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Report and Recommendations Concerning the Procurement and Use of Antibodies at Northwestern University

By Stephen F. Eisenman – President, Northwestern University Faculty Senate

"The following paper was prepared in response to inquiries made to me by representatives from two animal rights organizations concerning possible unethical sourcing of research antibodies at Northwestern University. Though I am indebted to the authorities cited in the acknowledgements and endnotes, the content is purely my responsibility. This paper does not represent the views of the Faculty Senate or Northwestern University."

Summary: 

Recent developments in regulation, biomedical science and ethics urge reform in the use of research antibodies. The current model, reliant upon obtaining monoclonal antibodies (mABs) produced in live animals (in vivo), should be replaced within three to five years by the procurement and use of recombinant antibodies (rABs) produced in the laboratory (in vitro). Recombinant antibodies are now widely acknowledged to be superior to the former in 1) speed of production; 2) target avidity/specificity; 3) convertibility into any antibody isotype; 4) reproducibility; and 5) ethical sourcing – no animals suffer or die in their production. 

The increase in availability of rABs from both commercial and non-commercial sources, combined with the scientific and moral consensus concerning their superiority, argues persuasively for the change.  Northwestern’s office of the Vice President for Research, Institutional Animal Care and Use Committee (IACUC), office of Purchasing Resource Services, and the offices of the dean of Feinberg and Weinberg Colleges, should quickly organize a team to evaluate the costs and explore the physical and administrative infrastructure needed for the change. Establishment of a dedicated rAB production facility at Northwestern is one possible option; another is collaboration with other universities or research centers; a third is procurement of rABs from commercial vendors.

Background:

Antibodies are large Y-shaped proteins produced by plasma cells.  They are essential to the healthy functioning of immune systems in humans and other animals because they bind to antigens (antibody generators) and neutralize them. Antibodies protect us from harmful viruses and bacteria. Upon infection or immunization, many different antibodies are generated against the infecting/immunizing target. The collection of such antibodies is called a “polyclonal antibody” and for purposes of research, such antibodies are usually generated from rabbits or goats. Any one of the generated antibodies, produced to homogeneity, is termed a “monoclonal antibody”. These are laboratory-generated antibodies that come from identical immune cells cloned from a unique parent. (The technology dates to the 1970s.) They are valuable in medicine and research because they bind to biological targets and can be used to identify them in scientific experiments. Furthermore, when they recognize particular cells they can be used to target and destroy them. They are also important in biomedical and toxicological research: Antibodies may be configured as platforms to analyze proteins changed by drugs, as reagents to identify and measure protein and drug levels in biological samples, and to register changes in cellular proteins after exposure to a toxic or other chemical agent. Monoclonal antibodies have played a crucial role in medical and other basic and applied research for more than 30 years.

mAbs are however flawed instruments in two significant respects:  1) In the process of manufacture and propagation, minute changes to mAB structure, or contamination (by irrelevant antibodies, plasma proteins, infectious agents, etc) may occur, rendering them non-antigen specific and thus less valuable for protein-binding.  In that circumstance, the result may be non-replicable science that retards rather than advances research objectives.  2) The process of obtaining mABs involves significant suffering of laboratory and other animals and thus violates widely held moral strictures – cross-cultural and trans-historical -- regarding animal protection and welfare.  Recent developments in cognitive ethology and changing public attitudes toward non-human animals additionally argue for reducing or eliminating the use of animals in antibody production. 

Violations of the Animal Welfare Act (AWA) by two of Northwestern’s chief mAB suppliers – Rockland Immunochemical and Santa Cruz Biotech (the latter exposed in an article in The New Yorker) -- are what initially brought the issue of research antibodies to the attention of the Faculty Senate leadership. In 2014, Rockland paid a fine of $32,000 for its multiple, serious violations; in recent months it has maintained a record of AWA compliance.  Santa Cruz however, because of its longer history of violation of the AWA and Northwestern’s own strict Purchase Order Terms and Agreement, has been given notice that its contract is in jeopardy. In fact, since that notice was sent in October, 2014, Santa Cruz has been charged by the USDA with more than a dozen additional violations of the AWA. So far, Northwestern has not severed its association with the firm. 

How antibodies are currently obtained:

Monoclonal antibodies are initially generated by immunizing mice, isolating antibody producing cells from their spleens (after killing the mice) and fusing them with cancer cells. This creates fusion cells termed “hybridomas” which immortalizes the antibody production of the fused spleen cell. Once hybridomas have been developed and tested for appropriate reactivity, antibodies can be produced in one of two ways: 1) in vivo, by growing hybridomas (comprised of antibody producing B cells and myelomas) in the abdomen of a mouse or other animal, or 2) in vitro after the initial generation of a small number of hybridoma cells in a mouse spleen. The first process (called the ascites method) is now banned (with limited exceptions) in the United Kingdom, Australia, the Netherlands, Germany, Switzerland and elsewhere.  It remains legal in the U.S., but its use is discouraged by the National Institute of Health and the U.S. Department of Agriculture.  Santa Cruz Biotech and Rockland Immunochemical, among many other biotechnology companies, make extensive use of the ascites method even though it may induce in animals anorexia, dehydration, paralysis, respiratory distress, circulatory shock, peritonitis and significant pain. In order to try to avoid the latter, IACUC protocols require euthanizing animals before abdominal distension exceeds 20% of body weight. To make polyclonals rabbits or goats are immunized and their blood harvested at regular intervals. The quality of the antibodies produced can be variable, which is one of the reasons there is a preference for monoclonals.

In response to these challenges, a new method of antibody production was developed. Since the early 1990s, it has been possible to create synthetic or recombinant antibodies (rAbs) using antibody genes manufactured in a laboratory or derived from human cells. Absent its animal derivation, these antibodies 1) are essentially invariable and thus ideal for replicable scientific research; 2) involve no animals and thus raise no ethical concerns; and 3) eliminate the problem of animal to human translation. rAbs can be used in all applications in which traditional mAbs are used.

Recombinant antibody production:

rABs are produced by: 1) creating large, cell libraries drawn from humans or  manufactured through gene synthesis; 2) displaying the library on cell or bacteriophage (virus) surfaces; 3) isolating antibodies that bind to specific targets; 4) improving antibodies (if necessary) by mutagenesis for affinity or specificity and then sequencing their genes; and 5) reproducing antibodies to desired scale through growth in tissue culture cells, bacteria, or yeast. This multi-stage process can be accomplished in a single, standard, molecular biology laboratory.

No animals are required for rAB manufacture; this is one of their chief benefits. However if particularly rare or useful antibodies have in the past been created through mouse or other animal immunization, they can be rescued, preserved and reproduced in vitro. Dr. Andrew Bradbury, co-author of the recent essay in Nature (cited, note 2, above) outlined the importance of improving reproducibility through the use of rABs: “This has a number of advantages: the antibody is sequenced, so we have the "barcode"; and monoclonal antibodies produced recombinantly tend to be much better than those produced by traditional hybridomas because they are not contaminated by additional irrelevant antibodies.”  Moreover, once the gene sequence of an antibody is published, it is universally available to researchers and the antibody itself can be produced very inexpensively. Bradbury’s collaborator, Dr. Andreas Pluckthun summarizes: “Clearly, recombinant methods work well and have reached maturity. Especially, they generate defined reagents, which are "immortal", since the genetic information will allow us to recreate them forever, like a chemical formula. You have the blueprint, and this can be shared.”

The future of rABs:

The current, global antibody production and distribution system is a more than $1.6 billion business.  Santa Cruz Biotech and Rockland Immunochemical have a larger than average share of the market in mABs, but there are many smaller companies that produce them just as well, and with a strong, animal welfare commitment.  What is more important however, is that a greater and greater number of biotech companies now produce antibodies from both monoclonal or recombinant methods, or rABs alone.  That number is certain to increase as understanding of the crisis of research reproducibility grows, and as ethical guidelines are tightened due to public pressure.

Nevertheless, obstacles to universal conversion to rABs remain, the chief one of which is economic. Currently, if a recombinant antibody already exists (for example, if a company like AxioMx is selling it), it will be comparable in price to the animal-derived antibody. If the rAB is obtained from a non-profit research facility or university, (such as at Vanderbilt University), the cost will likely be still lower.  However, to buy a brand new recombinant antibody from a company that needs to develop it from scratch can cost in the range of $10-15,000 compared to the price of about $5-15,000 for a traditional monoclonal antibody created by immunization or $2-500 for an already-made recombinant or animal-derived product. Thus one obvious solution is for companies to make enough recombinant antibodies – produce a large enough library -- that the start-from-scratch cost for any individual purchase is overcome.

A second option at Northwestern for overcoming the cost of purchasing rABs that must be made from scratch is for the university to start a core facility to produce them in-house. This would require an initial investment of between $500,000 and $1 million; the cost, according to Bradbury, would include protein and antibody expression equipment, selection robots, flow cytometry and standard molecular biology equipment. There would obviously be an ongoing staffing cost.

A third way to overcome the cost hurdle, proposed by Bradbury and Pluckthun among others, is to establish consortia of industry, academia, government, and research non-profits (NGOs) to fund the development of rABs defined by their sequences. In private communication with the author, Bradbury has proposed the creation of a pilot project between NU and the Los Alamos Research Laboratory in which the latter would “supply recombinant sequenced antibodies to NU researchers in collaboration with them, and approach NIH to see if they would be willing to fund such a wider, joint project.” Other universities with existing rAB laboratories might be interested in similar joint ventures. Obviously, in the case that NIH was willing to fund the project, the cost to NU would be minimal.

A simple way to start the conversion from mABs to rABs at Northwestern would be: 1) for researchers to choose recombinant over monoclonal versions of the same antibody if the former already exists; 2) for the schools or central administration to dedicate a sum of money to allow selected researchers to use recombinant antibodies, even when they must be made from scratch; and 3) begin planning for an rAB facility at NU or work in collaboration with other universities to establish one.

Whatever the cost of rapid conversion to the exclusive use of rABs may be, it is less expensive than the status quo. Bradbury and Pluckthun note that nearly half of all US spending on protein binding reagents (about $700 million) is wasted – expended on bad antibodies.  That $350 million price tag overlooks the incalculably greater expense of wasted individual and institutional research time, as well as lives damaged or lost from medical or research failure. As one of the leading centers for biomedical research in the US, NU must be shouldering a large share of these costs. Because of the depth of its resources and the strength of its research program, Northwestern is well poised to make the modest investments in time and money to effect the conversion described here.

Acknowledgements: The author wishes to thank the following scientists for their generous expenditure of time answering questions via email, in person or by phone: Andrew Bradbury (Los Alamos National Research Laboratory), Thomas Brody (National Institute of Health), Michael Festing (Leicester University, ret.), Andre Luiz De Gouvea, (Northwestern University), Alan Goldberg (Johns Hopkins Bloomberg School of Public Health), Andreas Pluckthun (University of Zurich), and Hank Seifert (Northwestern University)


1 “Monoclonal antibodies,” Altweb, Johns Hopkins, Bloomberg School of Public Health, 2015. http://altweb.jhsph.edu/mabs/mabs.html

2 Jennifer Bordeaux, et al, “Antibody validation,” BioTechniques, Vol. 48, No. 3, March 2010, pp. 197–209; Andrew Bradbury and Andreas Plucthun, “Reproducibility: Standardize antibodies used in research,” Nature, vol. 518, no. 7537, February 5, 2015, pp. 27-29; “Protein Binder Woes,” Nature Methods, vol. 12, no. 5, May 2015, p. 373; J.l.A. Voskul, “Commercial Antibodies and their Validation,” [v2; ref status: indexed, http://f1000r.es/4jp] F1000Research 2014, 3:232 (doi: 10.12688/f1000research.4966.2)

3 A conference the author attended at the National Academy of Sciences in 2014 addressed what has been called “the replication crisis: ”The missing “R”: Reproducibility in a Changing Research Landscape,” NAS Workshop, June 4-5, 2014; C. Glenn Begley and Lee M. Ellis, “Drug development: Raise standards for peclinical cancer research,” Nature, vol. 483, no. 7391, May 2012, pp. 531-533; J.M. Schooler, "Metascience could rescue the 'replication crisis'", Nature, vol. 515, no. 7525), Nov. 2014, p. 9.

4 John McArdle, Alternatives to Ascites Production of Monoclonal Antibodies, Animal Welfare Information Center, Vol. 8, no. 304, Winter 1997-8, pp. 14-36. National Research Council (US) Committee on Methods of Producing Monoclonal Antibodies. Monoclonal Antibody Production. Washington (DC): National Academies Press (US); 1999. 6, Animal-Welfare Issues Related to the Ascites Method for Producing Monoclonal Antibodies. Available from:

http://www.ncbi.nlm.nih.gov/books/NBK100190/. And see, for example: Steiner, Gary, Animals and the Moral Community: Mental Life, Moral Status and Kinship (New York: Columbia University Press), 2008; and Stephen F. Eisenman, The Cry of Nature: Art and the Making of Animal Rights, (London and Chicago: Reaktion Books/University of Chicago Press), 2013.

5 Donald R.Griffin, Animal Minds: Beyond Cognition to Consciousness (Chicago and London: University of Chicago Press), 2001).

6 http://www.newyorker.com/online/blogs/elements/2014/02/valuable-antibodies-at-a-grisly-cost.html?mobify=0

7 Email correspondence from Jay Walsh, VP for Research, Friday, October 17, 2014 12:40 PM
:
“Dear Research Community:
I am writing to you to make you aware of a troublesome situation regarding one of Northwestern’s Preferred Vendors, Santa Cruz Biotechnology.  We have been informed and have verified that Santa Cruz Biotechnology has been cited with several violations of the Animal Welfare Act by the USDA.  You are receiving this letter because your lab purchases animal antibodies from Santa Cruz Biotechnology.
We have been in communication with Santa Cruz Biotechnology to notify the company of Northwestern’s expectations that they will correct these violations.
We will monitor Santa Cruz Biotechnology’s progress over the next six months.  If sufficient improvement has not occurred, we will request that researchers begin developing a plan to transition animal antibody purchases to other vendors.  We are working with Purchasing Resource Services to make sure we have an agreement with an appropriate number of vendor options to meet your needs for the products you currently buy from Santa Cruz Biotechnology.  Northwestern is committed to compliance with animal welfare regulations and does not wish to do business with a vendor that consistently does not comply with the regulations.  Further, these violations represent a potential risk to the supply of materials from this company for your research and via this message we are telling you of this potential risk.
The production of custom antibodies involves the use of live, vertebrate animals.  Thus, as you likely know, researchers ordering custom antibodies from commercial sources are required to contact the IACUC office 3-9339 acuc@northwestern.edu before placing any orders. If you have any questions or would like to discuss, please contact Sue Kallay at s-kallay@northwestern.edu.”  Also see: Calvin Men, “USDA Files Second Complaint in Seven Years Against Santa Cruz Biotech,” Santa Cruz Sentinel, November 13,, 2014: http://www.santacruzsentinel.com/general-news/20141113/usda-files-second-complaint-in-seven-years-against-santa-cruz-biotechnology.

Concerning Rockland, the record is only slightly less damning:
Between March 13, 2013 to October 21, 2013, the United States Department of Agriculture (USDA) cited Rockland for 29 violations of the AWA. Many of these were repeat violations. They include:

  • Staff failed to update treatment cards on rabbits’ cages, leading to missed treatments required by the protocol that had been approved by the IACUC.
  • Experimenters used animals in protocols that had not been approved by IACUC, undermining institutional oversight at the company. Such violations are considered by the USDA to be gravely serious.
  • Rockland’s IACUC approved an incomplete proposal and an IACUC subcommittee approved a proposal that had not been submitted to each committee member, thereby preventing full committee review.
  • Rabbits were housed in broken, unsafe, and unsanitary enclosures and their food containers were found to be unsanitary and in disrepair.
  • Guinea pigs were housed in cages that were below the minimum size requirement to allow for normal postural adjustments and adequate freedom of movement as required by law.
  • Poisonous plants were found in the grazing areas for donkeys, llamas, and cows.
  • Rockland failed to provide adequate veterinary care to goats who were described by the inspectors as “lame” from “front hooves that were overgrown.”
  • Medication was stored improperly adjacent to the unwrapped body of a dead rat.
  • USDA inspectors noted multiple rabbits not being treated for a variety of health problems including: “yellowish discharge from the nose”; “thick nasal discharge”; “large mats and urine stains”; “watery discharge from both eyes”; “sore on the right side of its back”; and one rabbit who had “soiled wet fur and skin…did not move its rear legs in a normal manner”; another “was observed with a severe head tilt to the right and rolled over in its enclosure”; and another “was leaning against the side of the enclosure. The rabbit was listless and non-responsive.”
  • Rockland was also cited for systemic problems involving standard operating procedures for the use of anesthesia and humane euthanasia. The inspector noted that failure to properly anesthetize rabbits being used for cardiac blood draws “led to animals inadvertently waking during a painful procedure” and that employees were killing rabbits by cervical dislocation when the protocol specifies that cervical dislocation may only be used as a method “to complete euthanasia following the cessation of breathing in an unconscious animal.”

8 Cf, Guidelines for Monoclonal Antibody Production, National Health and Medical Research Council of Australia, 2008; https://www.nhmrc.gov.au/_files_nhmrc/publications/attachments/ea19.pdf

9 U.S. Department of Health and Human Services. Office of Laboratory Animal Welfare. Nov 1997. Production of monoclonal antibodies using mouse ascites method. http://grants.nih.gov/grants/olaw/references/dc98-01.htm. Also: “The NIH concurs with the findings and recommendations in the 1999 report of the National Research Council Monoclonal Antibody Production (PDF) which indicates that during the accumulation of ascites there is likely to be pain and distress, particularly when some cell lines that are tissue-invasive are used and in situations of significant ascites development. The Report concluded that there is and will continue to be scientific necessity for this method, but that as tissue-culture systems are further developed, tissue-culture methods for the production of monoclonal antibodies should be adopted as the routine method unless there is a clear reason why they cannot be used. Accordingly, IACUCs are expected to critically evaluate the proposed uses of the mouse ascites method. Prior to approval of such protocols, IACUCs must determine that (i) the proposed use is scientifically justified, (ii) methods that avoid or minimize discomfort, distress, and pain (including in vitro methods) have been considered, and (iii) the latter have been found unsuitable. http://grants.nih.gov/grants/olaw/faqs.htm#668

10 Email communication with the author, May 2, 2015.

11 Email communication with the author, May 2, 2015.

12 Christi Bird, “Antibodies Users Survey,” The Scientist, May 1, 2012: http://www.the-scientist.com/?articles.view/articleNo/32042/title/Antibodies-User-Survey/

13 See for example, ImmunoPrecise, Pocono Rabbit Farm and Laboratory, EMD-Millipore, and Bethyl Laboratories.

14 Sources offering premade recombinant antibodies (with links to their catalogs): AbD Serotec (host HuCAL); Creative Diagnostics (including Creative BioMart); University of Geneva
          

Sources offering custom recombinant antibodies (with links to their ordering processes): AbD Serotec; Abgent; Avantgen; AxioMx; University of Geneva

Sources offering premade aptamers (with links to their catalogs): Amsbio; Aptagen offers a database of aptamers; AptSci; Base Pair Biotechnologies; CD Genomics; Creative Biogene; GeneLink; OTC Biotech

Sources offering custom aptamers (with links to their ordering processes): AM Biotech; Aptagen; AptaMatrix; AptSci; Aptasol; Base Pair Biotechnologies; CD Genomics; Creative Biogene; GeneLink; LC Sciences; NeoVentures Biotechnology; SomaLogic; TriLink

Universities that have Established a Recombinant Antibody Laboratory Include:
University of California San Francisco; University of Colorado (See Drs. Jeffrey Bennett, Mark Burgoon, and Gregory Owens); Duke; University of Geneva; University of Illinois at Chicago; University of Nevada; University of Nevada Reno; University of Pennsylvania ; University of Texas

Universities that have Established an Aptamer Laboratory Include: Auburn University; Carleton University; McMaster University; University of Albany; University of California Santa Barbara; University of California at Irvine; University of Florida

University of Illinois at Urbana-Champaign; University of Iowa; University of Miami; University of Missouri; University of Texas; University of Wisconsin-Madison; Yale 

15 https://www4.vanderbilt.edu/vapr/home

16 Bradbury and Pluckthun, “Standardize Antibodies,” p. 29.

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