing systems, rheometers and viscometers. The company’s focus on biologics was furthered in 2012 when it created the Malvern Bioscience Development Initiative (BDI), an independent entity within Malvern. “It provides a two-fold advantage—a partnering vehicle through which biopharmaceutical companies can embed themselves in the development process, and a nimble approach to technology development that leapfrogs the traditional route to provide new and validated analytical tools,” stated Dr. E. Neil Lewis, CTO of Malvern and head of the BDI, and Dr. Linda Kidder of the BDI. The BDI currently employs 16 and is working on four principle projects.

The BDI addresses specific analytical challenges related to biologics. As Dr. Lewis told IBO, “One of the biggest of these is to determine not simply what can be measured, but which attributes are critical to quality, and that will provide meaningful predictive information.” The BDI partners with end-users and technology developers.

Among the analytical challenges for biologics development cited by Drs. Lewis and Kidder are the small amounts of sample and workflow bottlenecks in pre-formulation and formulation development. One area in which Malvern’s technology is employed is the identification and characterization of protein aggregates. “Technologies that can characterize the presence, kinetics and thermodynamics of oligomeric forms as a function of formulation conditions can provide significant insight into the driving forces governing stability and possibly shelf life of the product,” they said. Malvern’s new Zetasizer Helix combines dynamic light scattering and Raman spectroscopy to measure protein size and structure for monitoring changes in secondary and tertiary protein structures, which can affect aggregation. Malvern’s Raman spectroscopy–based Morphologi G3-ID imaging platform is also used for the study of aggregation.

To address new developments in the administration of biologics to patients, BDI is developing technology to facilitate analysis of high-concentration samples, including the ability to apply multiple characterization modalities to a single sample volume. “High-concentration samples impose a variety of constraints that can accelerate the formation of aggregates during storage, or the development of formulations in which the viscosity is too high for the product to be manufactured or delivered by syringe,” said Dr. Kidder. “Other issues are simply a factor of current tools not being able to easily work with high-concentration or small-volume samples, or to extract relevant information from complex formulations that optimize the stability of high-concentration samples.” Malvern’s Viscosizer 200 viscosity- and size-analysis system is designed for sample volumes down to 10 µL. The Zetasizer Helix is also suited for high-concentration samples. For development of antibody drug conjugates, it can track changes in antibody structure due to the addition of the conjugated small molecule.

Agilent provides several products for biologics analysis, including LC, MS and CE systems, LC columns and services. As Taegen Clary, senior director of Marketing for Agilent’s Pharmaceutical segment, told IBO, biologics development includes developing processes for scale-up, purification and formulation. “In all three cases, the intended molecule needs to remain consistent in structure and modifications and needs to be stable.” Areas of analysis include peptide sequence, post-translational modifications and, for stability, aggregation and degradation. “The analytical challenge continues to be the complexity of the instruments and methods being used to assess these biologic attributes,” he said. “The tools being used have come of age, but their usability across a wider population with varying experience continues to be lacking.”

Agilent is focused on increasing usability, according to Mr. Clary. “Our customers working with biologics have made it clear to us that they want to make their analytical workflows accessible to more biologist, biochemist and protein-engineering groups. They want to take some of the routine analytical burden off the expert protein analytical scientists.” To do so, one area Agilent has emphasized is automated sample preparation, including for HPLC and LC/MS workflows. The company recently released the AssayMAP Bravo automated sample preparation solution for glycan analysis and peptide mapping with a guided-user interface. For simplifying the use of LC/MS, informatics is key. “We are also working with our customers to make Q-TOF and TOF MS usable by biologists and biochemists that have never used an LC/MS system before. Similar to walk-up LC single-quadrupole and NMR systems in a medicinal chemistry lab, we are using our MassHunter Walkup software in combination with our BioConfirm software, for biologic characterization, to provide an end-to-end walk-up LC/MS solution.”

In the manufacturing of biotech drugs, analytical techniques are used to monitor biological characteristics. “A high majority of these quality methods are run on HPLC systems due to their simplicity and reliability,” explained Mr. Clary. “We see more and more customers turning to MS to identify their impurities in order to more rapidly identify where they came from.” Agilent’s support for biologics manufacturing also includes compliance services and collaborations to implement LC/MS.

Agilent is also addressing the development of new types of biologics, such as antibody-drug conjugates. “Measuring stability of conjugates requires the use of both qualitative MS analysis of highly heterogeneous large molecules and quantitative MS quantitation of the small molecules that may become free,” said Mr. Clary. In addition to systems, Agilent provides services for such applications. “Agilent’s Nucleic Acid Solutions Division provides contract-manufacturing and analytical services for companies developing oligonucleotide therapeutics. We are able to leverage the expertise from our CMO/CRO team to better serve our customers using our analytical platforms for oligonucleotide characterization.”

AB SCIEX is also a provider of LC, MS and CE technologies. Its products for biologics development include the new TripleTOF 6600 MS with SWATH Acquisition 2.0 (see page 3) and BioPharmaView software for automating workflows. Last year, AB SCIEX added the CE technology of another Danaher firm, Beckman Coulter, and launched the CESI 8000 (see IBO 3/15/14). The CESI 8000 was partly the result of the company’s BiologicsFocus Initiative, which was launched in 2012.

Discussing the analytical challenges of biologics development, Joseph Fox, senior director, Pharmaceutical Business, at AB SCIEX, told IBO that, for small-molecule developers transitioning to biologics development, the firm provides expertise to develop new workflows. Established biotech companies have different needs. “For [them,] the biggest challenges are, and will be, transitioning some of their assays into a far more efficient mechanism—for example, ligand-binding assays, where we lead the way in the adoption of MS as a complimentary, or in some cases as a new primary technique,” he commented. “That is where the focus on making many of our tools much more routine and accessible to nonspecialists plays a significant role.“ He also noted the role of collaborations, citing the development of the SWATH workflow for Host Cell Protein analysis.

Analytical challenges for biologics development include the use of MS/MS in routine workflows. “One of the continuing challenges is the incorporation of MS/MS—i.e., the information from tandem MS and fragmentation patterns —into everyday workflows,” said Mr. Fox. Informatics has been important in such advances, according to him, noting the use of SWATH 2.0 for such applications.

MS has also found greater adoption for biologics manufacturing. “I think it is fair to say that, up until just two or three years ago, people were more timid about the adoption of MS as a routine tool in biologics manufacturing—now they can see that it is much closer to reality with the use of software, routine and generic acquisition techniques, and the incorporation of ion mobility without fuss,” said Mr. Fox.

Another area that AB SCIEX is addressing is antibody drug conjugates. “We’ve seen the combination of the TripleTOF and our 6500—not least of which because of the fluid transfer of data and methodology between the two—quickly adopted in this area,” he said. “CESI is also uniquely positioned to add value here, and I would only say to watch that space as we work on some very interesting applications unique to our solution that we will bring to market.” In general, the development of new types of biologics generates more complex samples and data, according to Mr. Fox. The complexity can result in greater reliance on analytical tools. “The combination of heterogeneous molecules with hydrophobic cytotoxic drugs, and the additional challenge of dealing with labile linkers, means we have to track through the software multiple different categories all at once. That’s why you have seen developments, such as BioPharmaView, to track all aspects of the biomolecule.”