The past couple of decades have witnessed significant advances in upstream bioprocess technologies and approaches. Since its establishment, BPI has been a facilitator of discussion both in print and at professional conferences, as well as in webcasts and news online. To mark the 20th anniversary of the publication, we surveyed articles published over the past two decades and found hundreds that highlight significant advances in both emerging and established themes in biopharmaceutical production:
â€˘ â€śhardwareâ€ť technology (e.g., analytical instrumentation, bioreactors, and facilities) â€” Part 1 (1)
â€˘ â€śsoftwareâ€ť technology and knowledge (e.g., process controls, quality by design (QbD), and process analytical technology (PATs)) â€” Part 1
â€˘ â€śwetwareâ€ť technology (e.g., expression systems, cell-line development, culture media, and emerging modalities such as cell and gene therapies) â€” Part 2.
Note that the references listed as Further Reading are but a small sampling of the hundreds we could have provided on these topics from the BioProcess International archives so far. The topics often overlap, so many articles also cover more than one type of advance. You can find them all in the issue-archives section of BPIâ€™s websiteÂ here.
|20 Areas of Advancement in 20 Years
1 Facility considerations: ballroom and modular manufacturing concepts, equipment installation, technology transfer, instrument qualification, and process validation2 Closed, modular cell-culture isolator systems and related advances in aseptic processing3 Single-cell cloning and selection systems4 Bioreactor systems, with options proliferating for mode of operation (fed-batch/continuous, suspension/adherent), mixing technologies, capacity for high-density and extended-viability cultures, sampling (at/on-line) and monitoring technologies, and process controls5 Perfusion cell-culture systems that operate across scales and products
Software6 Options for integration into cell-culture systems7 Capabilities for quality by design (QbD), statistical design of experiments (DoE), high-throughput screening, and scale-down modeling8 Automation and process analytical technology (PAT) for improved design, analysis, and control of critical quality attributes (CQAs) through measurement of critical process parameters (CPPs)Wetware
9 Pathogen safety and contamination control
10 Culture-media components: serum-free and chemically defined formulations and supplements coupled with mammalian host-cell and cell-adaptation approaches
11 Culture-media preparation: media batching, media-powder hydration, media cartridges, and tools for agglomeration prevention
12 Cell banking and recovery for seeding robustness
13 Mammalian cell-line development: improvements to transfection efficiency and metabolic engineering; development of expression hosts for increased product expression levels with consistent product quality attributes (PQAs)
14 Cell-therapy culture systems (autologous and allogeneic) and viral production systems for gene therapies
15 Genetic engineering game changers (targeted expression and genome editing)
16 Bacterial protein expression: improvements to Escherichia coli and Pseudomonas fluorescens expression systems, inclusion-body solubilization, product secretion, and overall recovery
17 Alternative expression systems: growing application of yeast cells, insect cells with the baculovirus expression-vector system (BEVS), plant cell-culture systems, cell-free expression, and whole-organism production (e.g., proteins expressed in transgenic-animal milk and secretions from whole plants)
19 Improvements in product quality control and impurity detection, including for host-cell proteins (HCPs)
20 The bottom line: reduced costs, increased efficiencies, shortened timelines, and consistent product quality
Although the term had been used in niche scientific papers since the 1950s, science-fiction writers popularized the concept of â€śwetwareâ€ť in cyberpunk novels such as Bruce Sterlingâ€™s Schismatrix, published in 1985. When applied to biotechnology, we use it similarly to the â€śwet chemistryâ€ť description of some laboratories. Living cells, tissues, and organisms are â€śwetâ€ť by nature in that they are highly water-based entities, and bioprocessing takes place in fluid environments.
Mammalian Cell-Line Development: Widely touted improvements in expression titers, especially for monoclonal antibodies (MAbs), that the industry achieved around the turn of the 21st century primarily came from process optimization work and increasing culture densities. Since then, however, cell-line engineering has come to the forefront as the means by which the industry is increasing production yields even further for a breadth of proteins. Along with proven metabolic engineering methods, such work has taken what were highly impressive ~1Â g/L titers of target proteins in Chinese hamster ovary (CHO) cell cultures 20Â years ago up to double-digit levels now. Thanks to risk management and QbD enabled by advancing analytical technologies, that feat has been accomplished alongside measurement, monitoring, and maintenance of product quality attributes as required by good manufacturing practice (GMP) guidelines.
In the past few years especially, BPI authors have chronicled such advancements and highlighted the instrumentation thatâ€™s helped make them possible. Chief among those have been the ClonePix colony picker from Molecular Devices, the Beacon system from Berkeley Lights, and Ambr microbioreactors and Octet label-free biolayer interferometry systems from Sartorius. As speed to clinic has become a widespread goal among biopharmaceutical companies, the contract development and manufacturing organizations (CDMOs) that serve them have reported on workflow improvements to shorten cell-line development times from months to weeks. Transient transfection has provided another avenue for moving products quickly into testing while traditional engineering and development of a stable cell line continues in parallel toward eventual large-scale production.
Genetic Engineering: Without a doubt, cell-line engineering is in the midst of a revolution. Genome-editing technologies are changing the game. As early as 2006, Morphotek authors were describing a â€śwhole-genome evolutionâ€ť approach in BPI. Soon after, we began to hear talk at conferences about the potential for zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) technologies for cell-line engineering. But things really heated up with the emergence of clustered, regularly interspaced, short palindromic repeats (CRISPR) and the CRISPR-associated protein Cas9, which together provide a powerful system for altering cellular chromosomal sequences in situ.
Unlike ZFNs and TALENs before it, the CRISPR-Cas9 system is relatively straightforward and inexpensive to use â€” despite the legal complications of its related intellectual property, which are as yet unresolved. When those inevitable difficulties are settled, we expect to see CRISPR use expand throughout the industry. Meanwhile, expression cassette engineering and genetic knock-out technologies continue to provide improved sequences for targeting and controlling expression and even some posttranslational modifications (PTMs) such as N-linked glycosylation.
Single-Cell Cloning and Selection: Significant advances have been made in the technology of cell cloning and selection since BPIâ€™s early days. Historically, serum-containing media were used to facilitate cell adhesion in such work, and single cells were cloned upon serial dilutions over multiple rounds of selection and plating using pipettes. Efforts to wean cells off the need for serum and adapt them to suspension culture would have to follow â€” significantly extending development timelines. Additional drawbacks included the potential safety risks of serum and a lack of faithful traceability to ensure single-cell clonality as the basis of production cell lines. Consider too that those clones ranking best in productivity, growth, and quality of expressed proteins often would not exhibit the same performance and stability when removed from serum-containing media and adapted to serum-free conditions.
Many of the significant increases in cell-specific productivity and volumetric product titers (while maintaining stable and reproducible product quality) that were achieved in the past couple of decades can be attributed to improved efficiencies and throughput of clone screening and selection systems. Other contributors include targeted (rather than random) genome integration and hostâ€“cell-line engineering as mentioned above. And although some solutions such as the million-dollar Berkeley Lights systems can be out of reach for small start-up laboratories, many can be reached through contract services and partnerships.
Cell Banking: No matter the cell type, once a clone has been selected under aseptic conditions, through screening for critical criteria such as productivity, viability, growth, and product quality attributes, that single cell is cultured by expansion methods for banking. A master cell bank (MCB) is initially produced, from which vials can be thawed and further expanded to generate multiple working cell banks (WCBs) for long-term storage of vials that can be used for each future production campaign. Significant advancements over recent years have improved technologies for assuring and documenting single-cellâ€“derived clonality for long-term consistency of current good manufacturing practice (CGMP) drug-substance production. As technology has advanced, regulatory expectations have evolved to emphasize clonality as well.
Other improvements have made it possible for WCBs to meet increasingly higher standards upon thaw and release for use through extensive characterization and testing: e.g., for purity, identity, genetic stability, and the presence of potential pathogens or adventitious agents. Until use, cells are kept stored in cryopreservation media, typically in freezers set to temperatures below â€“70 Â°C or in vapor-phase liquid nitrogen tanks. To mitigate risks that could compromise quality/performance, cell banks are stored in multiple controlled-access locations. When needed, vials of cells are removed from storage and shipped under conditions that are controlled and recorded until each shipmentâ€™s arrival at its point of use. ICH guidelines (Q5 a, b, d) provide help for assuring consistency and quality of product lots that will be manufactured throughout the life cycle of a drug product.
Culture Media and Supplementation: Mammalian cells are the prevalent hosts for production of protein therapeutics for which human-like PTMs (e.g., glycosylation) and complex three-dimensional structures are critical to effective function, increased stability, and reduced immunogenicity. CHO cells are the most commonly used host, especially for MAb production. Twenty years ago, animal serum was still commonly used in cloning for its adhesion-promoting effects and in production media to facilitate growth and productivity. However, safety concerns â€” e.g., related to viruses and transmissible spongiform encephalopathies (TSEs) â€” and the ill-defined nature and inherent variability of sera led to a shift toward serum-free and chemically defined media formulations. That has been enabled largely by cell-line adaptation methods and innovative cloning strategies that no longer rely on cell adhesion.
Often during process development, specific media components are added individually for optimization purposes â€” as process engineers seek to tailor a final culture-media formulation to the production cell line and process. Because of ever-tightening timelines, media batching methods often have been carried over from early development into commercial manufacturing with minimal changes. Streamlining media batching operations can simplify operations in fed-batch cultures and even more so in perfusion processes that rely on continuous large-scale media batching. Whether using proprietary/in-house media formulations or off-the-shelf commercial options, upstream groups find that investments in simplifying media preparation processes can return significant savings and operational advantages. Newer technologies in dry-media manufacturing, packaging, and feeding of culture vessels â€” increasingly developed through tight partnerships between biomanufacturers and media/service providers â€” are bringing the industry closer than ever to its goal of closed, continuous manufacturing.
Microbial Expression: Despite the current dominance of mammalian cell culture â€” particularly using CHO cells â€” microbial expression systems continue to play an important role in biopharmaceutical production. With applications reaching back to the early days of biotechnology, Escherichia coli remains the producer of choice for hormones and other small proteins without glycosylation requirements. Wherever it is practical, bacterial expression is preferred for a number of reasons: high expression levels and low development costs, with simplicity of genetic engineering based on many available versatile plasmid vectors and host strains.
Wherever production is impractical for bacteria, eukaryotic microbes such as Saccharomyces cerevisiae and Pichia pastoris yeasts might suffice. Such has been the case for insulin products approved in 2017 and 2018 as well as a recently developed hepatitis B vaccine. Fungi offer many of the same advantages as bacterial production systems in addition to an ability to perform important PTMs on expressed proteins. Production levels arenâ€™t as high as they are for bacteria, however, because overproduction of recombinant products can stress fungal cells. When it comes to relatively simple proteins that are better characterized as peptides, bacteria reign supreme.
An evolving number of bacterial and fungal strains are available now and variously designed to express recombinant proteins, peptides, and plasmid DNA â€” some even able to secrete soluble proteins in their biologically active form into cell culture medium (rather than as inclusion bodies that must be denatured before the proteins of interest can be refolded and further processed). With the expanding range of biopharmaceutical product modalities, microbial bioprocessing is here to stay and ready to advance with the times.
Other Expression Systems: Even though mammalian cell culture represents about two-thirds of all biopharmaceutical production, and microbial manufacturing accounts for about a quarter, the remaining few percent is both varied and innovative. Some early BPI authors touted the potential of transgenic whole-organism sourcing (e.g., from animal milk and plant tissue), and although that approach did not revolutionize the industry to the extent that its pioneers predicted, recent articles show that some companies have found real applications for it. And plant cell culture approaches may be more promising these days than whole-organism transgenics.
Meanwhile, transient transfection of insect cells using the baculovirus expression vector system (BEVS) has established a niche in production of viral vectors and vaccines such as those used for immunizing people against the human papillomavirus.
Finally, the idea of cell-free synthesis (CFS) of complex proteins â€” once considered all but impossible â€” is presenting an increasingly viable alternative. If anything could prove to be truly disruptive to bioproduction, then CFS could do so in the future.
Product Quality: Many important product quality attributes (PQAs) for drug substances are directly affected by upstream bioprocessing conditions. Chosen cell lines, media/raw materials, and culture process parameters and conditions can influence PQAs and serve as potential sources of impurities such as host-cell proteins (HCPs). Desirable product quality attributes are achieved increasingly by design through comprehensive screening during cell-line selection and development. This became achievable through advances in reducing volume requirements for critical attribute testing with improved detection technologies. Consistency of media and processes from clone selection through process development to commercial production have improved the predictability of drug substances meeting their PQAs â€” based on using the same media throughout development and using scale-down systems (e.g., Amber microbioreactor systems from Sartorius) that can reproduce the conditions of full-scale manufacturing. Even perfusion systems, once a challenge to scale down, now have been successfully scaled down to improve predictability during development of products to meet PQAs and other targets.
Impurities can come from the culture environment (e.g., carryover of spent media components) or from host cells themselves. Product-related impurities generally take the form of incorrectly formed (e.g., truncated or multimered) proteins that can affect biological activity, safety, or efficacy. Impurities from host cells can include nucleic acids, proteins, and other cellular components. Of particular concern is the potential for HCPs to copurify with protein products and ultimately trigger undesirable immune responses in patients. Thus, limits are set to control patient exposure to HCPs.
It is important to establish reliable methods for HCP detection and estimation, another field that has seen great advances in recent years. Several complementary methods often are used. For example, antibodies raised against a parental cell line and/or host strain form the basis of enzyme-linked immunosorbent assays (ELISAs). Chromatographic, mass-spectroscopic, and multidimensional separation, among other technologies, help analysts identify and quantify HCPs for the purposes of impurity characterization and control.
|Advances Related to Cell-Therapy Systems
A key difference between protein biologics (which are isolated from culture, then purified and formulated) and bioprocess-produced cell therapies is that bioproduced viable cells themselves constitute the product. Allogeneic cell therapies originate from donors who are not their recipients, with a production process that is similar to common biologics production processes in that it is initiated from an â€śoff-the-shelfâ€ť cell bank.With autologous cell therapies, the cells originate from the same individual who will be receiving them after they are manipulated and scaled up â€” reducing the chance of patients developing immunogenic responses or rejection. In essence, the number of products equals the number of patients, which greatly limits and complicates production and testing and makes these treatments highly variable.In both autologous and allogeneic processes, isolation, purification, formulation, characterization, and testing all happen at the cell level rather than the protein level. Both types of cell therapies usually can be cryopreserved until use. The past couple of decades have brought significant advances in optimization of these activities and their regulation. For example, cell-harvest techniques have improved through use of continuous centrifugation processes that leverage density cushions to maintain much higher cell viabilities than are possible with previous harvesting methods. Ongoing research and development in cryopreservation and cell banking should improve bioefficacy upon cell thawing and allow for storage and shipment at higher temperatures. The latter would be important for certain applications such as field treatment â€” a realistic scenario in certain regions of the world and in military applications, where deep refrigeration is not available â€” or if the cells are meant to treat mass-casualty events (e.g., exposure to nuclear radiation).Further Reading
Macdonald G. Room for Both Allogeneic and Autologous in Cell Therapy Space. BioProcess Insider 19 October 2021; https://bioprocessintl.com/bioprocess-insider/therapeutic-class/room-for-both-allogeneic-and-autologous-in-cell-therapy-space.
Walters P. Scalability in Cell and Gene Therapy Facilities: How Todayâ€™s Developers Are Preparing for Tomorrowâ€™s Commercial Success. BioProcess Int. 20(4) 2022: 14â€“18; https://bioprocessintl.com/manufacturing/facility-design-engineering/scalability-in-cell-and-gene-therapy-manufacture-how-todays-developers-are-preparing-for-tomorrows-commercial-success.
The Bottom Line
From upstream to downstream, drug substance to drug product, process development to clinical testing, every group working in the biopharmaceutical industry faces the same mandates from company management: reduce costs, increase efficiencies, and shorten timelines, all without compromising on quality. Those goals are not only imposed from outside â€” as investors want more return for their money, regulators demand safety and efficacy, and patients need treatments that they can afford to use â€” but also arise organically from within companies. And such trends are reflected throughout the advancements highlighted herein. As problems have arisen on the way to meeting those objectives, technology providers have helped to provide solutions that moved upstream bioprocess work forward.
Past achievements can shed light on near-term progress; however, unforeseen â€śdisruptiveâ€ť technologies can produce significant advances that change the course of future progress. We have seen examples of that in each category covered above. In hardware, the influence of single-use technologies and associated ancillary technologies extended beyond upstream and downstream unit operations per se, altering approaches to cleaning and validation operations, scale-up/down, facility design, technology transfer, and process economics. In the rapidly advancing software area, combining QbD with PAT supported by statistical, risk-, and knowledge-based approaches have accelerated process development timelines, leading to higher yields and improved efficiencies in production operations as well as increased control and assurance of meeting target PQAs. Wetware game-changers such as CRISPR and cloning-system technologies have in turn helped cell-line developers achieve long-sought goals such as targeted integration and documented single-cell cloning. And after initial high-profile setbacks, advanced-therapy (gene, cell, and tissue-based) medicinal products have become recognized by industry and regulators for their revolutionary potential to treat or cure diseases. The ability of ATMPs to address unmet medical needs is evident from many companiesâ€™ current pipelines as well as recent and imminent publication of specific guidelines.
Although it is nearly impossible to predict what future technologies could be developed or invented, one thing is certain for the future of upstream bioprocessing: The field is in the midst of a transformational era that will continue to draw the brightest fresh minds working for the benefit of society. If the past is prologue, then the future is likely to bring better and faster processes to make more affordable biologics for the world. We look forward to the exciting journey ahead by contributing within the roles we play in the industry and in facilitating the exchange of groundbreaking information and knowledge.
1 Shimoni Y, Scott C. Hardware, Software, and Wetware: 20 Years of Advancements in Biopharmaceutical Production, Part 1. BioProcess Int. Julyâ€“August 2022: 34â€“37.
Further Reading, Part 2
You can find all of these and more in the issue-archive section of BPIâ€™s website at http://www.bioprocessintl.com.
Broedel SE, Papciak SM. The Case for Serum-Free Media. February 2003
Chatrathi K. Metabolic Cooling Capacity of Fermentors. April 2004
Scott C. Chapter 3: Commercial Production in Insect Cells. June 2004 (supplement)
Jerums M, Yang X. Chapter 4: Optimization of Cell Culture Media. June 2005 (supplement)
Whitford WG. Chapter 3: Supplementation of Animal Cell Culture Media. June 2005 (supplement)
Tholudur A, et al. Using Design of Experiments To Assess Escherichia coli Fermentation Robustness. October 2005
Julien C. Production of Humanlike Recombinant Proteins in Pichia pastoris: From Expression Vector to Fermentation Strategy. January 2006
Kim HY. Improved Expression Vector Activity Using Insulators and Scaffold/Matrix-Attachment Regions for Enhancing Recombinant Protein Production. May 2006 (supplement)
Ludwig DL. Mammalian Expression Cassette Engineering for High-Level Protein Production. May 2006 (supplement)
Shi J, Yang J. Transient Gene Silencing in NS/0 Suspension Cell Culture By siRNA. October 2007
Gerber MA, et al. Integrated Strategies for Clone and Media Formulation Selection. January 2008
Peacock L, Auton KA. Comparing Shaker Flasks with a Single-Use Bioreactor for Growing Yeast Seed Cultures. January 2008
Kunert R, Gach J, Katinger H. Expression of a Fab Fragment in CHO and Pichia pastoris. June 2008
Manzi AE. Carbohydrates and Their Analysis, Part Three: Sensitive Markers and Tools for Bioprocess Monitoring. June 2008
Paul WC. Maintaining Product Titer While Replacing Undefined Components in a CHO Culture System. September 2009
Simula T, Grosvenor S, Scott C. Rethinking Media Performance: Optimizing with Defined, Animal-Free Supplements. September 2009
Langer ES. New Plant Expression Systems Drive Vaccine Innovation and Opportunity. April 2011
Dhulipala P. Differential Cell Culture Media for Single-Cell Cloning. December 2011
Everett K, et al. Development of a Plant-Made Pharmaceutical Production Platform. January 2012
Bogli NC, et al. Large-Scale, Insect-Cellâ€“Based Vaccine Development. May 2012
Keil KM, Tilkins ML. Screening Yeastolate Raw Material Used in Insect Cell Culture Media. January 2013
Marconi PL, et al. Mathematical Model for Production of Recombinant Antibody 14D9 By Nicotiana tabacum Cell Suspension Batch Culture. January 2014
Langer ES, Rader RA. Powders and Bulk Liquids: Economics of Large-Scale Culture Media and Buffer Preparation are Changing. March 2014
Dhanasekharan K, et al. Rapid Development and Scale-Up Through Strategic Partnership: Case Study of an Integrated Approach to Cell-Line and Process Development for Therapeutic Antibodies. June 2014
Oâ€™Kennedy RD. Multivariate Analysis of Biological Additives for Growth Media and Feeds. March 2016
Leupold M, et al. A Stirred, Single-Use, Small-Scale Process Development System: Evaluation for Microbial Cultivation. November 2017 (supplement)
Cheng G, et al. Addressing Quality in Cell-Line Development â€” Direct Analysis of Bioreactor Harvest for Clone Selection and Process Optimization. February 2018 (eBook)
Cherney B, et al. Production Cell-Line Development and Control of Product Consistency During Cultivation â€” Myths, Risks, and Best Practices. April 2018 (eBook)
Scott C. Science Guiding Technology: Cell Line Development and Engineering 2018. September 2018 (supplement)
Yamagata M. Creating Novel Cell Lines By Genome Editing: Simplifying Cell-Based Assays and Improving Production of Biomolecules. September 2019
Scott C. Microbial Expression: Continuing Relevance. March 2020 (eBook)
Gill J. Get to IND Faster: Accelerated and High-Performance Cell-Line Development. June 2020 (supplement)
Mora A, Ezzyat Y. Anticipating Cell-Line Challenges to Drive CMC Readiness. June 2020 (supplement)
Melinek B, et al. Toward a Roadmap for Cell-Free Synthesis in Bioprocessing. September 2020
Gazaille B, et al. Plant-Cell Cultures and Cell Lines for Recombinant Protein Expression. September 2020 (supplement)
Piestun D. Use of CRISPR and Other Gene-Editing Tools in Cell Line Development and Engineering. September 2020 (supplement)
Taron CH, Samuelson JC, Morrison L. Microbial Expression and Purification: One Companyâ€™s Historical Perspective. September 2020 (supplement)
Shimoni Y, Moehrle V. Compounded Media Powder Streamlines Cell Culture Media Preparation Operations. March 2021
Winkler M, et al. Viral Clearance in AAV Purification: Case Study. April 2021
Macdonald G, et al. Engineering Alternatives: Modern Technology Enables Expression System Developers to Think Beyond CHO Cells. May 2021
Scott C. Avenues for Innovation: The Latest in Cell-Line Engineering and Development. May 2021 (supplement)
Scott C, Melinek B. Cell-Free Expression: A Technology with Truly Disruptive Potential. May 2021 (supplement)
Scott C, Tian F. Increasing Expression Titers: New Technologies Could Help Other Cell Lines Catch Up to CHO. May 2021 (supplement)
Gazaille B, Castillo F, Nims RW. Cell Banking in the Spotlight: Advising Biologics Developers About Cell Bank Preparation and Characterization. June 2021 (supplement)
Montgomery SA, Wheelwright S, Poon HF. Contractor Perspectives: Best Practices for Transfer, Handling, Testing, and Storage of Cell Banks. June 2021 (supplement)
Boulanger R, et al. Viral Contamination of Cell Cultures. September 2021
Allard G, et al. Risk Determination of Potential Mycotoxin Exposure to Patients: Testing Recombinant Human Factor VII from Transgenic Rabbits. October 2021
Ding Y, et al. Expression of Recombinant Antibody Fragments: High-Density Fermentation in Multiuse and Single-Use Systems. Januaryâ€“February 2022
Allard A, Evans S. Mycotoxin Risk Determination: Measuring the Potential for Patient Exposure with Antithrombin Alfa Sourced from Transgenic Goat Milk. March 2022
Scott C, Krawitz D. HCP Assay Development: Managing Risks with Evolving Technologies. March 2022 (supplement)
Cossar D, McLean MD, Don Stewart D. Plant-Based Protein Expression: Emerging Systems Bring Viable Approaches to Biopharmaceutical Manufacturing. May 2022
Scott C. Introduction: Cell-Line Engineering and Development at BPI West. May 2022 (supplement)
Scott C, Haley B. Genome Editing for Cell-Line Development. May 2022 (supplement)
Yuval Shimoni is associate director and product quality leader at BioMarin Pharmaceutical, 105 Digital Drive, Novato, CA 94949; email@example.com. Cheryl Scott is cofounder and senior technical editor of BioProcess International, part of Informa Connect Life Sciences, 1-212-600-3429, firstname.lastname@example.org.