Daniel I.C. Wang, an MIT Institute Professor who was considered one of the founding fathers of the field of biochemical engineering, died Saturday in Cambridge, Massachusetts. He was 84.

During his long career at MIT, Wang contributed to many aspects of biochemical engineering — a field that involves genetically engineering microbes and human cells to produce useful proteins. His research spanned all phases of the process, including fermentation, monitoring and control of bioprocesses, enzyme technology, product purification, and protein folding.

In 1985, Wang was the driving force behind the launch of MIT’s Biotechnology Process Engineering Center (BPEC), which was founded as a multidisciplinary research center bringing together faculty from the departments of Biology, Chemistry, and Chemical Engineering.

“Danny’s work and impact in the field of biochemical engineering were profound, and led to a major shift in the growth of chemical engineering at the interface with biology,” says Paula T. Hammond, the David H. Koch Professor and head of the MIT Department of Chemical Engineering. “He extended chemical engineering concepts to bioreactors and the first efforts in bioprocesses, enzyme technology, and mammalian cell cultures, among many other accomplishments. Chemical engineering has lost a giant, and the department has lost a good friend and incredible mentor to our faculty, researchers, and numerous alumni.”

The establishment of MIT’s BPEC coincided with the overall emergence of biotechnology as an industry and a research field. Wang and other early pioneers developed ways to use emerging knowledge about the genetics of microbes to engineer them to produce useful products. Among many other projects, Wang worked on engineering E. coli to increase their production of desired recombinant proteins. He also studied techniques for increasing yields of therapeutic recombinant proteins and monoclonal antibodies from mammalian cells.

He also worked closely with the biotechnology industry and was one of the original members of Biogen’s scientific board, where he was instrumental in the company’s development of the manufacturing of complex biopharmaceuticals.

Born in Nanking, China, Wang worked to establish international ties between MIT and universities in other countries, particularly in Asia. He established a joint program in molecular engineering of biological and chemical systems with the National University of Singapore, which became part of the Singapore-MIT Alliance for Research and Technology (SMART).

Wang, who served as the Chevron Professor of Chemical Engineering before being named an Institute Professor, was also known for his dedication to his students. Noubar Afeyan, a former student of Wang’s who is now the CEO of Flagship Pioneering and a member of the MIT Corporation, described him as a friend and cherished mentor.

“Danny touched thousands all over the world by inspiring generations of students, industrial collaborators, and fellow professors. He was confident yet humble, tough yet caring, serious yet playful, with an insatiable appetite for good Chinese food. We will miss Danny and work hard to make his legacy proud,” Afeyan says.

In recognition of Wang’s pioneering research, MIT’s Frontiers of Biotechnology Lectureship was renamed for him in 2014. Now known as the Daniel I.C. Wang Lecture, the lectureship honors achievements at the frontiers of biotechnology, and the distinguished scientists and engineers responsible for them.

“Dan Wang’s influence as a teacher, mentor, researcher, and friend has been monumental to so many people who have become the leaders in building a biotech industry and biochemical engineering as a profession,” says Charles Cooney, the Robert T. Haslam Professor of Chemical Engineering at MIT. “Though saddened by his passing, we celebrate his legacy of unwavering nurturing of students and colleagues to address challenging problems with innovative solutions.”

Wang earned two degrees from MIT — a BS in 1959 and an MS in 1961. In 1963, he earned a PhD in chemical engineering from the University of Pennsylvania. He joined the MIT faculty in 1965 and was named an Institute Professor, MIT’s highest faculty honor, in 1995. He received numerous honors and awards, including the Amgen Biochemical Engineering Award in 1995 and the William H. Walker Award from the American Institute of Chemical Engineers in 1994. He was also a member both of the National Academy of Engineering and the American Academy of Arts and Sciences.

In 2019, the American Institute of Chemical Engineers established an award in his honor — the D.I.C. Wang Award for Excellence in Biochemical Engineering. The award is given annually and “recognizes individuals for their contributions to the field and to the practice of biochemical engineering through their position in industry or academia as exemplified by Professor Wang in his 50 years of contributions,” according to the AIChE.

Wang also contributed to national efforts in biotechnology, as chair of the Membership Committee of the National Academy of Engineering, a member of the National Biotechnology Policy Board at the National Institute of Health, a member of the National Research Council Committee on Bioprocess Engineering, a member of the National Research Council Committee on Biotechnology, and a member of the Board of Biology of the National Research Council.

He also co-authored five books, published more than 250 papers, and was awarded 15 patents.

Wang is survived by his wife, Victoria; his son, Keith; his daughter-in-law Katherine; his two granddaughters, Veronica and Emily; his sister, Judy, and her family; and his sister-in-law, Cecile. Plans for a memorial will be announced at a later date.

The Beat Goes On

[Let] us examine the case of a new company, now just three years old, and in the newest of emerging technological fields, biotechnology. PerSeptive Biosystems is too young to "prove" anything, but its formative years again show the uncertainty and sometimes even turmoil that need resolution as a start-up finds its own direction and moves forward.

Noubar Afeyan and PerSeptive Biosystems, Inc. Beirut, Lebanon, is the starting point of the story. Noubar Afeyan was born there in 1962 in the Armenian section, the youngest of three sons. His father had had an architecture education in Bulgaria and worked at that profession in Lebanon during the day, but started an import/ export business at night two years before Noubar was born. While Noubar was growing up, his father's business, now a full-time occupation, grew too and Noubar remembers well his childhood impressions of warehouses, the port, unloading ships, rounding up day workers by truck. His childhood was not exactly typical for a to-be biotechnology entrepreneur, with continuing outbreaks of violence eroding the quality of life. But in that polyglot community Noubar received a good education, becoming fluent in Arabic, Armenian, English, and French, while also developing a "street smart" sense. In 1975, the civil war in Lebanon had erupted again and Noubar's parents decided to move the family out of the country. They departed for Montreal, where cousins were already living. One indicator of the trauma they were experiencing is that the Beirut airport closed two days after the Afeyans departed, not to reopen for four years.

During their first year in Montreal, the boys entered Jesuit school while their father tried to start a manufacturing business. Months of frustration led to the elder Afeyan reestablishing his import/export enterprise, which five years later, in 1981, finally led into the manufacture of plastic-covered furniture and related products. Noubar, his brothers, and his mother actively worked at the business whenever needed.

Noubar thought about applying to MIT when he was finishing high school. But concern about leaving his then 86-year-old great-aunt, who had lived with the family throughout his childhood, led Noubar to enroll in McGill University, just one block down the street from his house. Choosing chemical rather than electrical engineering for his undergraduate major was influenced by his father's encouragement that chemical engineering might at least relate somewhat to his plastics activities. The first semi-serious discussions about starting his own company took place regularly with Noubar's undergraduate friend Dave Rich. Together they were going to create ARC-Afeyan-Rich Company, to undertake a variety of creative businesses but nothing real came of these many chats. Noubar's rather remarkable great-aunt, 97 years old in 1990, reminds him that he was always talking about running his own business even when he was a child.

Despite an attractive job offer from Dow Chemical Noubar decided, in 1983, to apply, and got accepted, to MIT's new Ph.D. program in biotechnology process engineering. His work primarily with Professors [Dr. Daniel I-Chyau Wang (born 1936)] and Charles Cooney exposed him to their growing networks of consulting relationships with large and small companies in the United States and abroad, as well as venture capital firms. Noubar generated lots of ideas, leading to impressive experimental findings and he published papers with several MIT faculty. But he had no interest at all in becoming a professor; he clearly wanted all along to be a key player in some company. One of the MIT faculty, Raymond Baddour, gave a seminar on all the alumni of the MIT Chemical Engineering department who had achieved outstanding success (several in association with Baddour's own multiple entrepreneurial efforts; see Appendix, Track 1). That seemed great to Noubar and he wanted to become part of that list.

In 1986, Afeyan enrolled in the New Enterprises course in the Sloan School of Management and had to prepare a business plan. He worked out a semiserious plan, without the financials, for one of his ideas on a protein purification system that he named CARE-Continuous Affinity Recycle Extraction-doing the project alone because he was afraid someone might try to take the idea and run with it. Noubar reports feeling that the Sloan School was entirely foreign to him-the jargon, costume, aspirations of business school students were almost alien to the engineer. Later that year he entered into negotiations with the giant Swedish firms Alfa-Laval and Pharmacia to turn that CARE idea into an actual company, but the deal evaporated when the two large companies had a falling out with each other. The negotiations were not without some lasting benefit; Noubar married one of the women Alfa-Laval had sent over to MIT during the discussions.

In spring 1987, as he was nearing completion of his Ph.D. dissertation, Noubar came to see me for permission to take my course on Corporate Strategies for New Business Development. I initially refused him, as he obviously lacked the formal prerequisite subjects. I told him that I doubted that he could write an acceptable term paper without lots of Sloan School background education. Afeyan's persistence overcame my stubbornness and in the end he prepared one of the best papers in the course, contrasting entry strategies into the biotechnology field of several major chemical companies.

At the same time Noubar had tired of trying to get his own ideas translated into the basis for a new firm. He reluctantly accepted the notion that he seemed to be hearing from everyone that he was too young and inexperienced and needed industry seasoning and was on the verge of accepting a job in industry. Fortuitously, Professor [Dr. Daniel I-Chyau Wang (born 1936)], his thesis chairman, introduced Afeyan to a much older experienced multicompany entrepreneur who was being pushed out by investors from his latest company creation. Noubar, then 24 years old, and the older entrepreneur, 60 years old and wiser from his several company start-ups, hit it off immediately, and by the end of one intensive day of discussions they had more-or-less agreed to start a new company in the area of biotechnology processing. On the very next day several people began warning Noubar that this relationship would not work out, that his partner was too inflexible and had a reputation of being tough to work with. Noubar had concluded that they were a good complementary match. Now he had the chance to translate his many ideas into a real company. Wistfully thinking back to that time, Noubar recalls, "I thought he knew his limitations and that therefore we'd be able to get along. I could create and promote and he could manage the technology development."

In August 1987, Noubar Afeyan became the first Ph.D. graduate from MIT's Center for Bioprocess Engineering, and he was already hard at work getting the new company underway with his partner. During October Afeyan brought me a copy of their first business plan, a proposal for launching Synosys Corporation as a rather generic developer and producer of biotechnology processing hardware, with one of Noubar's system ideas as product number 1. He invited me to become a director and an initial stockholder, but after reading the plan I declined. They hoped to enlist major corporations as their principal financiers and collaborators in a series of strategic alliances. Neither the plan nor its timing was great; the stock market's Black Monday occurred one week after I saw Noubar. Among other effects of the market crash, venture capital companies became more conservative and especially skeptical of new companies focused on capital equipment markets, such as Synosys. Despite the apparent problems Synosys was incorporated at the end of November, the two founders each owning half the company, with the bills being paid by loans to the firm from the older partner.

Months dragged by as they presented their plan to numerous companies and prospective investors. In the meantime Professor Fred Regnier of Purdue University, one of the world's leading experts in separations technology, was recruited to become an advisor to Synosys and began working with Noubar on new approaches to porous materials for biotechnology separations processes. Noubar took a part-time job at MIT in January 1988, working as the technology transfer manager for the BioProcess Engineering Center under Danny Wang's directorship. Much more time was going to be needed to raise the capital to get going and they could use the income and the contacts the job would produce. They also rented a small office in American Twine Office Park, a converted old mill filled with MIT high-technology spin-offs, located just behind MIT in East Cambridge.

With all the critical feedback on their first business plan Noubar wrote a new one in March, aimed at venture capitalists not corporations as the potential funders and following to the letter the guidelines in Jeff Timmons' textbook, New Venture Creation (1985). This plan had a dual focus: the porous separations materials and a biotechnology processing hardware system, aimed at new product developers. Noubar started sending around the new plan and visiting venture capital firms. I received my copy in April 1988, read it and decided to visit the founders in their offices. As I entered I was offered a bench to sit on, just being hammered into completion by Danny Wang's son who was working there part time. The co-founders and I talked at length, especially about my feelings that the materials business was attractive by itself and that the hardware system was a confusing distraction. We also talked about the roles of the two co-founders and whether they would be willing to bring in a more experienced partner as CEO or Executive VP (reminiscent of AR&D's hesitation with DEC). Despite some reservations, upon return to my office I called the biotech specialist at First Stage Capital, a venture capital firm I co-founded and serve as a General Partner, and suggested he look into Synosys in depth. First Stage began working closely with Synosys, criticizing many aspects of their plans but also encouraging their overall efforts.

[Dr. Daniel I-Chyau Wang (born 1936)] began to get more involved and technical progress was being made with the porous materials. But funding decisions dragged on. In June First Stage Capital turned down Synosys because of our unwillingness to fund the hardware part of the company. Noubar borrowed some money from his father and began to pay the bills; his partner was running out of funds and was looking for alternative employment in academia. In August, Wang and several friends invested $200,000 and suddenly things seemed a bit brighter. In September an agency of the Canadian government with which Noubar's father had good relationships indicated that it would be willing to invest $2 million in the company, provided that Synosys would move its hardware activities to Canada. Despite uncertainty as to whether Synosys should or would accept the Canadian funds, that change in prospects was enough to renew First Stage Capital's interest in financing the materials portion of the company.

By November, still without the major capital infusion needed to move ahead decisively, the Synosys team and its consultants developed "perfusion chromatography", an approach to protein purification that could produce a tenfold speed advantage over existing technologies. Finally, or so it seemed, in mid-January 1989 Synosys reached agreement with First Stage Capital and Noubar signed off on a detailed term sheet. Two days later his older partner suddenly announced he was through, claiming family pressures, thus beginning several months of fighting over stock, roles, compensation. Terry Loucks from Rothschild Ventures, who had met Noubar during the fund-raising period, agreed to come in as full-time chairman and CEO, with Noubar becoming president and chief technical officer. On April 1, hopefully not to be remembered later as April Fools' Day, the checks were signed for $1 million for one-third of the company, First Stage Capital being joined in the investment by Raytheon Ventures and 3i, a large British venture capital fund with an office in Boston. In search of a new identity the firm was reincorporated as PerSeptive Biosystems, trying to put the trauma of Synosys Corporation in the background. Two years had elapsed since the day Noubar and his now former older partner had agreed to start a company. The new team now featured Noubar Afeyan, Terry Loucks, and Fred Regnier from Purdue.

People began to be hired, several new Ph.D.s coming in from Purdue and MIT. Patents were filed on the concepts, the materials, and the designs for new processing equipment. The now named "Poros" materials began to generate amazing results in lab tests. In November the PerSeptive team stole the show at a technical symposium in Philadelphia, presenting several papers on the technology and its performance and generating instant sample orders from several large companies.

As of September 1990 PerSeptive Biosystems had 26 employees, a growing group of enthusiastic customers, and newly delivered checks from its second round of venture capital, $3.3 million from its original venture capital investors plus Venrock and Bessemer Securities, the funds needed for expansion of the company and for development of its second product line, an instrument system for automating use of the Poros materials in developmental applications. I asked Noubar what he wanted to accomplish now. "I want to create an analog of Hewlett Packard-tools for a new breed of engineers, bio-process engineers. If the industry grows the way it's expected to, we should be able to reach $40-50 million in sales in five to six years. By the way, becoming rich wouldn't hurt", he added. "There's nothing more motivating to succeed than having to beg for money to get started." N oubar Afeyan was 28 years old.

Compared with the cases presented throughout this book Afeyan shows strong continuity of the earlier patterns. Noubar's father was a professional and an entrepreneur, and Noubar gained much experience with the process of business development while he was growing up. After receiving his MIT degree he had worked only in an MIT lab before setting up his own company at a young age. In fact, Noubar really started PerSeptive on a part-time basis while he was still in graduate school, and his general interests in running his own firm stem from childhood. The technology was transferred directly from his MIT education and lab work as well as from Fred Regnier's work at Purdue. Initial funds came from his co-founder's savings, then from his family and friends, and only later in two large rounds of investments from venture capital firms. [...]

Daniel I.C. Wang: A Tribute to an Inspirational Leader and Colleague

T. Alan Hatton

Department of Chemical Engineering,

Massachusetts Institute of Technology, Cambridge,

Published online 24 August 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.21103

Abstract: Daniel I.C. Wang has been an influential leader of the biotechnology industry over the past four decades through his inspirational research activities, the legions of students and other researchers that have studied under him, his development of many research and educational initiatives, both nationally and internationally, and the advice he has given worldwide to companies, research institutions, universities, and governments.He has played an important role in the mentoring and nurturing of junior faculty members, and has been a supportive collaborator in research and teaching. This two-part article provides a brief overview of Danny Wang’s many contributions to furthering the global development of biotechnology, with particular emphasis on his recent activities in Asia, and concludes with an account of his research collaborations with the author over the past two decades.

INTRODUCTION

Few people are destined to have as major and long-lasting an impact on their profession and on the people around them as has Daniel I.C. Wang—he has made his mark on this world not only through his fathering of the field of biotechnology but also through his significant contributions to the education of generations of undergraduate and graduate students, and through his nurturing of graduate student advisees (often through ‘‘tough love’’) and mentoring of junior faculty. It was my fortune to be one of those faculty members, and while I do not share Danny’s passion for tennis, poker, and Chinese food (although I like them well enough), I have had the privilege of sharing many research interests, graduate students, and professional activities with Danny over the past quarter of a century. This article is a personal reflection on and appreciation of Danny’s collegiality and contributions over the years, and emphasizes the significant impact he has had on the evolution of biotechnology in Asia.

PROGRAMS AND EDUCATIONAL INITIATIVES

Danny Wang has had an unusually strong presence locally and globally, and his contributions to biotechnology and education have been widely recognized. His appointment as one of only 12 Institute Professors at MIT is a significant endorsement of the importance of his teaching, research, and service contributions to the Institute and to the nation, while his early induction into the National Academy of Engineering similarly reflects the esteem others hold for him. He has also garnered numerous other awards and honors from professional institutions, from other institutes of higher learning and from foreign governments and organizations.

DannyWang was the driving force behind the Biotechnology Process Engineering Center (BPEC) at MIT, one of the first, and certainly the most successful and enduring, of the Engineering Centers supported by the National Science Foundation (and discussed at greater length in other articles in this commemorative issue). The remarkable ongoing success of this center two decades later is itself a tribute to the extraordinary vision Danny had for the biotechnology industry and the role academics could play in the evolving industry. Danny was also not averse to including a young and very junior faculty member in on policy deliberations and internal proposal evaluations under this program, an early indication of the care and nurturing that he would provide to a number of other junior faculty members over the ensuing years. Others in this commemorative issue have addressed Danny’s many contributions to the field of biotechnology education and practice in the US, including BPEC, so the focus of this article will be primarily on Danny’s considerable presence and influence in Asia, in particular in Singapore, China, and Taiwan, as well as Hong Kong, Thailand, and Malaysia.

I was fortunate to have worked with Danny (and our colleagues Ken Smith, Jackie Ying, and Paul Laibinis) on the first visits and discussions with various parties in Singapore as we fleshed out our joint program on Molecular Engineering of Biological and Chemical Systems (MEBCS) which was to be one of the five programs under the Singapore-MIT Alliance (SMA). (One of our earliest successes was to persuade Miranda Yap, a long-time Singaporean colleague, tennis partner and friend of Danny’s to take the lead on the Singapore side.) This alliance heralded a bold, new approach to international collaboration in graduate research and education in areas of strategic importance to academia, research institutions, and industry, through both real-time distance education and on-the-ground personal interactions. Undoubtedly, Danny’s keen knowledge and understanding of

the political, industrial, and academic landscapes in Singapore helped us forge a strong program in this area, and his tireless devotion to education resulted in not only his teaching of scores of SMAstudents under this program, often through long-distance education, but also his co-supervision of a number of joint, Singapore-based Ph.D. students in the program. As we begin to enter Phase 2 of the SMA program (now renamed Chemical and Pharmaceutical Engineering), recognition should be given to Danny’s significant imprint on the program structure, and his determination to continue participating as vigorously as ever with no signs of stepping down in the foreseeable future.

During this exciting period, Danny was also appointed to the prestigious Temasek Professorship at the National University of Singapore, and utilized the associated 2-year research grant to develop synergistic research activities between his lab back at MIT (he spent 4–5 months each year in Singapore) and the newly formed Bioprocessing Technology Institute (BTI, an outgrowth of the former Bioprocessing Technology Center, for which he had been an advisor, and which was directed by Miranda Yap). This research was focused on the production, stabilization and formulation of recombinant proteins using mammalian cells, and on glycoprotein quality. Danny has served as Chairman of the Scientific Advisory Board of the BTI for the past 6 years, playing an active role in developing a strategic road map for its future research and development activities, and is on the Board of Directors of BTI-spin-off company, A-Bio Pharma Pte. Ltd. Other influential roles that Danny has played in Singapore include being on the scientific advisory boards of the National Research Council (NRC) and more recently of ICES (the Institute of Chemical and Engineering Sciences), as well as serving as an ad hoc advisor to the Economic Development Board (EDB) on their investments in the biotechnology area.

Even prior to the SMA program, Danny was involved with the NUS, having served on their Search Committee for the Head of the Chemical Engineering Department, and subsequently leading a trio of MIT faculty members that included colleague Ken Smith and myself as an Advisory Group for the Dean of Engineering at NUS to examine the education and research programs for the Department of Chemical Engineering. This was my first introduction to Singapore, and Danny’s insight and understanding of the city-state made it an enriching experience, both culturally and professionally.

But Singapore is not the only country in Asia in which Danny has played a significant role. He continues to be active in promoting ties between universities on the Chinese mainland and MIT, and has delivered his short courses on Biotechnology and Bioprocess Engineering at a large number of these institutions, and keynote lectures at several of them; his contributions have been recognized through the bestowment of Honorary Professorships at three Chinese universities. As advisor to the China National Biotechnology Center, Danny has assisted in its research and development programs in biotechnology, while his interactions with the private sector in China have been instrumental in transferring a US company’s technology into the pharmaceutical manufacturing industry in China, partly through organizing technical seminars, one in Beijing and the other in Shanghai, to over 50 Chinese pharmaceutical companies. He also worked to establish a joint venture between a US company and China, which required an examination of the technical capabilities in genomics and proteomics in that country where collaborations might be achieved through such a venture. Finally, Danny has been involved in several start-up companies founded by his former students and post-docs by assisting them in networking and finding equity funding; clearly, he never relinquishes his role as mentor to all who have been fortunate to have worked with him.

Across the straits, Danny played an influential role in the Taiwanese public sector also, having participated in a number of strategic analyses of the biotechnology industries there, at the requests of both the Minister of State and the Vice Minister of Economic Affairs. These analyses identified the strengths and weaknesses in these sectors, and noted in which areas joint ventures with US companies would most likely be fruitful. As a member of the Technical Advisory Group of the Biomedical Engineering Center, ITRI, he was responsible for leading similar strategic assessments of the research and development activities of this center, and established close research interactions between the Center and MIT.

Hong Kong’s public and private sectors were also not immune to Danny’s considerable influence (and charm!) as he headed an MIT team commissioned to analyze the various commercial sectors in that city. These studies resulted in the book ‘‘Made in Hong Kong,’’ in which the biotechnology section recommended that Hong Kong focus on developing the field of Chinese medicine; as a direct consequence of this advice, the Hong Kong Jockey Club provided funding for a new Institute of Chinese Medicine to support research to modernize this field. For these efforts, Danny was awarded an Honorary Doctorate in Engineering by the Hong Kong University of Science and Technology.

I have had the opportunity to work with Danny in Thailand as well, where he, together with our colleague Greg McRae, opened the way for us to assist King Mongut’s University of Technology, Thonburi (KMUTT) in establishing a very successful Chemical Engineering Practice School program modeled after the 90-year-old program at MIT.

COLLABORATIVE RESEARCH INTERESTS

The contributions Danny Wang has made to the global biotechnology sector through his advisory services, consulting and educational initiatives are impressive. But the foundations for his deep appreciation and understanding of these sectors must derive from the vigorous research program he has pursued over the past four decades or more. It is difficult, if not impossible, to capture in a short piece the wide range and scope of the research areas that Danny has tackled over the years, so this article provides only a rather personal overview of a limited subset of his vast research program, restricted to those areas on which Danny and I have collaborated and co-supervised students and post-docs over the past two decades. In many respects, our joint work on harnessing colloids to effect potential large-scale processing of biological systems, has helped shape my appreciation of the field of biotechnology, and just as importantly, the man behind the field. I apologize in advance that no specific references are given to the often-significant prior work of others that have paved the way for the research discussed here; these works can be found in the literature cited in the references in our papers.

RECOVERY OF AMINO ACIDS

The recovery of primary and secondary metabolites has long of been of interest to the bioprocessing industries, with solvent extraction recognized as a potentially competitive approach relative to other methods such as adsorption, precipitation, chromatography, distillation, membrane separation, crystallization, ion exchange, and electrodialysis. As Danny was well aware, it becomes all the more imperative to remove and recover the product in situ from the fermentation medium in which it is produced when the product can be inhibitory to its own production, as in the case of phenylalanine. He pointed out that significant advantages in production rates and final titer could be anticipated if the phenylalanine were to be removed and concentrated as it was formed, so that the concentration in the fermentation medium was maintained at a sufficiently low level as not to be inhibitory. We felt that perhaps solvent extraction using the emulsion liquid membranes originally developed by Norman Li when at Exxon might be an effective way to approach this problem.

Typical solvent extraction processes require first the selective extraction of one component in an extraction step, followed by contacting with a second, stripping phase to recover the purified product. If conditions are selected appropriately, then some degree of concentration of the product can be achieved in the stripping step. Conventional solvent extraction is limited by the capacity of the solvent phase for the solutes of interest, and would be enhanced dramatically if the extracted solute could be removed immediately by stripping into a second phase, that is, simultaneous extraction and stripping with an added bonus of concentrating the product during the process, rather than by conducting this stripping operation in a second stage.

Liquid emulsion membranes were considered to be a potentially powerful method for the simultaneous recovery and concentration of bioproducts in a single-step extractionstripping process. Together with graduate student Michael Thien (Itoh et al., 1990a,b; Thien and Hatton, 1988; Thien et al., 1986, 1988), we investigated the utility of such systems for the selective recovery and simultaneous concentration of amino acids. Specifically, an emulsion liquid membrane system consists of a water-in-oil emulsion dispersed as droplets within an aqueous phase. The solute to be recovered is extracted by the oil layers separating the inner and outer aqueous phases and transported by diffusion to the inner aqueous droplets, where the solute is stripped from the oil and, if conditions are selected appropriately, concentrated therein. The driving force for the process can either be simply conversion of the transported species by chemical reaction within the aqueous droplets to an oil-insoluble form (e.g., the conversion of phenol to the phenolate ion by a strong base within the droplets), or the transport can be facilitated by a carrier that complexes with the solute on the feed side, and then is stripped of this solute by a reverse reaction within the droplets. In all cases, it is this continual stripping of the oil phase by the internal aqueous droplets that overcomes the typical solubility capacity limitations of solvents in traditional liquid-liquid extraction operations, and that maintains a large mass transfer driving force into the emulsion droplets; the extraction process is now limited by the capacity of the internal aqueous droplets for the solutes, which is determined by the concentration of an appropriate reagent in these droplets—this reagent could be a strong base, such as NaOH in the case of phenol extraction, or a salt such as NaCl to drive the reverse reaction in carrier facilitated transport processes. In the latter case, it is the counter-transport of the chloride ion that drives the solute transport and the initial concentration of the chloride ion in the aqueous droplets that eventually limits the extent of extraction of the solute. In many cases, the final solute concentration in the droplets can be many-fold higher than the solute feed concentration, and thus the process consists of extraction, stripping, and solute concentration in a single unit operation that is more efficient than either of the extraction or stripping units in conventional liquid-liquid extraction operations. This advantage comes at a price, of course, as reflected in the need for other ancillary equipment that is not normally required for conventional applications— it is necessary to first form the emulsion phase before dispersing it within the feed stream, and then to break the emulsion to permit recovery of the solute entrapped within the aqueous droplets.

The initial solute fluxes into the emulsion droplets were found to increase with increasing carrier concentration, but so too was the rate at which water was transferred to the inner receiving phase droplets under an osmotic driving force. This latter ‘‘swelling’’ effect was mediated also by the surfactant used to stabilize the emulsion, and the water fluxes were generally noted to remain unchanged during the extraction/ stripping process, while the solute flux decreased over time as the capacity of the internal droplets for the solute was approached. These observations, coupled with the results reported by others in the field, allowed for the formulation of specific guidelines for the selection of the carrier, the solvent, the counterion and the emulsion-stabilizing surfactant to maximize solute uptake while minimizing the swelling that inevitably occurs owing to hydration of the transported species, carriers, and surfactants.We noted that the extraction efficiency correlated directly with the hydrophobicity of the amino acid residue, and that it could be reduced by the cotransport of inorganic anions in the feed phase that competed with the target amino acids for complexation with the carrier molecules. But, with careful design of the carrier molecules, and selection of the emulsion-forming components, these problems should be containable.

PROTEIN RECOVERY AND PROCESSING USING REVERSED MICELLES

At about this time the genetic engineering of microorganisms to produce recombinant proteins in non-native hosts was a rapidly developing technology ripe for exploitation, and there was a great interest in the recovery of recombinant proteins from the milieux in which they were produced, either in their active folded form, or as inclusion bodies which needed to dissolved before the proteins could be recovered as individual entities and refolded to their native, active state. Liquid-liquid extraction was considered a potentially viable approach for the continuous, large-scale recovery of proteins. The usual class of organic solvents so well used in the metallurgical, chemical, and petrochemical industries, and in the production of antibiotics, was not directly suitable for recovery of proteins, since these biomolecules were generally not soluble in such solvents, and were often denatured by exposure to them. There had been extensive studies on the use of two-phase aqueous polymer systems for protein separations by, for example, research groups at Lund University and Maria Kula’s group at Julich, but we adopted a different approach, stimulated by the early work of Luisi at ETH who noted that under certain conditions proteins can be transferred from an aqueous phase and solubilized in organic solvents by encapsulation within reversed micelles. Our initial studies in this area were focused on demonstrating that this approach could be used effectively for the large-scale extraction of proteins, with an emphasis on modifying the specificity of the extraction process by exploiting electrostatic, hydrophobic, and size exclusion interactions, but it was quickly realized that we should be able to enhance the selectivity by incorporating affinity ligands in the reversed micellar phase. This was accomplished by developing cosurfactants with hydrophobic tail groups and hydrophilic headgroups having particular affinity for a targeted protein. One of the issues to be considered was the thermodynamic description of the binding equilibria in such systems where the distribution of the affinity ligand itself between the two phases (aqueous feed and reversed micellar extractant) was affected by the attachment of the protein to the headgroup, while incorporation of the co-surfactant into the reversed micelle itself could alter the structure of the aggregates, hence also altering the hosting environment for the protein in the reversed micelle water pool. The difficulty was to unravel the different effects on the protein partitioning between the two phases. Our joint student, Brian Kelley (Kelley et al., 1993a,b), considered the extraction of concanavalin-A by octyl glucosides, myelin basic protein by natural amphiphiles such as lecithin, and chymotrypsin by alkyl boronic acids, showing that small amounts of the affinity cosurfactants added to the AOT reversed micellar phase both increased the amount of protein extracted and expanded the pH and salt range over which the proteins could be solubilized, with the interactions between the protein and the reversed micellar phase being stronger in the presence of the cosurfactant than in its absence. At lower salt concentrations, the reversed micellar droplets were larger, and able to accommodate even non-specific proteins well; the addition of the cosurfactant at any particular salt concentration expanded the capacity of the reversed micelles for both the specific and non-specific proteins, and thus higher overall protein transfers were obtained at low-salt concentrations, while greater specificity in the protein extraction toward the targeted protein was observed at higher salt concentrations. Through various control studies, it was demonstrated that the interaction between the affinity ligand and the protein was crucial for the enhanced specificity of the process.

Other factors also come into play when considering the extraction efficiency of the affinity cosurfactant systems, not the least of which is the effect of the partitioning of the cosurfactant itself between the two phases. Kelley developed a model for the affinity partitioning process that accounted for interactions between the cosurfactant and the protein, and the interfacial activity of both the free cosurfactant and the protein–cosurfactant complex. Under normal circumstances, it is not possible to estimate the individual partitioning coefficients for the different components, in particular for the protein–cosurfactant complex, by simple partitioning experiments. Kelley overcame this problem by covalently attaching the protein to the affinity surfactant, and was then able to determine precisely the effects of the different interactions on the distribution of the affinity co-surfactant and its complex with the protein between the two phases. The model predicted that there would be an optimum tail length for the affinity cosurfactant— if the tail were too short, then the hydrophobic force pushing the affinity ligand to the reversed micellar phase would be too weak, and the ligand would partition primarily to the aqueous phase. If the tail were too long, then the ligand would partition strongly to the reversed micellar phase and not be as accessible to the targeted protein. The model predictions agreed well with experimental measurements of the partitioning of a-chymotrypsin using boric acid as the affinity ligand attached to alkyl tails of varying length. Again, guidelines were developed for the selection of appropriate protein-ligand systems, with the importance of having smaller dissociation constants being emphasized. For larger dissociation constants, good enhancements in extraction are realized if more water-soluble cosurfactants are used, but these advantages are counteracted by the unacceptable losses of the cosurfactant to the discarded aqueous phase.

PROTEIN REFOLDING IN REVERSED MICELLES

One of the major processing problems faced when expressing proteins in non-natural hosts is that the organisms do not have the appropriate machinery for the proper folding of the proteins, and thus the protein products are often recovered as inclusion bodies, or large aggregates of the unfolded proteins stabilized by hydrophobic effects and often cross-linked by mismatched disulfide bonds. The accepted method for the dissolution of these aggregates and the subsequent refolding and recovery of the proteins as active compounds is to reduce the disulfide links using appropriate reducing agents, and to dissolve the aggregates in strong urea or guanidine chloride solutions. The refolding is achieved by slowly reducing the concentration of the denaturants allowing the proteins to fold naturally to their native state, and then forming the correct disulfide linkages by introduction of appropriate oxidizing agents. It is usually necessary to perform these refolding operations under very dilute conditions to minimize the possibility of two or more proteins interacting to form undesired small oligomeric aggregates, which greatly compromises the volumetric efficiency of these processes. Danny theorized that if we could sequester individual proteins in such a way that their interactions with each other were sterically hindered, so that they were not able to form these aggregates, then maybe we could refold them effectively at higher concentrations than typically possible using traditional approaches. And what better way to do this, he continued, than to encapsulate the proteins individually within the water pools of reversed micelles dispersed in an organic solvent. The reversed micelles themselves do interchange low molecular weight solutes through the continual collision, partial coalescence, and redispersion of the reversed micellar droplets, and this effect could be exploited to modify the conditions to which the protein is exposed by both injection of small amounts of the desired aqueous reagents directly into the organic solution, and by contacting the reversed micellar organic phase with a bulk aqueous solution. The translation of these ideas into practice fell to graduate student Anna Hagen (Hagen et al., 1990a,b), who was persistent in overcoming the many obstacles to the successful completion of this project, but with Danny’s strong support and encouragement, complete it successfully she did. She showed that denatured and reduced proteins (ribonuclease-A in this case) transferred readily from a buffered solution containing guanidine hydrochloride to a reversed micelle phase at conditions under which the native protein was completely excluded by the reversed micelles. The denaturant concentration within the reversed micellar water pools was reduced in a series of extractive steps by contacting the loaded reversed micellar phase with denaturant- free aqueous solutions, and upon the addition of a mixture of reduced and oxidized glutathione added to reoxidize the disulfide bonds, the proteins were found to recover full activity over 24 h. Finally, the refolded and active protein was recovered from the reversed micellar phase by extraction back into a 1.0MKCl aqueous solution containing ethyl acetate. More detailed studies showed that the efficiency of the activity recovery depended strongly on the size of the reversed micelles, as reflected in the water-tosurfactant molar ratio, W0, (the reversed micelle diameter is directly proportional to this quantity), with poor recovery being attained at W0¼5.7, and recoveries similar to those obtained in aqueous solution at W0 approaching 10. In all cases the recovery was compromised at higher concentrations of the denatured proteins owing to the increasing level of inter-protein interactions, even when the proteins were sequestered within the reversed micelles, because of the continual collisions, partial coalescence and re-dispersion of the droplets (indeed, it is similar processes that are needed to enable the proteins to be extracted by and stripped from the reversed micellar phase, for the denaturant to be removed from the reversed micelles and the oxidizing reagents to be added to the water pools). Other complications included the strong interactions of hydrophobic proteins with the reversed micellar components that inhibited the refolding of such classes of proteins. Thus, while the processing advantages originally envisaged for reversed micellar-assisted refolding of proteins were not forthcoming, this rather unique approach was shown to be feasible, and demonstrated once again Danny’s ability to conceive and execute novel concepts in bioprocessing operations, and to motivate students to think out of the box and to be driven to succeed.

ENZYMATIC CATALYSIS IN REVERSED MICELLES

Modification of the catalytic reaction profiles and changes in substrate specificity of enzymes can expand and enhance their utility in a wide range of applications. One way in which we can attain these goals is to modify the environment of the proteins to either control the structural attributes of the proteins or to modulate their access to substrates, co-factors, and the like. The encapsulation and sequestration of enzymes within the constrained environment of reversed micellar water pools had been recognized by a number of workers, most prominently those in the groups of Luisi at ETH and Martinek in Moscow, as being a possible route to enhancing the activity of these biocatalysts. In many cases, there was a lack of in-depth understanding of the factors responsible for the modified catalytic activity. With Andy Bommarius (Bommarius et al., 1990, 1995), we investigated the reactivity of xanthine oxidase in reversed micellar systems by looking at the oxidation of a range of substituted benzaldehydes of differing electronic and hydrophobic properties, correlating the relevant parameters in terms of linear free energy relationships (LFERs). The kcat was found to depend mostly on the substituent constant while the 1/KMA correlated well with the partitioning constant p.

MAGNETIC NANOPARTICLES FOR PROTEIN RECOVERY

In our more recent endeavors, we have explored the use of magnetic nanoparticles in biological processing applications, both for protein separations and for the enhancement of oxygen mass transfer rates in fermentation processes (this latter application to be discussed later). Functionalized nanoparticles have the desirable property that they can be dispersed as a stable colloidal suspension within an aqueous feed phase to provide an effective single-phase system hydrodynamically (and thereby avoid the problems with multiphase flows and contacting efficiencies), but with the adsorption characteristics of a two-phase system of large surface area per unit adsorbent volume. Since the interparticle distances are on the order of the particle diameter, mass transfer resistances from the bulk fluid phase to the particles are negligible. The recovery of the particles would normally be a problem, however, as the typical methods of nanofiltration or ultracentrifugation would counteract the advantages of using the nanoparticles in the first place. But, if the nanoparticles are superparamagnetic,we argued, they can be recovered by high-gradient magnetic separation methods that are well established in the metallurgical industries, albeit for micro- rather than nanoparticles. Through a series of experiments and theoretical analyses, we were able to demonstrate that while individually dispersed 8–10 nm magnetite particles cannot be recovered effectively, when they are clustered into aggregates of size 70–100 nm, they can easily be removed by HGMS, even at high overall flow rates through the column. The issue was then how we should prepare these nanoclusters so that they have a controlled size, are colloidally stable over a wide range of conditions, and have the desired selectivity and capacity for proteins of interest. This task fell to Andre Ditsch (Ditsch et al., 2005a,b; 2006), a Ph.D. CEP student co-supervised with DannyWang, who tenaciously overcame many obstacles in developing ways to synthesize these clusters. The particles were prepared by the common method of chemical co-precipitation of ferrous and ferric oxides on addition of ammonium hydroxide at an elevated temperature, in the presence of a stabilizing polymer that attached to the particles as they formed to arrest their growth and to stabilize them against aggregation and precipitation.We used copolymers of acrylic acid (carboxyl groups coordinate with iron atoms on the particle surface to anchor the polymer coating), vinyl sulfonic acid (to provide the desired cation exchange properties) and styrene sulfonic acid (which, in addition to cation exchange properties, also allowed for hydrophobic interactions). It was discovered that the size of the clusters could be controlled by using a stoichiometric deficiency of the polymer, which yielded incipiently unstable clusters; these clusters could be stabilized against further changes in solution conditions (particularly high-salt concentrations) by the addition of a secondary polymer that coordinated with the free surface and provided additional stabilization against agglomeration. For stabilization against 5 M monovalent salts, low-molecular weight acrylic acid was sufficient as the secondary polymer, but for harsher conditions as found in fermentation media, a graft copolymer of poy(ethylene oxide) arms attached to an acrylic acid backbone was found to be a superior polymer for the stabilization of the nanoclusters. These nanoclusters were found to have high capacity for proteins, which decreased with increasing ionic strength owing to electrostatic screening effects. At highionic strengths, however, the adsorption of hydrophobic proteins increased owing to salting out effects, providing another handle for selective separation of proteins. It was then demonstrated that the nanoclusters could be used effectively for the recovery and purification of a small protein, drosomycin, from a clarified fermentation broth.

ENHANCEMENT IN FERMENTATION PROCESSES

It has long been recognized that the rate of oxygen uptake by cells during fermentation processes is often limited not by the metabolic rates of reaction, but by the rate at which oxygen can be supplied to the fermentation medium from the oxygen source, and various means for enhancing the overall transport rates have been proposed in the past. One approach was to add a second, water-immiscible phase with higher absorptive capacity for oxygen than that of water to the fermentation medium itself, although generally water-in-solvent type dispersions had been used. One particularly important class of solvents is the perfluorocarbons, which have 20 times the oxygen capacity of water, and oxygen diffusivities up to twice those observed for water. Danny argued that small droplets of the perfluorocarbon dispersed within the aqueous medium should provide better operational control on the oxygen transfer rates, since the droplets should adsorb at the air bubble-water interface, quickly pick up oxygen, and then detach from the bubbles and redisperse within the aqueous medium, where they could release the oxygen as required by its metabolic consumption rate. The questions raised were threefold: (i) whether this concept was valid; (ii) what were the theoretical underpinnings for the enhancement of oxygen mass transfer for this process; and (iii) how was one to determine accurately the actual oxygen tensions in the aqueous phase under the dynamic conditions of the fermentation process. In the latter case, oxygen electrodes, in which an electrochemical cell was separated from the fermentation broth by a semipermeable membrane, were fine for use under fairly stable oxygen conditions, but were found to have insufficient dynamic response to capture rapidly changing dissolved oxygen concentrations, and thus Danny suggested that we look to new, faster ways to measure the oxygen concentrations dynamically and in situ. And so it was left to Beth Junker (Junker et al., 1988, 1990a,b) to develop a successful fluorescence-based probe to satisfy these requirements, and to exploit her newly found capability to provide true measurements of mass transfer coefficients in airsparged fermentation vessels. This required the introduction of specific oxygen-sensitive fluorophores into either the fermentation medium or the perfluorocarbon phase, optical excitation of these probes within the dispersion, and in situ measurement of the fluorescence emissions using fiber optic probes.With these probes, Junker was able to show that mass transfer enhancement factors for the dispersions relative to those in the absence of the dispersed perfluorocarbons ranged from 1 to 4 over the perfluorocarbon loading range of 0%–50%, where the perfluorocarbon was dispersed as drops within the aqueous phase, with dramatic increases of up to 25-fold in the enhancements at higher solvent loadings when the perfluorocarbon was the continuous phase. These strong enhancement gains for water-dispersed systems must be weighed against the loss in volumetric productivity associated with the smaller aqueous phase volumes for the same total fermentor volume, however. Existing models were inadequate to describe the overall enhancements, and thus Junker worked on developing more detailed mathematical models that were able to capture the trends in oxygen transfer enhancement with perfluorocarbon loading. For fermentations of interest, an enhancement factor of about 3 or 4 is generally adequate to sustain the maximum growth rate of cells in typical fermentations, which indicated that a dispersed phase volume fraction of about 30%–40% of the perfluorocarbon would be needed to ensure no oxygen transport limitations. One of the realizations was that the large dispersed phase droplets penetrated the mass transfer boundary layer around the gas bubbles to a limited extent only, and hence their full capacity was not completely utilized during the time they were exposed to the bubbles. As is his wont, Danny did not let this problem go away, and, triggered by work that we were doing using functionalized magnetic nanoparticles to capture biological and nonbiological compounds, followed by their recovery using high-gradient magnetic separations (HGMS), he posited that it should be possible to enhance the overall mass transfer rates using a much more finely dispersed phase in the form of appropriately functionalized nanoparticles that were much smaller in size than the mass transfer boundary layers so that the entire nanoparticle could be utilized for the oxygen uptake, and, being colloidal in nature, would have additional mobility within the boundary layer to enhance the transfer rates. Preliminary experiments using a surface aerated stirred cell with magnetite nanoparticles coated with a perfluorocarbons conducted by post doc Seyda Bucak showed that even at low-particle concentrations one could obtain about a 40%–60% increase in oxygen transfer rates, while largerscale experiments done in collaboration with post doc JinYin using the nanoparticles synthesized by Research Scientist Lev Bromberg showed up to a fourfold increase in oxygen transfer rates, with a commensurate increase in cell production and growth rates. Bernat Olle (Olle et al., 2006), a joint Ph.D. CEP student, then took it upon himself to understand the observed transport behavior, and through a series of experiments showed that the very large increases in overall oxygen mass transport rates could be attributed partially to the mere presence of nanoparticles, but primarily due to the increase in interfacial area caused by the change in the air-water interfacial properties owing to adsorption of the nanoparticles at the bubble surface. The effect of the nanoparticles themselves, which accounted for about a 20%–50% mass transport enhancement, is still very much an open question, however, as it is in the heat transfer literature, where similar enhancements in overall heat transfer coefficients have been observed on addition of nanoparticles to the working fluid. This is currently an intriguing area of research, with many unanswered questions, and reflects well Danny’s penchant for looking at old problems in new ways, and posing difficult challenges to his students and colleagues alike. This project was also an important indication of Danny’s continuing efforts to ensure our research programs can also have benefits in the education of the next generation of biological engineers, as he has introduced these approaches into the undergraduate laboratory course on Biological Engineering in the Chemical Engineering Department at MIT.

CONCLUSION

Danny Wang’s irrepressible dynamism and energy show no signs of abating as he completes his seventh decade, and at an age in which most people have long retired, he continues to contribute vigorously to our educational, research, and international programs, championing and furthering the cause of biotechnology and biological engineering. He is still actively involved in research, and in transferring new research results to undergraduate teaching. His contributions to the Singapore-MIT Alliance have been a major factor in the continuing success of our programswith Singapore, as he actively travels globally to promote the program and recruit students. He is highly sought after as a consultant by universities, research institutions, and governments alike, particularly in Asia, and plays a major role in the directions their programs take. But most importantly, Danny continues to be a caring and thoughtful mentor to our young faculty, encouraging and supporting them in their early years; for some of us, this mentoring role never changes, and, even after 25 years, I still rely on Danny for his wisdom and sage advice, for his friendship and collegiality, and for his continuing inspiration and encouragement.

Thank you from all whom you have influenced over the years, Danny!

References

• Bommarius AS, Holzwarth JF, Wang DIC, Hatton TA. 1990. Coalescence and solubilizate exchange in a cationic 4-component reversed micellar system. J Phys Chem 94(18):7232–7239.
• Bommarius AS, Hatton TA,Wang DIC. 1995. Xanthine-oxidase reactivity in reversed micellar systems—A contribution to the prediction of enzymatic-activity in organized media. J Am Chem Soc 117(16): 4515–4523.
• Ditsch A, Lindenmann S, Laibinis PE,Wang DIC, Hatton TA. 2005a. Highgradient magnetic separation of magnetic nanoclusters. Ind Eng Chem Res 44(17):6824–6836.
• Ditsch A, Laibinis PE,Wang DIC, Hatton TA. 2005b. Controlled clustering and enhanced stability of polymer-coated magnetic nanoparticles. Langmuir 21(13):6006–6018.
• Ditsch A, Yin J, Laibinis PE, Wang A, Hatton TA. 2006. Ion-exchange purification of proteins using magnetic nanoclusters. Biotech Progr
• Hagen AJ, Hatton TA, Wang DIC. 1990a. Protein refolding in reversed micelles—Interactions of the protein with micelle components. Biotechnol Bioeng 35(10):966–975.
• Hagen AJ, Hatton TA, Wang DIC. 1990b. Protein refolding in reversed micelles. Biotechnol Bioeng 35(10):955–965.
• Itoh H, Thien MP, Hatton TA, Wang DIC. 1990a. A liquid emulsion membrane process for the separation of amino-acids. Biotechnol Bioeng 35(9):853–860.
• Itoh H, ThienMP, Hatton TA,Wang DIC. 1990b.Water transport mechanism in liquid emulsion membrane process for the separation of amino-acids. J Membr Sci 51(3):309–322.
• JunkerBH,Wang DIC, Hatton TA. 1988. Fluorescence sensing of fermentation parameters using fiber optics. Biotechnol Bioeng 32(1):55–63.
• Junker BH, Hatton TA,Wang DIC. 1990a. Oxygen-transfer enhancement in aqueous perfluorocarbon fermentation systems. 1. Experimentalobservations. Biotechnol Bioeng 35(6):578–585.
• Junker BH,Wang DIC, Hatton TA. 1990b. Oxygen-transfer enhancement in aqueous perfluorocarbon fermentation systems. 2. Theoretical-analysis. Biotechnol Bioeng 35(6):586–597.
• Kelley BD, Wang DIC, Hatton TA. 1993a. Affinity-based reversed micellar protein extraction. 2. Effect of cosurfactant tail length. Biotechnol Bioeng 42(10):1209–1217.
• Kelley BD,Wang DIC, Hatton TA. 1993b. Affinity-based reversed micellar protein extraction. 1. Principles and protein ligand systems. Biotechnol Bioeng 42(10):1199–1208.
• Olle B, Bucak S, Holmes TC, Bromberg L, Hatton TA, Wang DIC. 2006. Enhancement of oxygen mass transfer using functionalized magnetic nanoparticles. Ind Eng Chem Res 45(12):4355–4363.
• Thien MP, Hatton TA. 1988. Liquid emulsion membranes and their applications in biochemical processing. Separation Sci Technol 23 (8–9):819–853.
• Thien MP, Hatton TA, Wang DIC. 1986. Liquid emulsion membranes and their applications in biochemical separations. ACS Symp Ser 314:67–77.
• Thien MP, Hatton TA, Wang DIC. 1988. Separation and concentration of amino-acids using liquid emulsion membranes. Biotechnol Bioeng 32(5):604–615.

Biochemical engineering at MIT emerged in the 1950’s with a focus on the use of fermentation technology for traditional food and beverage processing and the increasing demands of antibiotics production. New discoveries in natural products were creating a need for improvements in large-scale fermentation as an enabling technology. In this context, the biochemical engineering program became a collaboration between biology, chemical engineering and the newer department of nutrition and food science. Its curriculum embraced fundamentals from these three disciplines. With its multidisciplinary roots, the Department of Nutrition and Food Science, home to the Biochemical Engineering Program, reached out in 1965 to hire a young Ph.D. Chemical Engineer from the University of Pennsylvania, Daniel I. C. Wang. After completing his Ph.D. research with Prof. Arthur E. Humphrey on high-temperature shorttime sterilization, the new Dr. Wang spent 2 years in the US Army doing bioprocess research at the Fort Dietrich Biological Research Laboratories. This post-doctoral experience significantly broadened Wang’s experience into fermentation and the nascent technology of animal cell culture.

It was in a backdrop of rapidly emerging scientific discoveries in biology providing a technology push and an increasing appreciation within multiple industries creating a technology pull, that Daniel I. C. Wang joined the Department of Nutrition and Food Science at MIT as an Assistant Professor of Biochemical Engineering. During the next 40 years he would become the primary driver of innovation in both education and multidisciplinary research initiatives that have defined modern Biochemical Engineering. It is interesting to reflect on the evolution of our discipline over these past 40 years as it has changed substantially in many ways while being invariant in the vision of ‘‘engineering of biochemical systems and components over multiple scales’’.

1965–75—ESTABLISHING THE FOUNDATION FOR BIOCHEMICAL ENGINEERING IN FOOD AND FEED PRODUCTION

By the mid 1960’s, when the young Dan Wang joined the Department, the price of corn and soy beans was rising rapidly while global food and feed resources were poorly distributed. The Green revolution was beginning to take effect but projections of food and feed shortages were calling for innovative solutions in production. The concept of single cell protein (SCP) or protein derived from microbial sources emerged as a promising solution to this global problem. Economic SCP production required low-cost, large-scale technologies and created an opportunity to move not only fermentation technology but also cell and protein recovery technology to a higher plane through improved understanding and innovation. During this period we saw research from Wang’s lab on the airlift fermentor, the use of flocculation and membrane processes for cell and protein recovery, the fermentation of formose sugar syrups and hydrocarbons for SCP, and the elucidation of principles for cell disruption by high-pressure homogenization to name a few of the contributions. Keep in mind that while predictions of food prices were escalating rapidly, prices for energy resources such as gas and petroleum were declining, thus, making the conversion of methane, methanol, and n-alkanes to protein very attractive. This work established the platform on which many other researchers began to build the discipline. One could also see interesting excursions into areas that would later become critical to the field; for example, his early work on cell culture on centrifugation of animal cells in 1968 and the recovery of viruses with ultra filtration membranes in 1971 in collaboration with Anthony J. Sinskey.

1975–85—THE ERA OF ENZYME TECHNOLOGY, COMPUTER CONTROL, AND CELLULOSIC BIOMASS CONVERSION

By 1975, the prices of corn and soybean had begun to stabilize and the Green Revolution was beginning to have positive impact on global supplies of corn and soybean. The need for SCP declined but the lessons learned from the drive to improve the fundamentals of fermentation and recovery technologies were of great benefit to the bioprocess industry at large. Furthermore, the education and training of a new cadre of leaders in the discipline of biochemical engineering had a broad confidence building effect on other applications of this technology. Some of Dan’s early students—Charles Cooney, Larry Gasner, Henry Wang, and Richard Mudgett—had become academicians and were building on a multidisciplinary educational paradigm that Wang has established. Furthermore by this time, the Fermentation Technology summer course had moved into its second decade with its focus on fundamental principles with topical applications to industrial problems.

In this period, there was great fascination with the concept of building biochemical systems from purified enzymes. With Wang’s leadership, a large multidisciplinary initiative on the use of enzymes for in vitro, non-ribosomal peptide synthesis was successfully taken to the National Science Foundation for funding. This was a significant accomplishment for several reasons. First, the proposal to NSF was large relative to the more common single investigator project and very risky because no one had set forth to synthesize a molecule as structurally complex as Gramicidin S, with its attendant need for multiple co-factor regeneration, before. NSF was ready for the risk and the scale of the problem. The project required multiple disciplines—chemical engineering, chemistry, and biology—working closely together; this established a close collaboration with George Whitesides, Charles Cooney, Anthony Sinskey, Arnold Demain, and Clark Colton and their students and post-docs. The success of this initiativewas seen in the ability to meet the target of gram quantity synthesis of Gramicidin S, regeneration of bothATP and NADH, and the production and purification of large enzyme complexes.

During this same period, an energy crisis emerged and expectations for petroleum and gas prices reversed themselves as predictions of grave shortages of these much needed commodities generated rapid increases in prices. One solution was to ferment renewable resources as a means to convert solar power, converted into plant material, to liquid fuels. The social and political implications of converting food and feed resources, primarily sugar and starch, to fuel drove the thinking to focus on cellulosic biomass. Once again, under Wang’s leadership, a multidisciplinary team was created to address this large-scale problem with innovative solutions. The concept of direct conversion of cellulosic biomass containing cellulose, hemicellulose, and lignin emerged as a unifying theme as we entered the early 1980s. The Department of Energy was receptive to funding this venture and a team was formed. Many lessons had been learned about large-scale fermentation, enzyme production and purification, and genetic manipulations from prior work in the laboratories of Wang and his collaborators at MIT. These lessons now needed to be applied to a new set of microorganisms growing under strict anaerobic conditions. The research focused not only on ethanol but also on chemicals such as acetic acid, butyric acid, butanol, and acetone. It was necessary to develop the means to genetically modify and improve the performance of obligate anaerobes for cellulose production and product tolerance. The cellulose system for lignocellulose degradation was poorly understood and fundamental research to elucidate the rate-limiting steps was needed. In the end, there was substantial success in demonstrating the direct fermentation of cellulosic biomass to ethanol and other chemical products. Application of the technology at scale was precluded by a decline in energy prices. Now 20 years later interest in this strategy has again emerged as fuel prices rise.

Fermentation emerged as the core technology able to address multiple industrial needs ranging from health care products such as antibiotics; food products such as protein, amino acids, and vitamins; liquid fuels and chemicals such as ethanol and acetic acid; and industrial enzymes. Research continued to address the needs of this core technology in the labs of Wang and his collaborators. Improvement in mass transfer for non-Newtonian mycelial fermentations; understanding the underlying principles of the air-lift fermentor; introduction of novel sensors to monitor performance; and the introduction of computer control to facilitate understanding and enhance process performance were amongst the contributions of this period.

By 1980, recombinant DNA technology was seen as a transformational event and the potential for production of many new human therapeutic products was becoming real. With humble beginnings in the production of heterologous human proteins in bacterial systems, the concept quickly advanced to animal cell culture. It is notable that Wang’s interest in animal cell culture had continued to thrive in the background of major multidisciplinary programs on enzyme synthesis and cellulosic biomass conversion. In 1977, he published on the use of microcarriers for not only animal cell cultivation but also virus production as well as interferon synthesis. Clearly there were lessons to be learned by extending knowledge of traditional fermentation to cell culture andWang’s students and collaborators were building a platform for the future.

1985–95—ADVANCES IN ANIMAL CELL CULTURE, PROTEIN REFOLDING, AND THE BIOTECHNOLOGY PROCESS ENGINEERING CENTER (BPEC)

The events of the early 1980’s were indeed transformational. The biochemical engineering program had become a joint Afeyan and Cooney: Professor Daniel I.C. Wang: A Legacy 207 Biotechnology and Bioengineering. DOI 10.1002/bit program between the Departments of Chemical Engineering and Applied Biology (the new name for the former Department of Nutrition and Food Science); the Biotechnology Process Engineering Center (BPEC) was formed with major funding as an Engineering Research Center from the National Science Foundation with significant support from industry; recombinant DNA technology led to creation of a new Biotechnology industry; multidisciplinary research initiatives became the normal strategy to address large important problems and students were thirsty for multidisciplinary education.

The early recombinant proteins made in Escherichia coli, were overexpressed in large quantity but often accumulated as insoluble and inactive aggregates of improperly folded proteins. Recognizing the need for an engineering solution to this problem, Wang and his collaborators, which included Alan Hatton and Jonathan King, investigated multiple alternatives to refolding proteins in vitro. It became clear that to address the real problems of manufacturing biotherapeutics, one needed increasingly powerful analytical techniques that could track molecular scale events associated with protein synthesis, folding and post-translational modification. The problems of protein folding with bacterialderived recombinant proteins as well as the inability of bacteria to properly catalyze post-translational events such as glycosylation further emphasized the need to use animal cells as a manufacturing method for recombinant proteins.

The platform that DanielWang began in the late 1960’s on biochemical engineering of animal cell culture became a platform for manufacturing many of the important biologics in use today. From the mid 1980’s onward,Wang’s laboratory became a hotbed of innovation in cell culture technology. There was a focus on improving cell cultivation with microcarrier technology, growth medium design, process monitoring, and control and novel bioreactor design. An understanding of mass transfer needs and effects of mechanical shear led to significant improvement in operation. The wide array of approaches and the in depth understanding that evolved from these studies are seen in the numerous publications form this period. An important consequence of this work was the training of many students, not only in animal cell technology, but also in how to bring multidisciplinary and innovative solutions to important problems. The impact these students have had on the nascent biotechnology industry has been very important to the delivery of healthcare globally.

1995–PRESENT—MULTISCALE APPROACHES TO MANUFACTURING BIOTHERAPEUTICS

By 1995, the stage had been set for new paradigms in biochemical engineering research and education. Throughout his 40-year career, Wang with his students and collaborators strove to take a broad view of biochemical engineering that embraced the engineering of biochemical systems and components. Moving into the mid 1990’s, our understanding of biology and the introduction of new analytical tools allowed us to move to the molecular scale to both seek understanding and develop new solutions to important problems. Fundamental approaches to engineering of biochemical systems moved from the reactor to the cell to the metabolic pathway to the proteins themselves. This is seen in the work to evolve from Wang’s laboratory that addresses elucidation of how process operation affects posttranslational modification of proteins and cellular behavior. This is done with the goal of improving manufacturing and thus delivery of important products to improve global health care. The work with collaborators, which included Philip Sharp, Greg Stephanopoulos, Bernhardt Trout, Harvey Lodish, Daniel Blankstein, and Paul Libinis added to those already mentioned speaks to how he reached out to embrace multidisciplinary approaches to complex problems. This has set an example for an untold number of students that not only says reach beyond your areas of scientific comfort but also teach to others what you know and understand.

The story does not end here. As we speak, Prof. Daniel Wang’s laboratory continues to embrace important problems, especially in animal cell culture for biotherapeutics production. As energy prices rise, there is new interest in direct conversion of cellulosic biomass, albeit with the introduction of new molecular-scale understanding and technology that will enable major improvement in the potential for this technology. The lessons learned in biochemical engineering have become the fundamentals of the discipline on which we all continue to build. The students trained have become the next generation of teachers and the industrial leaders. For all this we are grateful and we look forward to more to come.

PUBLICATION HISTORY

• . Food Processing Technologies/Single Cell Proteins
• . Downstream Processing/Bioseparations
• . Fermentation/Biochemical Engineering
• . Bioconversion/Enzyme Technology
• . Cell Culture Technology
• . Protein Analysis/Product Characterization

LOOKING AHEAD

The Symposium to Honor Daniel I.C. Wang took place on April 22, 2006 at MIT (Fig. 4 is from the symposium announcement). It brought together over 150 participants representing academia, large pharmaceutical, and chemical companies, mid-size biotech companies and start-ups. Attendees came from China, Japan, Singapore, Australia, New Zealand, Europe, South America, and North America. During this symposium, participants were invited to work together in groups identifying current challenges and offering predictions for the future within several sectors. Four groups were formed covering Cell Culture, Downstream Processing, Industrial and Enzyme Biotechnology, and Education. The following is a summary report from these working group sessions.

Bioseparations

The challenges foreseen in the Downstream Processing arena included very large-scale production of biotherapeutics through cell culture. Without productivity increases in bioseparations, the upstream improvements in throughput will not to translate into overall gains. Another challenge identified by this working group is the one posed by rapid development cycles required to enter into clinical trials. Specifically, the industry anticipates requirements for a 6–8 weeks development cycle for purification processes to produce clinical grade material for Phase I trials. The last challenge in this area was viewed to be the extreme conservatism towards adopting new technologies and innovations that exists in major biotechnology and pharmaceutical companies.

The Bioseparations working group predicted several solutions would emerge to address the current challenges. These include the development of very high-capacity chromatographic resins (e.g., Protein A resins with 150 g/L binding capacity), high-throughput screening of diverse chromatographic resins for rapid methods development, very large-scale separation methods such as liquid extraction or precipitation applied to proteins, magnetic nanoparticles, fast and precise analytical techniques, and highly specific affinity adsorbents.

Cell Culture

The working group on cell culture technologies identified as the major challenges in their sector, capacity, cost, speed, and quality. As the production scale of certain biologic drugs has increased during the past decade, there is a need for more efficient and scaleable technologies. Given the regulatory environment, it is difficult to move lab innovations into a production setting without extensive validation trials with the attendant cost and time investments. Biogenerics and followon biologics are also expected to pose new challenges for cell culture processes. Post-translational modifications of biologics derived from cell culture continue to represent a significant challenge.

These challenges will represent opportunities for future generations of biochemical engineers. The tools available to them range from engineering cells to better control glycosilation or enabling non-mammalian production systems, to higher yield cell lines and better cell culture devices. In addition, with the advent of better and faster analytical techniques, the development of such processes will dramatically accelerate. There remains a significant need for training related to cell culture processes within industry and academia as well as within the FDA, as the number and complexity of glycosylated biotherapeutics increases rapidly.

Industrial and Enzyme Biotechnology

Several challenges were identified within the area of industrial biotechnology. These include the relative lack of robustness of enzymes and microbial strains as compared to heterogeneous catalysts; the lack of reliable design capabilities for protein catalysts or cells with modified metabolic pathways; and challenges in fermentation of mixed sugars (e.g., pentose and hexose sugars derived from lignocellulosic biomass). Solutions to these challenges are expected to come from hybrid biological and chemical processes, engineered crops with superior properties for the intended use, and systems biology approaches applied to microbial systems to facilitate engineering of pathways.

The industrial biotechnology working group foresaw opportunities in integrating cellulosic feedstock into starchto- ethanol plants. In addition, significant reduction in process development times will be needed for this sector to meet the growing needs for biofuels and other specialty chemicals derived from agricultural products and waste. Also needed are significant improvements in pretreatment and ability to handle multiple feedstocks as well as in the waste treatment aspects of such bioprocesses. The group predicted that within 10 years designed enzymes and organisms would be in common use and molecules derived from renewable feedstocks such as carbohydrates and lipids would displace many petroleum-derived products in use today.

The industrial biotechnology area is one in which Prof. Wang made seminal contributions three decades ago. With the recent increase in the price of gasoline to exceed $70 per barrel, many of the technologies developed decades ago are now being rediscovered, improved upon, and applied at large scale. In addition to the renewed commercial attractiveness of such processes, molecular biology advances during the intervening period have made industrial biotechnology more predictable and productive than ever before.

A final note of caution was provided by the industrial biotechnology working group concerning the potential shortage of trained biochemical engineers who can work on engineering better enzymes, cells, and processes to capitalize on the emerging opportunities. The overwhelming majority of life sciences research funding in universities during the past decades has focused on health care applications.Without a significant re-emphasis on traditional biochemical engineering education, this shortage will be a major competitive disadvantage to the US.

Education

The educational challenges within Biochemical Engineering were viewed by the working group to stem from the integration of ‘‘bio’’ within all components of engineering and science schools. As a result, undergraduates considering this field are often confused as are administrators within universities. In particular, incoming students often are not presented with sufficient information to distinguish biochemical engineering, biomedical engineering, biological engineering, and chemical engineering when choosing a major.

Instead of building a core curriculum that simply borrows courses from other disciplines, the group recommended that a core, integrated biochemical engineering curriculum based on shared principles, concepts and models be developed and taught.With the explosive increases in biological knowledge and innovative technologies available today, the textbooks, curriculum, and approaches to training experts in this field will need to be constantly updated. While doing this, it will be also important to maintain a clear set of core courses, including such foundational topics as fermentation and cell culture, which can be invariant even while new applications emerge over time. A strong educational foundation will be especially important to US educated personnel to be able to compete effectively within the current trend towards increasingly outsourcing the design and manufacture of biochemical products to China and India.

Educating students as well as academic and industrial colleagues is perhaps the most significant of the contributions made to the field by Prof. Wang. The challenge and responsibility of educating future generations of biochemical engineers lay on the shoulders of Prof. Wang’s academic family and friends. The highest form of compliment this community can pay to his legacy is a renewed commitment to excellence in educating the future, so-called ‘‘Danny Wangs’’, and following the path Prof.Wang has ably laid for us during the past four decades.

REFERENENCES

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• Ditsch A, Lindermann S, Laibinis P, Wang DIC, Hatton TA. 2005a. Highgradient magnetic separation of magnetic nanoclusters. Ind Eng Chem Research 44:6824–6836.
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• Fox SR,YapM,Wang DIC. 2004. Maximizing interferon-gamma by chinese hamster ovary cells through temperature shift optimization. Biotechnol Bioeng 85(2):177–184.
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• Nestaas E, Wang DIC. 1981b. A new sensor, ‘‘the filtration probe’’, for quantitative characterization of the penicillin fermentation: I. Mycelial morphology and culture activity. Biotechnol Bioeng 23:2803.
• Nestaas E, Wang DIC. 1983a. Computer control of the penicillin fermentation using the filtration probe in conjunction with a structured process model. Biotechnol Bioeng 25:781.
• Nestaas E, Wang DIC. 1983b. A new sensor, the ‘‘filtration probe’’, for quantitative characterization of the penicillin fermentation: III. An automatically operating probe. Biotechnol Bioeng 25:1981.
• Nestaas E, Wang DIC, Suzuki H, Evans LB. 1981. A new sensor, ‘‘the filtration probe’’, for the quantitative characterization of the penicillin fermentation: II. The monitor of mycelial growth. Biotechnol Bioeng 23:2105.
• Nyberg GB, Balcarcel RR, Follstad BD, Stephanopolous G, Wang DIC. 1998a. Metabolism of peptide amino acids by Chinese hamster ovary cells grown in a complex medium. Biotechnol Bioeng 62:324–335.
• Nyberg GB, Balcarcel RR, Follstad BD, Stephanopolous G, Wang DIC. 1998b. Metabolic effects on recombinant interferon-g glycosylation in continuous culture of Chinese hamster ovary cells. Biotechnol Bioeng 63:336–347.
• Paliwal SK, Nadler TK, Wang DIC, Regnier FE. 1993. Automated process monitoring of monoclonal antibody production. Ana Chem 65:3363– 3367.
• Park HW, Wang DIC, Yarmush ML. 1992. A rapid, simple immunofluorometric assay: Development and characterization. Biotechnol Bioeng 37: 40, 313.
• Perry SD,Wang DIC. 1989. Fiber bed reactor design for animal cell culture. Biotechnol Bioeng 34:1.
• Rangel-Yagui CO, Lam H, Kamei DT, Wang DIC, Pessoa-Jr. A, Blankschtein D. 2003. Glucose-6-phosphate dehydrogenase partitioning in two-phase aqueous mixed (nonionic/cationic) micellar systems. Biotechnol Bioeng 82:445–456.
• Robinson DK, Wang DIC. 1987. A novel bioreactor for biopolymer production. Ann NYAcad Sci 506:229.
• Robinson DK, Wang DIC. 1988. A transport controlled bioreactor for the simultaneous production and concentration of Xanthan gum. Biotech Progress 4:231.
• Schilling BM, Alvarez LM, Wang DIC, Cooney CL. 2002. Continuous desulfurization of dibenzothiophene with Rhodococcus rhodochrous IGTS8 (ATCC 53968). Biotechnol Prog 18:1207–1213.
• Shabtai J, Wang DIC. 1990. Production of emulsion in a fermentation process using soy bean oil in a carbon nitrogen coordinated feed. Biotechnol Bioeng 35:753.
• Singhvi R, Stephanopoulos GN, Wang DIC. 1992. Effect of substratum morphology on animal cell adhesion and behavior. Material Res Soc Proc 252:237.
• Singhvi R, Stephanopoulos GN, Wang DIC. 1994a. Effect of substratum morphology on cell physiology. Biotechnol Bioeng 43:764–771.
• Singhvi R,Kumar A, LopezGP, Stephanopoulos GN,Wang DIC, Whitesides GM, Ingbar DE. 1994b. Engineering cell shape and function. Science 264:696–698.
• Singhvi R, Schorr C, O’Hara C, Xie L, Wang DIC. 1996. Clarification of animal cell culture process fluids using depth microfiltration. BioPharm 10:35–41.
• Sinskey AJ, Chu G, Wang DIC. 1971. Recovery and purification of viruses through ultrafiltration. Chem Eng Symposium Ser 67(108):75.
• Sinskey AJ, Fleischaker RJ,TyoMA,Giard DJ,Wang DIC. 1981. Production of cell-derived products:Virus and interferon. AnnNYAcad Sci 369:47.
• Smiley A, Hu WS, Wang DIC. 1989. Production of human interferon by recombinant mammalian cells cultivated on microcarriers. Biotechnol Bioeng 33:1181.
• Speed MA, Wang DIC, King JA. 1995. Multimeric intermediates in the pathway to the aggregated inclusion body state for P22 tailspike polypeptide chains. Protein Sci 4:900–908.
• Speed MA, Wang DIC, King JA, 1996. Specific aggregation of partially folded polypeptide chains: The molecular basis of inclusion body composition. Nat Biotechnol 14:1283–1287.
• Speed MA, King JA, Wang DIC. 1997a. Polymerization mechanism of polypeptide chain aggregation. Biotechnol Bioeng 54:333–343.
• Speed MA, Morshead T, Wang DIC, King JA. 1997b. Conformation of P22 tailspike folding and aggregation intermediaries probed by monoclonal antibodies. Protein Sci 6:99–108.
• Stramando JG, Avgerinos GC, Costa JM, Colton CK, Wang DIC. 1981. Inhibition and enzyme destabilization. In: Vezina C, editor. Advances in biotechnology, Vol. 3. London: Pergamon Press, 101.
• Stramondo JG, Solomon BA, Colton CK,Wang DIC. 1978. ATP Utilization during gramicidin S synthesis. AIChE Symp Ser 74(172):1.
• ThienMP, Hatton TA,Wang DIC. 1987a. The separation of amino acids from fermentation broth using liquid emulsion membranes. Proceedings of ISEC’86, Munich, Germany.
• Thien MP, Hatton TA, Wang DIC. 1987b. Liquid emulsion membranes and their applications in biochemical separations. Am Chem Soc Symp Ser 314:67.
• Thien MP, Hatton TA, Wang DIC. 1989. Separation and concentration of amino acids using liquid emulsion membranes. Biotechnol Bioeng 32:604.
• Tyo M, Wang DIC. 1981. Engineering characterization of animal cell and virus production using controlled charge microcarriers. In: Moo Young M, RobinsonCW, Vezina C, editors. Advances in biotechnology, Vol. 1.
• London: Pergamon Press, 141. Tzeng CH, Thrasher KD, Montgomery JP, Hamilton BK,Wang DIC. 1975. High productivity tank fermentation for gramicidin S synthesis. Biotechnol Bioeng 17:143.
• Van Dyke D, Wang DIC, Goldblith SA. 1969. The dielectric loss factor of reconstituted ground beef. The effect of chemical composition. Food Technol 22:1266.
• Wang DIC. 1968a. In: Mateles RI, Tannenbaum SR, editors. Cell recovery in single-cell protein. Cambridge, MA: MIT Press.
• Wang DIC. 1968b. Protein from petroleum. Chem Eng 75(18):99.
• Wang DIC. 1969a. Protein recovery problems, in engineering of unconventional protein production. Bieber H, ed., CEP Symposium Series, 95, 66.
• Wang DIC. 1969b. Introduction to the symposium on engineering aspects of fermentation. Biotechnol Bioeng 11:581.
• Wang DIC. 1982. Production of biofuels by anaerobic fermentation. Proceedings of the 1st ASEANWorkshop on Fermentation Technology, 430.
• Wang DIC. 1984. Biotechnology: Past, present and future. In: Grunwald H, editor. Chemistry for the future. New York: Pergamon Press, 41.
• Wang DIC. 1985a. Production of bulk chemicals through bioconversion rates. In: Ghose TH, editor. Biotechnology and bioprocess engineering. New Delhi: United Indian Press, 515.
• Wang DIC. 1985b. Animal cell cultivation: Its biotechnology implications. In: ‘‘The World Biotech Report, 1985’’, 3, 217.
• Wang DIC. 1986. Biotechnology process engineering center. In: The new engineering research center.Washington, DC: National Academy Press, 107.
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• Wang DIC. 1987b. Designing and implementing an engineering research center. Proceedings on the National Conference on Collaborative Initiatives in Biotechnology, Montgomery High Technology Council, 191.
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• Wang DIC. 1988. Biotechnology: Status and perspectives. AIChE Monograph Ser 84(18):1–22.
• Wang DIC. 1991. Bioprocess engineering in agro-biotechnology. Biotechnologie avanzate e agricultura. Italy: Bologna Feirara.
• Wang DIC. 1992a. Putting biotechnology to work: Bioprocess engineering. Washington, DC: National Academy of Sciences.
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• Wang DIC. 1993a. Sensors for bioprocess monitoring and control: bioproducts and bioprocesses 2. Yoshida T, Tanner RD, editors. New York: Springer-Verlag, pp. 167–178.
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• Wang DIC, ChiouTW. 1990. Animal cell culture engineering. ‘‘Proceedings of the Asia-Pacific Biochemical Engineering Conference 090’’, Kjungju, Korea, p. 3.
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• Wang DIC, Gbewonyo K. 1982. Modern concepts in biotechnologyenhanced oxygen transfer in mycelial fermentations. Proceedings of the 1st ASEAN Workshop on Fermentation Technology, 311.
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• Wang DIC, Hagen AJ. 1990. Protein Refolding Process, Korean Institute of Chemical Engineering Symposium Series, 90–93, 105.
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• Wang DIC, Humphrey AE. 1969. Biochemical engineering. Chem Eng 76(27):108.
• Wang DIC, Ochoa A. 1972. Measurements on the interfacial areas of hydrocarbons in yeast fermentations and relationships to specific growth rates. Biotechnol Bioeng 14:345.
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• Wang G,Wang DIC. 1984. Effect of acetic acid on the growth of Clostridium thermoaceticum. Appl Environ Microbiol 47:294.
• Wang SD, Wang DIC. 1989a. Pore dimension effects in cell loading in a porous carrier. Biotechnol Bioeng 33:915.
• Wang SD, Wang DIC. 1989b. Cell adsorption and local accumulation of extracellular polysaccharides in an immobilized acinetobacter calcoaceticus system. Biotechnol Bioeng 34:1261.
• Wang SD, Wang DIC. 1990. Mechanisms for biopolymer accumulation in immobilized acinetobacter calcoaceticus system. Biotechnol Bioeng 36:402.
• Wang DIC, Scharer J, Humphrey AE. 1964a. Kinetics of death of bacterial spores at elevated temperatures. Appl Microbiol 12:451.
• Wang DIC, Humphrey AE, Eagleton LC. 1964b. Measurement of the kinetics of biological systems at elevated temperatures utilizing flow techniques. Biotechnol Bioeng 6:367.
• Wang DIC, Sonoyama T, Mateles RI. 1968a. Enzyme and bacteriophage concentration by membrane filtration. Anal Biochem 26:277.
• Wang DIC, Sinskey AJ, Gerner R, DeFilippi RP. 1968b. Effect of centrifugation on the viability of Burkett lymphoma cells. Biotechnol Bioeng 10:641.
• Wang DIC, Sinskey AJ, Sonoyama T. 1969a. Recovery of biological materials through ultrafiltration. Biotechnol Bioeng 11:987.
• Wang DIC, Sinskey AJ, Butterworth TA. 1969b. Enzyme processing using ultrafiltration membranes. In: Flinn JE, editor. Membrane science and technology. New York: Plenum Press, pp. 98–119.
• Wang DIC, Hatch RT, Cuevas C. 1971. Engineering aspects of single-cell protein production from hydrocarbon substrates: The airlift fermentor. Proceedings of the 8th World Petroleum Congress, PD 21, Moscow, USSR, Elsevier Publishing Co., Ltd., Essex, England.
• Wang HY, Cooney CL, Wang DIC. 1977a. Computer-aided baker’s yeast fermentation. Biotechnol Bioeng 19:69.
• Wang DIC, Stramondo J, Fleischaker R. 1977b. Exploitation of multienzyme systems for synthesis. In: Bohak Z, Sharon N, editors. Biotechnological applications of enzymes and proteins. New York, NY: Academic Press, 183.
• Wang DIC, Fleischaker RJ,WangGY. 1978.Anovel route for the production of acetic acid by fermentation. AIChE Symp Ser 74(182):105.
• WangHY, Cooney CL,Wang DIC. 1979a. Computer control of baker’s yeast production. Biotechnol Bioeng 21:977.
• Wang DIC, Cooney CL, Wang SD, Gordon J, Wang GY. 1979b. Anaerobic biomass degradation to produce sugars, fuels and chemicals, In: Shuster WW, editor. Proceedings from the Second Annual Fuels from Biomass. Troy, NY: RPI, 537.
• Wang HY, Cooney CL, Wang DIC. 1979c. On-line analysis for material balancing and control, in ‘‘Computer Applications in Fermentation Technology’’, Biotechnology and Bioengineering Symposium No. 9, Armiger WB, ed., Interscience publication, 13.
• Wang DIC, Biocic I, Fang HY, Wang SD. 1979d. Direct microbiological conversion of cellulosic biomass to ethanol. 3rd Annual Biomass Energy Systems Conference Proceedings, 61.
• Wang DIC,Avgerinos GC, Biocic I,Wang SD, FangHY. 1983. Ethanol from cellulosic biomass. Phil Trans R Soc Lond B300:323.
• Wang DIC, Meier J, Yokoyama K. 1984. Penicillin fermentation in a 200- liter tower fermentor using cells confined to microbeads. Appl Biochem Biotech 9:105.
• Wang DIC, Xie L, Nyberg G, Gu X, Li H, Mollborn F. 1997. Gammainterferon production and quality in stoichiometric fed-batch cultures of Chinese hamster ovary cell under serum-free conditions. Biotechnol Bioeng 56:577–582.
• Wilcox RP, Evans LB, Wang DIC. 1978. Experimental behavior and mathematical modeling of mixed cultures on mixed substrates. AIChE Symp Ser 74(172):236.
• Wise DL,Wang DIC, Mateles RI. 1969. Increased oxygen mass transfer rates from single bubbles in microbial systems at low Reynolds numbers. Biotechnol Bioeng 11:647.
• Wise DL,Wang DIC, Racicot HA. 1971.Anovel aerobic aeration process for standard secondary treatment plant. Chem Eng Prog Symp Ser 67:107.
• Xie L, Wang DIC. 1994a. Stoichiometric analysis of animal cell growth and its application in medium design. Biotechnol Bioeng 43:1164– 1174.
• Xie L, Wang DIC. 1994b. Fed-batch cultivation of animal cells using different medium design concepts and feeding strategies. Biotechnol Bioeng 43:1175–1189.
• Xie L, Wang DIC. 1995. Application of improved stoichiometric model in medium design and fed-batch cultivation of animal cells in bioreactor. Cytotechnology 15:17–29.
• Xie L, Wang DIC. 1996a. Material balance studies on animal cell culture metabolism using a stoichiometrically based reaction network. Biotechnol Bioeng 52:579–590.
• Xie L, Wang DIC. 1996b. High density and high monoclonal antibody production through medium design and rational control in a bioreactor. Biotechnol Bioeng 51:725–729.
• Xie L,Wang DIC. 1996c. Energy metabolism andATP balance in animal cell cultivation using a stoichiometrically based reaction network. Biotechnol Bioeng 52:591–601.
• Xie L, Wang DIC. 1997. Integrated approaches to the design of media and feeding strategies for fed-batch cultures of animal cells. Trends Biotechnol 15:109–113.
• Yabannavar VM, Wang DIC. 1987. New bioreactor systems using in situ advent extraction for organic acid production. Ann NY Acad Sci 506:523.
• Yabannavar VM, Wang DIC. 1991a. Extractive fermentation for lactic acid production. Biotechnol Bioeng 37:1095.
• Yabannavar VM, Wang DIC. 1991b. Strategies for reducing solvent toxicity in extractive fermentations. Biotechnol Bioeng 37:712.
• Yabannavar VM, Wang DIC. 1991c. Analysis of mass transfer for immobilized cells in an extractive lactic acid fermentation. Biotechnol Bioeng 37:544.
• Yin J, Bonner G,Wang DIC. 2002. A simple and rapid assay of recombinant collagen in a crude lysate from Escherichia coli. J Microbiol Methods
• 49:321–323. Yin J, Lin J-H, LiW-T,Wang DIC. 2003. Evaluation of different promoters and host strains for the high-level expression of collagen-like polymer in Escherichia coli. J Biotechnol 100:181–191.
• Yin J, Chu JW, Speed Ricci M, Brems DN, Wang DIC, Trout BL. 2004a. Effect of excipients on the hydrogen peroxide induced oxidation of methionine residues in granulocute colony-stimulating factor (G-GSF). Pharma Res 22:141–147.
• Yin J, Chu JW, Speed Ricci M, Brems DN, Wang DIC, Trout BL. 2004b. Effect of antioxidants on the non-site-specific oxidation of methionine residues in granulocyte colony-stimulating factor (G-CSF) and human parathyroid hormone (hPTH) fragment 13-34. Pharma Res 21:2377– 2383.
• Yin J, Chu J, Trout BL, Wang DIC. 2004c. An experimental study of oxidation of methionine in G-CSF in the presence of excipients. Biochemistry (in preparation).
• Yuk IHY, Wang DIC. 2002a. Changes in the overall extent of protein glycosylation by Chinese hamster ovary cells over the course of the batch cycle. Biotechnol Appl Biochem 36:1–7.
• Yuk IHY,Wang DIC. 2002b. Glycosylation by Chinese hamster ovary cells in dolichol phosphate supplemented cultures. Biotechnol Appl Biochem 36:8–16.
• Yuk IH, Wang DIC. 2002c. Impact of growth-limitation on expression of recombinant interferon-gamma by anchorage-dependent CHO cells. Biotechnol Appl Biochem (Submitted).
• Yuk IH,Wildt S,Wang DIC, Jolicoeur M, Stephanopoulos G. 2002. AGFPbased screen for growth-arrested recombinant protein-producing cells. An effective screen for growth-arrested protein production cell-lines. Biotechnol Bioeng 79:74–82.
• Zhang J, Wang DIC. 1998. Quantitative analysis and process monitoring of site-specific glycosylation microheterogeneity in recombinant human interferon-gamma from chinese hamster ovary cell culture by hydrophilic interaction chromatography. J Chromatogr B 712:73–82.