Biochemistry in an Industrial Context: Methods of Protein Purification and Downstream Processing Pamela J. Weathem Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA 01609
Applied Biochemistry: A Natural Fit for an Englneerlng College Industrial develonment of biotechnolow will reauire severa1 diverse groups of technical personnel including molecular hioloeists. immunoloeists. and bionrocess eneineers (14). Of p&ic"lar concerdin chis report is the av&abiliti of bio~rocesseneineers. snecialists in the develonment and scaie-up of mi;croorg&idm and higher cell culture, or in the separation and purification of the commercial products resuiting from these fermentations. The latter is also referred to as "downstream processing". As recognized by the Office of Technology Assessment and the National Science Foundation, limited availability of bioprocess engineers could prove catastrophic for the industry just as commercialization of the products begins (1, 2). Too few individuals are adeauatelv trained in these areas and esneciallv in downstream processing (protein chemistry and separation procedures). and too few educational institutions offer adeauate training in bioprocess engineering. During the last two years, the Department of Biology and Biotechnology a t Worcester Polytechnic Institute, in conjunction with Bay State Skills Corporation and the local hiotechnology industry, has developed and incorporated into the graduate curriculum a new course in bioprocess technology: Methods of Protein Purification and Downstream Processing. This course was designed to augment our curriculum in biotechnology by providing students with hands-on training in the methods necessary to assist in the design and implementation of product (protein) processing for the biotechnology industry. The techniques used for separation and purification in hio~rocessesare the asnect of bioprocess engineering most in need of attention, especially for novel producrs such as proteins (2).A well-rounded bioprocess engineer must straddle two disciplines: biochemical engineering and hiology (1, 2, 4). The overall program a t WPI has been designed with that in mind. A particularly novel feature of the course is the development of training exercises in learning the "principles" of scale-up. Once a process for the purification of a protein product has been demonstrated in the laboratorv. i t must be scaled up, frequently beyond 1000-fold, to achik;e production of a commerciallv viable nroduct. The product must be cost effective in its production and in ampie supply. Scaleup is not a prescribed method or an eauation. Rather. the approach tosolvingscale-up problems i ~ a ~ w i l ~ s d i f f e rfor ent each product and it is usualls driven bv, and selected for. cost effectiveness as well as burity andyield. As a result methods which work very well in the laboratory may be inapplicable for industry (e.g., sonication for cell breakage). This course is focused on those methods which are scalable. Until now, educational exercises did not exist to give students experience in this area (5);experience was gained on the job. A working knowledge of general chemistry, organic chemistry, basic biology, microbiology, and particularly biochemistry is expected prior to entry into the course, although
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Journal of Chemical Education
Syllabus lor Methods 01 Protein Purification and Downstream Processing. Lecture 1-3
4 5
6 7-6 9 10 11 12 13-14
15 16 17-18
Topic
introduction. Theory ot Separations, Properties of Proteins Buffers. Maintainance of Active Preps, Kinetics, Assays, Purity and Yleid, Pyrogens. Separation Methods: Solids from Liquids Tangential Flow Filtration Breakage: Cavitation. Abrasion. Enzymic. Chemical Edraction: Precipitation Solvent Extraction Chromatography: TLC. Gel, ion Exchange, Reverw Phase, Affinity. Atfinity Escort. HPLC immobiiiratians Electrophoretic Melhcds. Eiectrodialysis Exam Culturing as a SeparationTool Scale-UP Scale-Up C Lab: Use of Ceraflo Pilot Unit at Norton Co. Q&A Site Visit Or Exam
Last 4 Weeks: IndependentProjects
Lab 1-2
3 4
5 6-7 8 9 10 11 12 13 14
Topic Enzyme Assays; Yields. Buffers. 8-Gaiactosidase Assay. Kinetics: BGaiactosidase pH. T. Lactose Inhibition. K,, V,.. etc. Report DUB Harvesting Methods: E. mliFiltration vs. Cwhifugation Breakase Methods: E. mli(Sonic vs. Grinding vs. Enzymatic vs. FR. Pressure Cell) Ammonium Sulfate Ppt.; Dialysis Liquid Chromatography: Ion Exchange. Gel Filtration lsoelectri Focusing HPLC Report DUB Scale-Up A: HPLC acd Economic Analysis Scale-Up El: Filtration Scale-Uo C: Use of Ceraflo Pilot Unit at Norton Ca. scale-U'P Dstaanalysis. Site Visit
Report DUB Last 4 Weeks: independent Projects
chemical engineers have performed well. A t the beginning of the course, students are polled as to their occupational intentions so that the instr&tor is aware of special needs. T o be effective, the course is intensively laboratory oriented; thus, class size is limited to 12. The course has very flexible lectures designed to keep pace with the open laboratory sessions. Plan of Study This one-semester course as outlined in the table, begins with a review of basic enzvmoloev. followed bv the theorv of separations and the key require&nts n e e d e d b maintain an
active preparation of enzyme product. The critical necessity of assays for monitoring purity and yield are discussed along with issues of aualitv assurance. ovroeens. etc. (Pvroeens . . - are agents, often bacterial wall deb&, whichcause fever in humans.) After this len&hv introduction, methods of seoaration are discussed inhetail and according to the seq"ence logically used in processes: solids from liquids, concentrations, solutes from solvents, and final analysis. Special emphasis is placed on designing an optimum purification scheme based on unit operations especially critical to the hiotechnology industry: membrane filtrations and chromatography (3,6-8). 8-Galactosidase (8-gal), which hydrolyzes lactose to glucose and ealactose. is chosen as the orotein to nurifv because it is well-characterized in the literature, itLis made by a common industrial oreanism, Escherichia coli, i t is chemically quite stable, it has simile assays, and it is inexpensive to produce. Initially students are provided with crude !%gal to kstahlish the enzymic of the protein: pH and ,., changes in the presence of temperature optima,K, and V inhibitors, etc. These first two exercises also permit students to become familiar with the assays necessary for establishing purity and yield throughout the separation. After a thorough study of the proposed product to be are ourified. 30 L of E. coli. induced for B-eal . " oroduction. * provided to the students. Initially separation of solids from liquids (cells from erowth medium) is demonstrated for 24 L using a lab-scale sharpies bowl centrifuge (see figure). Each group of four students is also given 2 L of cell suspension to separate the cells by tangential flow filtration using a Millipore Miuitan filtration unit (0.2 pm filter). Students are expected to assemble the equipment and produce pressure excursion curves for their cell suspensions. The concentrated cells are nooled from all sources for the next exercise. Since 8-&Iis an intracellular product, the concentrated cells must be broken. Five methods of cell breakaee are tested and compared: sonication, grinding with alumina, osmotic lvsis usine lvsozvme. homoeenization with elass beads, and pressur~c&itaiionusinga-~rench~ressurei'ell. All are performed in 0.01 M Tris (hvdroxvethvl) aminoethane buffer, pH 7.5. The latter method is-demonstrated hy instructor and the other four methods are performed in small groups. Data are collected from each group and tabulated to determine the relative yield and purity for each method. All fractions are pooled and aliquots provided to groups of students for subsequent separation (see figure). An initial class value for total protein and specific activity of the enzyme is established. From this point it is the goal of each group to achieve the highest possible activity, purity, and yield of @gal after completion of all steps in the subsequent purification scheme. After breakage, cell debris is removed by centrifugation a t 10,000 g and the protein in the supernatant fraction is precipitated a t pH 7.5 with 60% (NH&30aand concentrated by centrifugation. The precipitate is dialyzed to remove the (NH4)zSOa. The dialyzate is applied to a cation exchanger, CM Sephadex (2-50, and fractions eluted with a 0-2% NaCl linear gradient a t pH 7.5 in Tris buffer. The &gal fraction is identified using both UV spectroscopy and specific enzyme assav. Fractions containine- , B-eal are concentrated bv.Lvonh.. ilization, reconstituted, and applied to a size exclusion column of Seohadex G-200 in the same buffer. The B-eal is again identified, pooled, and concentrated as before. Final purity is assessed not only by assay but also by isoelectric focusing, pH range 3.5-10, with associated colored marker proteins. Size exclusion using high-performance liquid chromatography (HPLC) follows the above purification scheme to compare HPLC to themethods used for possible improvements in purity, activity, and yield. At each step of the purification scheme, total protein and specific activity are measured to follow the increase (or decrease!) in purity and yield of P-gal.
BEWAtE: OWTIC LWS. GRIMD. SQHIUTE, FENCH PRESSURE CELL IN 0.01 11 TRIS, L 7.5
PRECIPITATE
I
SUWT1XI
PRmEIN PEUET I W t I DISSOLVE IN 1 VOL. 0.0111 TRlS BUFFER, DH 7.5 A I R DIALYE VS. 2 1 300 m1 CHMGES OF UM BUFFER
DlSUN
cnkm
BUFFER.
a 7.5
\
OTHER FRACTIONS
Lmvmm: m v TO SEPHIOEX 6-2W
I N SME BUFFER
DISCAN DIALYZED. DESdLTD PROTELU HPLC: UTES PROTEIN PA1 300 L I-U5 I N SERIES WITH ABDVE BUFFER
Purification scheme used in the course Methcds of Protein Purification and Downstream Processing to purity 8-galactosidase.
After the purification scheme is complete, the scale-up studies begin. Because the goal here is to acquaint the students with hands-on approaches to scale-up, 8-gal is not used; rather, samples are chosen that are simple, are already demonstrated for the desired methods, and are inexpensive to test in large quantity. Although chromatography scale-up is demonstrated using HPLC, scaling columns, and cephalosporin C (an antibiotic), lecture material also covers the theory and rules of open-column liquid chromatography. The procedure used for HPLC is that developed by Millioore Coro. . (9). . . An abbreviated loadine studv is conducted using cephalosporin C, and the data are assessed for cost effectiveness usine software develooed hv Millioore corooration. This program for economic a ~ l y s i ~ a l l othe w operator ~ to simulate and alter operating parameters such as input purity, solvent regeneration, 1a6oi and materials costs, eic., for an overall estimate of production cost. As a result, this exercise teaches students not only how to develop scale-up for HPLC, hut also how to determine what parameters might best reduce the cost of production. Subsequent exercises focus on scaling up tangential flow filtration usineboth nlate and frame devices (Pellican. Millipore Corp.) a& hollow tube ceramic filters ( ~ e r a f l oNorton , Co.). Pressure excursion curves are calculated for a soecific membrane area and the data projected forascale-up &twice that area. The apparatus is then reassembled using double the membrane area and the projected operating parameters are verified using the greater area. Suspensions (20 L) of yeast or of casein are used for these exercises. Volume 65
Number 10
October 1988
855
Throughout the course, as methods are explained in lecture, comments as to the advantages and disadvantages of the utility of each method for scale-up are discussed. When the exercises on scale-un beein. lectures encompass not onlv the theory of the methods h i t also the principlls of scale-up: alwavs nroceed from laboratorv feasibilitv throuah lab-scale demonstration, from trial stage to pilot stage-never move from laboratory t o pilot stage i n one step. On some occasion in the future, i t will become necessary for these process engineers to be able to argue convincingly with management or investors for the stepwise development of a process. The nontechnical person sees the potential lab-to-pilot short cut as potential cuts in expense. However, this is shortsighted and invariably leads to problems, often expensive ones, causing greater expense than the logical, sequential approach. Haste makes waste. Although not always possible due to proprietary prohlems, FDA visits, etc, a site visit to a working industrial o~erationis hiahlv - .recommended for the students. Such a visit usually requires not more than two hours of tour time hut provides invaluable exposure to the real world. There is no substitute for experiencing the entire realm of hioprocessina from fermentation to packaping the final product. ~ f t e completion r of all o i the programmed laboratory work, students are offered a list of independent projects from which they may choose a topic for a more in-depth study. These topics cover all areas of separations and include suggestions from the industrial partners. In some cases students have performed their independent projects on site for a narticular industw. all of the labbratory exercises and independent pro-iects.. detailed. written reports are required (see table). Besides monitoring the progress of ski& development in the laboratory, two exams are also given that test the students' understanding of the techniques and their ability to propose reasonable purification schemes for hypothetical productions.
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L
o or
Key Elements for a Successful Course Besides having a faculty member and a graduate assistant skilled in offering this course, there are three other key ingredients for successful, state-of-the-art operation. These are industry interest and input, possession of some expensive equipment, and on-going commitment from the university for expenses for repairs and expendables. Whereas expendables are not so difficult to ohtain, the procurement and
maintenance of equipment is. Finally, hut of the greatest importance, is the interaction with the local industrial comm&ity. ~ h e s are e the people who can offer invaluable advice about their needs, the best methods, and the vendors and may even provide some of the necessary equipment and supplies. In the development of the course a t WPI, the industrial participants donated equipment, provided training to upgrade the faculty, provided some lectures, provided site visits, engaged students for on-site projects, and advised the overall curriculum including the selection of the purification scheme. A special henefit of this offerine was the interaction of Bav ~tate'SkillsCorporation I H S S C ~a, nonprofit organization funded h v the Commonwealth of Massachusetts to upgrade the states rnanpowerskills.Through a 5050 marchinkgrant with industry, RSSC u,ill pnn.ide salaries and expendable supplies for new training opportunities. Without all of this assistance, this course could not have been developed to its full extent to provide the practical, state-of-the-art training needed for students to learn the skills necessary for entry into downstream processing in the biotechnology industry. Acknowledgment I wish to thank Millipore Corp. for their donation of HPLC, filtration equipment, software, and employee time, especially William Skea and Phillip Onigman. I also wish to thank Norton Co. and Repligen for site visits and Zymark Corp. for a robotics workshop. Gratitude is also extended to Pharmacia for donation of materials and personnel time and to Biopure, Integrated Genetics, Karyon Technologies, and Swartz Associates for invaluable participation as advisors. S ~ e c i athanks l to Edward Merrill. Massachusetts Institute o i ~ e c h n o l o ~and y , to Joseph ~ a g s h a wWorcester , Polytechnic Institute. who assisted in the initiation of this educational exercise. Llterature Clted 1. NSF Workshops: "PmspeM for Biofochnology", Univ. Virg.,Apr. 54,1982; Developing the Biotechnolopy Component of Engineering", N. Carolina Biofoehnol. Center, Apr.24-25,1963. 2. U.S.Government reoort. . . OTA-BA-218.1964 3. uanBrunt, J. Biotechnol. 1985,3,419-424. 4. Zsbonk~.O.R.:Zubris.0.K. USBiotechnolop~EnpinaamSlofusRmon1985:OMEC lnter&onsl: Washington. DC 1985. 5. Merrill, R. E., MIT: Skea, W.. Millipore, peraonal communication. 6. Dwuer. J. L. Biorerhnol 1984.2.9571JS4. ~~
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CHEM ED '89: Call for Papers CHEM ED '89 will be held August 13-18,1989 at Queen's University, Kingstgn, Ontario, Canada. In keeping with the concept "teachers sharing with teachers", the program committee is now accepting abstracts for presentation at CHEM ED. For information about presentations and an abstract form, please contact one of the following. In the United States: Bette Bridges, 221 Oak St., Box 94A, Brockton, MA 02401 David Lee, 70 W. New St., Rockaway, NJ 07866 Lois Fruen, 4510 Westwood Ln., Minneapolis, MN, 55416 In Canada: Majorie Allen, Department of Chemistry,Queen's University, Kingston, Ontario, Canada K7L 3N6 To he placed an the CHEM ED '89 mailing list write CHEM ED '89, Faculty of Education, Queen's University, Kingston, Ontario, Canada, KIL 3N6
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Journal of Chemical Education
