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Biomolecular Measurement

INTRODUCTION

The objective of the Biomolecular Measurement Division (BMD) is to develop measurement technology, standards, and data that can be used to determine the composition, structure, quantity, and function of biomolecules. The work focuses on applied genetics, bioanalytical science, biomolecular structure and function, bioprocess measurements, and mass spectrometry.

The BMD has the following groups and focus areas: the Mass Spectrometry Data Center develops mass spectral libraries for small molecules, metabolites, peptides, glycans, and proteins; the Applied Genetics Group develops standards and technology for deoxyribonucleic acid (DNA) testing to aid human identification and to support biometric, law enforcement, and clinical applications of genetic information; the Bioanalytical Science Group has a research focus in precision medicine and develops protein-based standard reference materials (SRMs) such as urine albumin, C-reactive protein (CRP), and troponin I; the Bioprocess Measurements Group develops methods and SRMs for biophysical characterization of biological particles (protein aggregates and protein-particle surrogate materials as SRMs); and the Biomolecular Structure and Function Group performs crystallography, neutron scattering, and nuclear magnetic resonance (NMR) to determine the structure of biomolecules including protein reference materials and biotherapeutics.

These five BMD research groups carry out their work at the National Institute of Standards and Technology (NIST) Gaithersburg campus and at the Institute for Bioscience and Biotechnology Research (IBBR) in Rockville, Maryland. The five groups have distinct goals but also work on complementary research topics and technical themes directed at the forensics and diagnostics of nucleic acids (DNA), peptides, proteins, and macromolecules—in addition to small molecules. The staff of the BMD that is located at the IBBR carries out work on the characterization of protein and biomolecular structure, which is of relevance to biopharmaceutical measurement and engineering, as part of a collaboration between NIST and the University of Maryland (UMD). The work at the IBBR is enabled, in part, by a 900-megahertz (MHz) NMR spectrometer housed in the W.M. Keck Structural Biology Laboratories. The NMR spectrometer is used for basic and applied research within the BMD related to the characterization of a range of proteins, nucleic acids, and nucleic-acid protein complexes. It is also used to develop models, standards, and databases.

The number of people associated with the research groups of the BMD are given in parentheses: Mass Spectrometry Data Center (32); Bioprocess Measurements Group (22); Biomolecular Structure and Function Group (20); Bioanalytical Science Group (13); Applied Genetics Group (12); and the Biomolecular Measurement Division headquarters (11). A total of 110 people work in the BMD, of which 79 are permanent federal employees (29 female and 50 male). Additional members of the BMD staff include guest researchers and postdoctoral fellows. Thirty-five percent of the division’s staff are guest researchers, and 65 percent are federal employees. The overall budget of the BMD in fiscal year (FY) 2016 was $26.9 million, an increase of 15 percent since FY2014 ($23.3 million). Sales through the NIST/Environmental Protection Agency (EPA)/National Institutes of Health (NIH) Mass Spectral Library contribute roughly $6 million to $7 million annually. There are three primary products of the BMD:

measurement science and protocols disseminated through scientific publications, reference materials and reference data, and mass spectral libraries.

ASSESSMENT OF TECHNICAL PROGRAMS

Accomplishments

The overall quality of the technical programs within the BMD is excellent. The BMD has a well-defined mission and focus comprising a portfolio of thrusts that blend established and recognized excellence in mass spectroscopic data libraries and DNA forensics with rapidly growing strengths in biopharmaceuticals and a vision for future impact in precision medicine. The division achieves its impact through an excellent balance between top-down alignment with the strategic goals of the MML and bottom-up freedom given to individual investigators to formulate their own programs consistent with the divisions goals.

The BMD programs have positively impacted U.S. industry by addressing critical measurement needs related to biomolecular technologies. The BMD is achieving much of its success and high impact by working closely with the varied stakeholders to ensure the relevancy of its efforts. This aspect of the division is outstanding.

The Mass Spectrometry Data Center Group is internationally recognized for its technical excellence and sets the gold standard for mass spectroscopy data libraries in the United States. In 2017 this group released and updated a library containing the spectra of more than 300,000 compounds. The group has also expanded its efforts from a historical focus on small molecules to immunoglobulin G (IgG) fragment libraries and peptides, including glycans. The latter focus on reference data sets to enable characterization of glycosylation patterns of proteins is timely and important, as patterns of glycosylation influence the functional properties of monoclonal antibody (mAb)-based pharmaceuticals and are notoriously difficult to characterize. A new initiative to develop libraries for federally controlled substances for forensics, which complements the leadership of the BMD in DNA forensics, is excellent. The large impact of this group is evidenced by the fact that the major U.S. manufacturers of mass spectrometry instrumentation sell the NIST mass spectrometry library installed on their instruments. The Mass Spectrometry Data Center Group is able to recover its costs through sale of the mass spectra reference data sets.

The BMD works with many partners that are involved in regulated industries, including the in vitro diagnostics industry and the biopharmaceuticals industry, as well as federal and academic laboratories. As a neutral third party, the BMD plays two vital roles for these partners: First, because NIST is a nonregulatory agency, the BMD is able to interact with industry, providing objective feedback on precompetitive technologies, without regulatory implications. Second, the BMD enables interlaboratory interactions, as a neutral third party, through the creation of SRMs that can be shared between partners to permit interlaboratory evaluation of measurement methods. These are nationally unique and important roles played by the BMD, and they are exemplified by the Mass Spectrometry Data Center Group in the context of the BMD’s development of an SRM mAb (NISTmAb) RM8671. Specifically, the Mass Spectrometry Data Center Group has used the NISTmAb to enable an interlaboratory study of glycosylation analysis involving over 100 laboratories across the world. The study compared the accuracy of mass spectroscopic methods for characterization of glycans. The participating laboratories sent their protocols and results to BMD researchers for analysis and interlaboratory comparisons. The outputs of the study were shared by the BMD with the participating laboratories, which provided the opportunity for the participating laboratories to improve the accuracy of their measurement methods. This type of standardization of measurement methods, which is enabled by the BMD, has the potential to create large sets of self-consistent biological data. This greatly enhances the opportunities to mine the data for information.

The Mass Spectrometry Data Center Group has achieved its impact through the measurement of fragmentation patterns and the dissemination of the results in libraries. The holy grail of mass

spectroscopy is the ability to predict how molecules will fragment. This capability is currently not possible, with the exception of peptides. This challenge, if solved, would be a breakthrough in mass spectroscopy. Tackling these sorts of high risk/high reward research veins would be an excellent complement to the more routine, yet important, development of mass spectroscopic libraries currently undertaken by the BMD at NIST. It would contribute to the division’s strategic goals of enabling the next generation of measurement tools.

The Applied Genetics Group, whose research focus is in DNA forensics, has developed extensive collaborations with U.S. genomics reagent companies. The group plays a critical role for this U.S. industry by providing objective and neutral assessments of precompetitive technologies. As previously mentioned, the neutrality of the BMD as a nonregulatory agency is a key enabler of this important role. For example, the Applied Genetics Group characterizes, prior to their release, commercial STR profiling technologies for human identification. The Applied Genetics Group also works with, and is funded by, the Federal Bureau of Investigation (FBI), the Department of Homeland Security (DHS), and the Department of Justice (DOJ) to develop and validate methods for human identification. They also have an important impact through the dissemination of SRMs for the forensics genetics community.

The Applied Genetics Group is leveraging its expertise toward DNA-based precision medicine, contributing to the strategic goals of the division related to enabling reliable measurements for precision medicine. The strong connections between this group and industry, in the context of DNA forensics, can serve as a potential model of best practices for the BMD’s nascent efforts in precision medicine.

The unified focus of the BMD’s Bioprocess Measurements Group and the Bioanalytical Science Group is addressing measurement challenges in biopharmaceutical manufacturing—this focus has blossomed over the past 3 years. These groups have been highly effective in coalescing their efforts around a well-defined set of industry-centric measurement needs, including critical challenges for the U.S. pharmaceutical industry related to protein aggregation and the standardization of characterization methods for mAb production. A key accomplishment of these groups has been the creation of the NISTmAb, which was donated to NIST by Medimmune. As noted above, the existence of this SRM for the pharmaceutical manufacturing industry is enabling interlaboratory studies that are benchmarking analytical methods of characterization of mAbs during production. The outcomes of these studies are being broadly disseminated, as illustrated by the co-editing and co-authoring of a three-volume monograph on the characterization of the NISTmAb, State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization,¹⁰ which was published by the American Chemical Society (ACS) in 2015-2016. In addition, the BMD is developing reference materials to enable accurate measurement of protein particles in protein-based therapeutics. Overall, the impact of this thrust within the BMD is large and growing rapidly. Key enablers of the success of the BMD in achieving high impact and value are physical proximity to and close interaction with biopharmaceutical companies; proximity to federal agencies such as the Food and Drug Administration (FDA) and NIH; and participation in the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL). The development of the mAb SRM has the potential to catalyze the development of biosimilars, decreasing the cost and increasing the availability of mAb-based therapeutics in the United States.

Opportunities and Challenges

Overall, the technical programs of the BMD are excellent. As noted above, key personnel and physical infrastructure in the BMD for protein physical characterization is located at the IBBR. The BMD has a clear vision and plan for the role of the IBBR in further development of measurement science and

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¹⁰ J.E. Schiel, D.L. Davis, and O.V. Borisov, State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization, Volume 1, Monoclonal Antibody Therapeutics: Structure, Function, and Regulatory Space, December 16, 2014, http://pubs.acs.org/isbn/9780841230262.

technology for biopharmaceutical manufacturing. Specifically, the BMD strategic plan identifies the opportunity to develop expertise and capabilities at the IBBR to produce mAbs from cell cultures, which are subsequently characterized at the IBBR. The vision is excellent and it integrates the role of NIST and the University of Maryland (UMD) in the NIIMBL. However, it remains unclear to what extent the leadership of the IBBR is committed to this shared vision. It will also be necessary to increase the engagement of UMD faculty with expertise relevant to the bioproduction of mAbs (or other biologics) to achieve the goals of the BMD at the IBBR in the thrust area of biomanufacturing. A roadmap is needed to ensure that this productive interaction between the BMD and UMD through the IBBR continues into the future in a manner aligned with the goals of the BMD. This roadmap needs to include the commitment of the UMD to place faculty with aligned research interests in the IBBR. It would also need to describe the role of the IBBR in the context of the strategic goals of the BMD in order to establish new capabilities and expertise to enable protein production, to contribute to the NIIMBL, and to broadly impact biopharmaceutical manufacturing initiatives through improved measurement.

The rationale for focusing efforts within the BMD on precision medicine is compelling, as the development of robust, reproducible, and accurate in vitro assays for protein biomarkers remains an important, yet largely unresolved, challenge. The development of SRMs that can be used to benchmark in vitro assays is an important and meritorious goal for the Bioanalytical Science Group within the BMD.

The Bioanalytical Science Group participates with national and international clinical chemistry organizations such as the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) to identify and prioritize development of SRMs for biomarkers, including human serum/urine albumin, CRP, and troponin I. Although these biomarkers have clinical relevancy, the rationale for their choice as initial targets was not well articulated during the review and the Bioanalytical Science Group was not able to provide a prioritized list of future targets. For example, CRP is not recommended by the American Heart Association for the general screening of heart disease. The impact of the Bioanalytical Science Group in precision medicine can likely be increased by engaging more closely with the clinical community and the in vitro diagnostics industry. It is important to develop a clear set of goals for research in precision medicine and a roadmap for achieving those goals. Such a roadmap would include the prioritization of target selection for relevancy (e.g., protein biomarkers) and maximizing opportunities for collaboration across NIST (e.g., with the BBD). The Applied Genetics Group and the Mass Spectrometry Data Center Group of the BMD provide good examples of best practices for engaging industry and maximizing relevancy to industrial and federal agencies. Engagement of in vitro diagnostic companies making kits for detection of CRP would, for example, ensure relevancy of the efforts of the Bioanalytical Science Group related to the creation of SRMs for CRP.

The Bioanalytical Science Group has been working to develop protein biomarker SRMs in simple buffers and complex matrices, such as patient-derived urine and serum. To date, it issued several protein SRMs in simple buffers, but it has not yet succeeded in developing SRMs based on patient-derived matrices. The accomplishments with simple buffers is good, but the challenge of making reference standards in urine and serum has not yet been overcome despite efforts ongoing since the previous National Academies Assessment in 2014. If the group does not succeed in making progress toward SRMs in complex matrices, an alternative approach would be to use defined, synthetic substitutes for complex matrices. Additionally, obtaining positive patient-derived samples in sufficient quantity for SRMs will be challenging. The use of SRMs based on recombinant proteins may also have merit.

In terms of possible future directions for the Bioanalytical Science Group, two potential opportunities are (1) the development of reference standards for in vitro diagnostics of mAbs, as companions to mAbs that are the focus of the BMD groups who are working on biopharmaceuticals, or (2) the development of reference methods and SRMs that permit the evaluation of the functional properties of proteins in in vitro assays (e.g., by benchmarking in vitro assays against reference methods based on liquid chromatography-mass spectrometry [LC-MS]). In addition, long-range research related to the development of function-structure relationships has the potential to enable future measurement methods for precision medicine.

The BMD also needs to continue investing in long-range research that enables future measurement materials, methods, and data—including high-risk research that could lead to potentially revolutionary

advances in future measurement. It was evident that some groups within the BMD were pursuing this type of long-range research (e.g., efforts aimed at the development of methods for single-molecule protein sequencing).

A broad area of potential long-term impact for the BMD is nano-medicine. Some efforts within the division already fall into this area, including characterization of exosomes, field-flow fractionation, liposome characterization, vaccine characterization, and synthesis of reference nanoparticles based on the cross-linking of amino acids in a flow focusing device.

Last, the BMD excels at addressing the critical measurement needs of the United States in key areas involving biomolecular technologies, including mass spectroscopy, DNA forensics, and biopharmaceuticals. This impact is derived from close interaction with industry and federal agencies. It is important for the division to balance this immediate focus against the upsides of long-term and high-impact research aimed at enabling the next generation of measurement tools.

PORTFOLIO OF SCIENTIFIC EXPERTISE

Accomplishments

The quality of scientific expertise within the BMD is excellent. This excellence is evidenced by the awards they have received over the past three and a half years, including the Gold Medal Award of the Department of Commerce (DOC) and the Mike Lynch Award for the conception and development of the International Chemical Identifier (InChI)—a chemical identifier that was adopted by the International Union of Pure and Applied Chemistry (IUPAC). The DOC has also awarded two Silver Medals—the first to the Applied Genetics Group in 2014 and the other to the Biomolecular Structure and Function Group in 2016. In addition, between June 1, 2014, and February 28, 2017, 16 BMD staff members have been recognized for their work by external organizations, including election as fellows of the American Academy of Forensic Sciences (AAFS) and the IUPAC.

Opportunities and Challenges

The BMD is particularly strong in the structural characterization of proteins. As identified in the strategic plan of the BMD, additional scientific expertise is needed to enable the establishment of protein-production capabilities at the IBBR. Meeting this need will require additional hiring in the BMD or engagement of faculty from the UMD, with expertise in cell culture and protein production. Expertise in cell culture and protein production exists in other divisions of the MML (e.g., the BBD). Interdivision collaboration may be a third element of an approach to address the challenge of establishing expertise in protein production.

Expertise in data management, including analysis and dissemination, is critical to the BMD achieving its impact. Although there is expertise in data informatics within individual groups within the BMD, an opportunity exists to maximize the impact of this expertise and share new data mining approaches (e.g., machine learning-based methods) by creating cross-division sharing of data informatics expertise. Additionally, expertise within the MML Office of Data and Informatics (ODI) can be leveraged by the BMD. As the ODI transitions from being outwardly focused to engaging in internal interactions, substantial benefits related to data management may be realized by pursuing efforts that cross division boundaries. Cross-division interactions also have the potential to benefit the ODI, as best practices established within groups within the BMD can be disseminated across the MML through the ODI.

Overall, as illustrated by the examples presented above related to cell culture expertise and data management expertise, there is an untapped opportunity to achieve impact within the BMD through enhanced intergroup (within the BMD) and interdivision interactions.

ADEQUACY OF FACILITIES, EQUIPMENT, AND HUMAN RESOURCES

Accomplishment

Human resource management within the BMD has been effective in retraining personnel, as the focus of research evolves within the division. For example, a staff member who worked previously on physical measurement standards has received training in biological science and engineering, and is now leading the development of SRMs for characterization of visible protein aggregates in biopharmaceuticals. In general, while requiring alignment with the strategic directions of the BMD, the leadership of the division has provided its scientists with sufficient freedom to define their own focus and contribution to the mission of the BMD. This is a highly effective management style, as evidenced by the high level of staff engagement and the ownership of projects expressed by the staff.

Opportunities and Challenges

The laboratory space occupied by the Bioprocess Measurements and Mass Spectrometry Data Center Groups in the BMD on the NIST campus in Gaithersburg, Maryland, was constructed in the early 1960s. In some of the facilities, evidence of leaking roofs was found. Accordingly, equipment in some laboratories used by the BMD is protected from potential damage from the leaking roofs by the placement of plastic sheets over the equipment. In other laboratory space occupied by the BMD, the ventilation system produces fine particles, which limit the range of research that can be performed in those laboratories. Until the BMD laboratory space is replaced by newer facilities, it can be expected that the laboratory space will require frequent repair and maintenance.

Currently, the time that it takes for repairs and renovation to occur after submission of a request is unacceptably slow. In contrast to the laboratory space occupied by the BMD at this NIST facility, the laboratories at the IBBR are excellent. Given the quality of space at the IBBR, the BMD may wish to explore placement of additional research programs and NIST staff there.

The equipment within the BMD is, in many cases, state-of-the-art for characterization of protein structure. This includes instrumentation for mass spectroscopy and NMR (e.g., the 900 MHz NMR, located at the IBBR). Some of the purchases, like the 900 MHz NMR, were enabled by one-time funding obtained through the 2009 American Recovery and Reinvestment Act.

There is excellent synergy between the Biomolecular Structure and Function Group within the BMD and the neutron scattering facilities of NIST. Both are particularly well suited to address characterization of concentrated protein solutions encountered during the production and formulation of protein-based therapeutics by the biopharmaceutical industry. The proposed purchase of cryogenic transmission electron microscopy facilities for single-protein structural analysis is excellent, and complementary to the neutron scattering and NMR capabilities at NIST.

Discussions with the staff within the BMD revealed a high level of satisfaction with support for acquisition processes, and for the establishment of new contracts of various types, such as the bilateral Cooperative Research and Development Agreements (CRADAs), Materials Transfer Agreements (MTAs), and nondisclosure agreements (NDAs). It was noted that some processes were slow, which reflected the requirements of the federal government, and not the inefficiency of processes within NIST. Overall, the staff expressed satisfaction with its work environment.

In general, the staff articulated a clear understanding of the long-term vision of the BMD. A small number of postdoctoral researchers, however, commented that they did not fully understand how their own professional development is aligned with the overall vision for the future of the division.

DISSEMINATION OF OUTPUTS

Accomplishments

The BMD has made a large and tangible impact on U.S. industry and other federal agencies (e.g., the NIH, the FDA, and the FBI) by providing SRMs, RMs, and standard reference data sets. The division has developed and maintains 14 SRMs/RMs and 5 SRDs, including the NIST/EPA/NIH Mass Spectral Library with Search Program, SRD 1A; a humanized IgG1k mAb (RM 8671); a Bovine Serum Albumin (SRM 927e); a Human DNA Quantitation Standard (SRM 2391c); and a Human DNA Quantitation Standard (SRM 2372). The NISTmAb (RM 8671) can be highlighted as an example of the impact of an SRM developed by the BMD. The NISTmAb (RM 8671) was shared and characterized from 2013 to 2015 by more than 100 participants from 11 biopharmaceutical companies, 5 instrument vendors, 5 universities, and the FDA.¹¹ As previously mentioned, the characterization data formed the basis of a three-volume ACS book series, State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization, published in 2015-2016.

Over 30 different analytical methods were used under industry best practices to characterize the NISTmAb (RM 8671). This partnership was highly successful and serves as a model for future SRM products. The NISTmAb (RM 8671) was issued on July 28, 2016, and as of April 4, 2017, over 350 units were purchased. The customers include nearly all top-20 global biopharmaceutical companies (30 percent), major analytical instrument companies (30 percent), and private contract research and manufacturing organizations that support biopharma (15 percent).

In addition to being assigned a concentration value traceable to ultraviolet–visible spectroscopy (UV-Vis) transmittance, assays were developed per International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guidelines for size, charge, identity, and stability that are included in the certificate for NISTmAb (RM 8671). The BMD has also disseminated important advances in measurement methods, through high-impact publications and presentations at conferences. Between June 1, 2014, and February 28, 2017, staff members in the BMD published 14 books and book chapters, 157 archival journals, 22 conference proceedings, and 13 NIST reports. As an example of the high impact of BMD publications, the BMD was the first to demonstrate that the structure of mAbs could be mapped by two-dimensional (2D) NMR techniques at natural abundance. The accomplishment was published in the journal Analytical Chemistry in 2015.¹² Since the fall of 2014, the Applied Genetics Group has generated 39 publications and 86 oral presentations.

The BMD achieves its high impact through close interactions with industry, which is evidenced by the large number of MTAs and CRADAs in which the BMD has participated. Between June 1, 2014, and February 28, 2017, the BMD executed a total of 177 total agreements (including 141 MTAs and 4 CRADAs) with industry partners.

Opportunities and Challenges

Overall, the dissemination of outputs within the BMD are excellent. Four additional observations are offered. First, as noted above, the BMD reference standards have had an important impact on the measurement capabilities of the United States. This high impact has occurred with minimal marketing by NIST. In particular, the website for SRMs is not organized around the needs of industry users. The website could be reorganized to present SRMs grouped according to the industries served by the RMs

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¹¹ NIST, “Made to Measure Building the Foundation for Tomorrow’s Innovation in the Biological, Chemical, and Materials Sciences,” p. 116, 2017.

¹² L.W. Arbogast, R.G. Brinson, J.P. Marino, Mapping monoclonal antibody structure by 2D C-13 NMR at natural abundance, Analytical Chemistry 87(7):3556-3561, 2015.

(clinical diagnostics, biopharmaceutical, etc.). This focus on marketing has the potential to increase the use of NIST SRMs, increase cost recovery for the preparation of SRMs, and communicate to stakeholders the important role that NIST is playing in enabling the competitiveness of U.S. industry and commerce. In addition, it will facilitate the adoption of SRMs that are new and not widely known to potential users. The potential exists for even greater impact through the optimized marketing of SRMs, in particular, to industry and federal agency stakeholders. An increase in the use of SRMs prepared by NIST, however, will also necessarily require additional investment of resources within NIST to ensure a reliable supply of SRMs (particularly, when they are integrated into regulated manufacturing processes) and the capacity to address customer problems and needs when they arise. Second, as SRMs produced by NIST are increasingly integrated into U.S. manufacturing processes, it will be important for NIST to provide sufficient infrastructure support to ensure that the supply of the standards is reliable. The absence of the timely provision of SRMs used in critical pharmaceutical manufacturing processes has the potential to halt production of important medicines.

Third, a key output and impact of the Applied Genetics Group, whose research focus is in DNA forensics, revolves around the evaluation of new technologies for forensic analysis. Developers of new reagents and instrumentation send their technologies to NIST for independent and objective third-party evaluations. This service plays a critical role in establishing the high quality of forensic technologies in the United States; however, it also occurs through an informal process that involves collaboration. This informality potentially limits access to this valuable resource, as it may be unknown to new companies developing DNA forensic technologies. Last, the dissemination of the mass spectroscopic libraries by the BMD has an important impact on measurement by mass spectroscopy worldwide. Substantial income in the form of cost recovery is derived from the sales of these libraries, but the pricing of the libraries is largely historical and not based on a pricing or cost study. Additional cost recovery has the potential to provide additional resources that will enable further development of the libraries.