Monoclonal Antibody Production: A Project-Based Laboratory

Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX

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Monoclonal Antibody Production: A Project-Based Laboratory Program for Final Year Biotechnology Undergraduate Students Andrew J. Lindsay* School of Biochemistry and Cell Biology, Biosciences Institute, University College Cork, Cork, Ireland

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S Supporting Information *

ABSTRACT: A semester-long, project-based series of laboratory exercises is described that provides practical experience of the upstream and downstream processes involved in monoclonal antibody production. This 6 week project is aimed at final year undergraduate students undertaking primary degrees in biotechnology, biochemistry, or related disciplines. Students develop subject matter knowledge of biologics production and practical skills related to cell culture, column chromatography, quality control, and information literacy. Students are assigned a vial containing a hybridoma cell line stored in liquid nitrogen. They thaw the vial and culture the hybridoma over the course of 4 weeks. They perform daily counts of live/dead cells and quantify glucose levels in the media. During the 4 weeks they will expand their cells from a single 60 mm dish into two large T-175 flasks, one for antibody production and one for generating stocks. They harvest the medium from the production flask and purify the antibody via Protein G affinity chromatography on an FPLC instrument. The purity of the antibody is tested by SDS-PAGE, and the concentration is determined on a Nanodrop instrument. Students present their results in the form of a project thesis, a product datasheet, and a public presentation. Learning was assessed relative to six learning objectives using a student survey administered at the beginning (presurvey) and end (postsurvey). The students who have undertaken this module demonstrated substantial learning gains. This laboratory project is highly adaptable with scope to adapt it for longer or shorter study periods. KEYWORDS: Upper-Division Undergraduate, Biochemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Biotechnology, Bioanalytical Chemistry, Biological Cells, Electrophoresis, Chromatography

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INTRODUCTION An important element of science education is laboratory instruction.1−3 There are four commonly used formats of laboratory instruction: expository, guided-inquiry, problembased, and discovery. They differ in the level of student input into the design and conduct of the laboratory. The expository style is the more traditional “cookbook” or recipe-based instructional method and is often a poor model of the practices of professional scientists. The guided-inquiry format requires input from the students into experimental design, data acquisition, and data analysis. The students are provided with a procedure containing information on what to do and the type of data to collect. The instructor then guides them along a clearly defined path with the aim of developing an understanding of the underlying concepts through data analysis, evaluation, interpretation, and discussions.4 There are many examples of such multiweek biochemistry laboratory courses with a unifying throughline.5−9 The global monoclonal antibody (mAb) therapeutics market was valued at greater than $100 billion in 2017 and is projected to double by 2023.10 With the number of approved monoclonal antibody therapies increasing each year, and the introduction of biosimilars onto the market, there is a growing need to train personnel in their production. The high © XXXX American Chemical Society and Division of Chemical Education, Inc.

specificity and affinity of mAbs for their targets, combined with the relative ease with which humanized and fully human antibodies can be developed, has led to an explosive growth in this class of biopharmaceuticals in the past 20 years. There are 73 FDA-approved monoclonal antibodies currently on the market, with over 300 in various stages of clinical development.10 They are used for a wide range of indications including oncology, autoimmune disorders, and rare diseases. Their hallmark is to bind to their target antigen (e.g., a cancer cell) with high specificity and mediate its destruction. Human and humanized therapeutic mAbs are typically generated using recombinant DNA engineering, and their production belongs to the area of bioprocess technology. Small-scale production can be readily transferred to large-scale manufacturing using specialized fermenters or bioreactors, yielding kilogram quantities of the biotherapeutic.11 In contrast to most small molecule drugs, which are chemically synthesized and have well-understood structures, recombinant monoclonal antibodies are large (∼ 150 kDa) and complex macromolecules possessing post-translational modifications. Received: January 10, 2019 Revised: June 21, 2019

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DOI: 10.1021/acs.jchemed.9b00024 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

media and plasticware (Supporting Information, p S20). If stored correctly, the columns can be reused over a number of years. (6) mAb production is directly relevant to the treatment of human disease, which promotes student engagement. (7) Each successful project produces large amounts of antibody that is >95% pure, which is often of use to research laboratories in the university.

This necessitates their cultivation in living systems such as Chinese hamster ovary (CHO) or mouse myeloma cell lines. The therapeutic mAb is secreted into the culture media, from which it is then purified. This is a complex and costly endeavor, and many factors have to be taken into consideration, including removal of impurities, robustness, scalability, and ready availability of raw materials for large-scale production. Described herein is a cost-efficient, 6 week, laboratory-based, guided-inquiry project aimed at providing students hands-on experience with the key processes involved in the upstream and downstream production of mAbs. This project is designed to give students the opportunity to develop their practical skills, understand how scientific experimentation is conducted, and recognize the complexity and ambiguity of empirical work. It involves a linear series of experiments with the products of each process immediately required for the next task. This project utilizes a pre-existing tissue culture facility to teach the students cell culture and aseptic technique. Column chromatography is performed on a dedicated benchtop FPLC instrument, and the purified product is evaluated by SDSPAGE. The pedagogical goals for the project are the following: (i) to teach the students standard cell culture and biochemical techniques; (ii) to train the students to operate standard chromatographic protocols within specification; (iii) give the students the opportunity to acquire and analyze quantitative data; (iv) to build teamwork and interpersonal communication skills; and (v) to guide the students to source, review, and critically assess scientific literature relevant to the project. In addition to the production and analysis of a final product, progress toward these goals is evaluated during the semester by means of a periodic student survey, and at the end of the module in the form of a project thesis and formal seminar presentation of their results (Supporting Information, p S21). By the end of the project the students will have been familiarized with modern cell biology/biochemistry techniques and challenged to reflect and critically evaluate their results and performance. This project offers a number of advantages over other final year research projects in similar disciplines. (1) The students in this course perform the same set of techniques, which allows the instructor to offer relevant prelaboratory training and to manage 10 or more students working independently, and provides the opportunity for peer learning. Furthermore, the longest phase of the project is the cultivation and expansion of cells into large T-flasks. I find the students quickly become adept at cell culture and comfortable with working independently in their dedicated cell culture facility. (2) Each student group is provided with a different hybridoma cell line, each of which has its own growth characteristics and produces a unique antibody. (3) Column chromatography is a core process used by the biopharmaceutical industry to purify biopharmaceutics, and the students gain direct hands-on experience with this critical technique. (4) The project is self-sustaining, in that the students prepare hybridoma stocks that are stored in liquid nitrogen and are used in the following year’s program. (5) After an initial financial outlay to purchase the FPLC instrument and Protein G columns, the yearly cost of running the program is minimal. The recurring costs are for

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CURRICULAR CONTEXT University College Cork, Ireland, is a publically funded university with approximately 21,000 students. The BSc Biotechnology is part of a four-year degree program jointly run by the School of Biochemistry and Cell Biology and the School of Microbiology. Students enter through the general Biological and Chemical Sciences degree course and specialize in Biotechnology in Years 3 and 4. There are currently 20 places in the course, and the research project is a core 10-credit module taken by the students in the first semester of year 4 (the students must complete 60 credits per year). Ten students are randomly assigned to the laboratory program described herein, which is run by the School of Biochemistry and Cell Biology. A parallel laboratory program is provided by the School of Microbiology for the other 10 students. Two of the main reasons for establishing this program were (1) to deliver a project that more closely aligned with the training needs of the biopharmaceutical industry, which has a large presence in Cork, and (2) to take the pressure off research laboratories to provide space and resources for the increasing number of students enrolled in the Biological and Chemical Sciences course. The students will have taken modules in chemistry, microbiology, and biochemistry, and more specialized modules in biotechnology and biochemical engineering. They will also have just returned from an industrial placement. Students work in groups of 2, and each group is assigned its own hybridoma cell line. The specific hybridomas used vary from year to year depending on whether a research group expressed a requirement for a particular antibody. A dedicated “wet lab” and adjacent tissue culture facility are made available for the semester. A university lecturer and up to two technicians (working part-time on the module) provide the training. In the cell culture phase of the project the students take daily cell counts and check glucose levels, which takes approximately 1 h. A booking system is in operation for the laminar flow hood which means there is usually only one group in the tissue culture facility at any one time. Column chromatography and SDS-PAGE typically take place in weeks 4−6, when the students tend to spend up to 5 h in the wet lab per day. Integrated into the module are a number of Skills Sessions provided by the Skills Centre, UCC. These are 1 h workshops on topics such as presentation skills, critical thinking, scientific writing, bioinnovation and bioreactors. Formal student assessment is through evaluation of their performance in the laboratory (40%), a formal thesis (30%), a literature review based on a topic related to the laboratory project (20%), and an oral presentation (10%). The thesis, literature review, and oral presentation are evaluated by a first and second examiner (university lecturers), and laboratory performance is evaluated by the first examiner and technicians.

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EXPERIMENTAL DETAILS Students receive an introductory lecture outlining the background and goals of the project. At the end of the lecture they B




tank, thaws it in tissue culture, centrifuges the cells, and transfers them in fresh warm media into a 60 mm tissue culture dish. For each of the remaining 3 days, 1 mL is aspirated from the dish (replaced with 1 mL of fresh media) with a sterile pipette, and a sample is taken to count the live and dead cells using the trypan blue exclusion assay and a hemocytometer. The glucose level in the media is assessed using a glucometer. The hybridoma is usually expanded into a larger flask once the live cell count reaches 0.5−1 × 106 cells/mL. The aim is to seed two T-175 flasks, a “Production” flask from which the media will be harvested, and a “Stock” flask which will be used to generate 4−6 vials for storage in liquid nitrogen. Most cell lines will reach this stage in about 4 weeks. During this time the students check their cells daily and typically become comfortable enough to perform their tasks without supervision by the middle of week 2. The hybridomas continuously secrete mAbs into the medium, and by week 5 the titer is usually high enough to proceed to the next step. The medium is harvested, and cells are removed by centrifugation. The supernatant is stored at 4 °C until the FPLC is available. Affinity chromatography is performed using a 1 mL Protein G column (GE) operated on an AKTA Start (GE) benchtop FPLC system. The AKTA Start is an ideal training instrument as it is robust and has a relatively small footprint and intuitive software that can be operated from either a laptop or a touchscreen on the instrument itself. Results are displayed in real time, and consequently, by the end of the run it can be determined if the project has been successful, or not. A standard preparative FPLC instrument may also be used. In the absence of an FPLC, the protocol could be adapted for use with Protein G affinity columns alone, although no chromatogram would be obtained. The FPLC protocol takes approximately 4 h per mAb purification, so two groups can be accommodated per day. There are 16 fractions collected from each purification, and samples of each fraction plus a sample of the supernatant are run alongside molecular weight markers on a polyacrylamide gel. The students prepare, load, run, Coomassie stain, and destain the SDS-PAGE gel. This takes 2 days per group and allows the students to directly observe the purity of their mAb preparation. Finally, the concentration of mAb in each fraction

are randomly assigned into groups of 2 and allocated a hybridoma cell line. The laboratory program takes place over 6 weeks (Table 1). Table 1. Overview of Experiments Week

Experiment

1

Prepare cell culture media Thaw hybridoma in 60 mm dish Live/dead cell counts Quantify glucose concentration in the media Live/dead cell counts Quantify glucose concentration in the media Expand cells into 100 mm dish Live/dead cell counts Quantify media glucose concentration Expand cells into T-75 flask Live/dead cell counts Quantify glucose concentration in the media Expand cells into 2 × T-175 flasks Harvest medium Prepare hybridoma stocks and store in liquid nitrogen Protein G affinity chromatography using an FPLC instrument Analyze the purity of the antibody fractions by SDS-PAGE Quantify antibodies on Nanodrop using Beer−Lambert law Collate results Store antibodies in −20 °C

2

3

4

5

6

Prior to commencing cell culture, the groups are shown brief introductory YouTube videos on cell culture and aseptic technique (Thermo Fisher). Their first hands-on cell culture experience is the preparation of their media. Each group is given a new bottle of RPMI 1640 (Roswell Park Memorial Institute 1640) cell culture medium which they supplement with fetal bovine serum, penicillin/streptomycin, and Lglutamine, using aseptic techniques in a laminar flow hood. This is prepared under the supervision of a technician or the lecturer and can take up to 1 h per group. The students get a feel for the cell culture facility and the challenges of working in a laminar flow hood. Any mistakes at this stage are not critical as no cells are involved. The following day, each group retrieves their hybridoma vial from a liquid nitrogen storage

Figure 1. Line graph depicting live cell (blue line) and dead cell (orange line) counts over the course of approximately 4 weeks. C




Figure 2. Akta Start FPLC system. The supernatant is stored in the 80 mL Duran bottle and applied to the system via the pump valve. The binding and elution buffers are placed in 900 mL Duran bottles on top of the instrument. Waste goes into the beaker, and antibody fractions are collected in 1.5 mL Eppendorf tubes inserted in the fraction collector.

Figure 3. Representative chromatogram from a single mAb purification. UV absorbance on the y-axis and volume on the x-axis. Fractions are indicated by the red text superimposed on the graph.

is quantified on a Nanodrop instrument using the Beer− Lambert law.

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and frostbite, and students must wear a face shield and liquid nitrogen certified gloves. Acrylamide, 2-mercaptoethanol (βME), and sodium dodecyl sulfate (SDS) are toxic if swallowed and should not come into contact with skin. Eye protection must be worn when handling these reagents. Coomassie stain is not hazardous but will stain skin and clothes. Bleach is a significant hazard in the cell culture work, as it is a skin irritant and can cause serious eye damage. The purified monoclonal antibody is not hazardous unless ingested.

HAZARDS

There are multiple hazards in this laboratory course. Students are given a lab safety talk at the beginning of the module and are required to wear gloves, laboratory coats, and safety glasses for all experiments. Liquid nitrogen can cause cryogenic burns D




Figure 4. Quality control step. Samples of each elution fraction from a mAb affinity chromatography purification were denatured and separated by SDS-PAGE and stained with Coomassie Blue. The heavy (IgG H) and light (IgG L) chains are indicated. * denotes an ∼150 kDa contaminating upper band.

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Beer−Lambert law: A = εcl (A, absorbance; ε, molar extinction coefficient (M−1 cm−1); l, path length (cm)). The fractions with the highest concentrations of antibodies are stored at −20 °C.

RESULTS The students compiled the data collected during the cell culture phase of the project (cell count and glucose concentration) into line graphs using Microsoft Excel or similar spreadsheet software (Figure 1). Initially, considerable variability in cell counts was observed from one day to the next, as witnessed by the “sawtooth” pattern on the graphs. This was explained to the students as likely due to counting errors and incorrect sampling. Over time the counting became more accurate and the graphs smoother. The mAb was purified from the harvested supernatant by Protein G affinity chromatography on a benchtop FPLC (Figure 2). This stage was performed under direct supervision from the technicians and/or lecturer. The students were supplied in advance with the product information for the Protein G column12 and prepared a manual protocol on the system software that would be suitable for purifying their antibody. The protocol is then modified in discussion with the instructor (if necessary) and used to run the supernatant through the system. The manual method takes approximately 4 h to set up and run, and between 12 and 16 500 μL elution fractions are typically collected. It is easy to determine which fractions contain the antibody by analyzing the chromatogram (Figure 3). At the end of this stage the students know if their project has been successful. In all cases to date, an antibody has been successfully purified; however, the yields tend to differ from group to group, and from hybridoma to hybridoma. The purity of the antibody fractions is determined through SDS-PAGE followed by Coomasie blue staining of the gel (Figure 4). The IgG heavy and light chains are clearly visible in the fractions corresponding to the elution peak on the chromatogram. A lower abundance high molecular weight band of approximately 150 kDa has been observed in every purification to date. In their project report the students are asked to speculate on the identity of this impurity and to suggest purification techniques that could be used to remove it. The final part of the project is to quantify the amount of antibody in each fraction by measuring protein absorbance at 280 nm using a microvolume UV−vis spectrophotometer. The concentration is manually calculated by the students using the

Student Response

Student surveys indicated that this laboratory program accomplished the objective of allowing the students to acquire advanced laboratory skills and to generate and analyze data, an important goal of many guided-inquiry laboratories.

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DISCUSSION This laboratory project has been in place for 2 years, with a class size of 10 each time. To date, all students have successfully purified and validated mAbs. In one instance, a hybridoma cell line failed to proliferate sufficiently to be seeded into T-175 flasks. This group was then provided with a CHO cell line secreting an anti-IL-8 mAb. This was recovered directly into a T-75 flask and could be quickly expanded into two T-175 flasks. The pedagogical goals established for this project were achieved, namely, to effectively train students in standard cell culture practice and to introduce them to techniques involved in mAb purification and analysis. The students acquired enough cell culture expertise within 2 weeks to be able to work unsupervised. No issues with contamination have been encountered to date. They also gained a comprehensive understanding of column chromatography techniques as evidenced by the production of a final product and their ability to explain the process steps they undertook in their oral presentations, which are usually scheduled 6−8 weeks after the end of the project. Furthermore, many students were able to suggest supplementary chromatography steps that could be undertaken to remove the impurities from their eluate fractions. Coordinating tasks with their group partner, and organizing access to equipment with the other groups, built on their teamwork and interpersonal communication skills. Finally, the individual literature reviews and project write-ups enhanced their ability to search and critically analyze the scientific literature. Formal assessment is based on their laboratory performance, final laboratory report, and an oral presentation of their work (Supporting Information, p S21) E




(12) GE HiTrap Protein G HiTrap Protein G HP, 1 and 5 mL. https://cdn.gelifesciences.com/dmm3bwsv3/AssetStream. aspx?mediaformatid=10061&destinationid=10016&assetid=11420 (accessed Jun 2019).

Students expressed strongly positive assessments of the guided-inquiry format in an end-of-module anonymous feedback form. I find that the format of the laboratory program led to improved engagement, as each student was obliged to interact closely with the instructors in order to design and learn the experimental techniques. Furthermore, anecdotal evidence suggests that the weaker students benefitted from the peer-group learning aspect. Potential additions to this program are outlined in the Instructors’ Notes in Supporting Information.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.9b00024.

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Laboratory manual and Instructors’ Notes (PDF, DOCX)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Andrew J. Lindsay: 0000-0001-9693-7022 Notes

The author declares no competing financial interest.

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ACKNOWLEDGMENTS The author would like to thank Eoin Fleming and Jerry Reen for guidance and help in designing the project, Gavin Kinsley and Orlagh Fennelly for their technical support, and our students for their helpful feedback.

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REFERENCES

(1) Elliott, M. J.; Stewart, K. K.; Lagowski, J. J. The Role of the Laboratory in Chemistry Instruction. J. Chem. Educ. 2008, 85 (1), 145−149. (2) Moore, J. W. Let’s Go for an A in Lab. J. Chem. Educ. 2006, 83 (4), 519. (3) National Research Council. A Framework for K-12 Science Education: Practices, Crosscutting Concepts, and Core Ideas; The National Academies Press: Washington, DC, 2012; p 400. (4) Domin, D. S. A Review of Laboratory Instruction Styles. J. Chem. Educ. 1999, 76 (4), 543−547. (5) Craig, P. A. A Project-Oriented Biochemistry Laboratory Course. J. Chem. Educ. 1999, 76 (8), 1130−1135. (6) Vincent, J. B.; Woski, S. A. Cytochrome c: A Biochemistry Laboratory Course. J. Chem. Educ. 2005, 82 (8), 1211−1214. (7) Taylor, E. V.; Fortune, J. A.; Drennan, C. L. A research-inspired laboratory sequence investigating acquired drug resistance. Biochem. Mol. Biol. Educ. 2010, 38 (4), 247−252. (8) Murthy, P. P. N.; Thompson, M.; Hungwe, K. Development of a Semester-Long, Inquiry-Based Laboratory Course in Upper-Level Biochemistry and Molecular Biology. J. Chem. Educ. 2014, 91 (11), 1909−1917. (9) Muth, G. W.; Chihade, J. W. A streamlined molecular biology module for undergraduate biochemistry labs. Biochem. Mol. Biol. Educ. 2008, 36 (3), 209−216. (10) Grilo, A. L.; Mantalaris, A. The Increasingly Human and Profitable Monoclonal Antibody Market. Trends Biotechnol. 2019, 37 (1), 9−16. (11) Bakhtiar, R. Therapeutic Recombinant Monoclonal Antibodies. J. Chem. Educ. 2012, 89 (12), 1537−1542. F


Monoclonal Antibody Production: A Project-Based Laboratory

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