Examining Protein Use in Biomedicals, Destruction of Avian Influenza

By Annel K. Greene, PhD, Center Director, Clemson University,
Animal Co-Products Research and Education Center

Immunologist Dr. Thomas R. Scott, a member of the Clemson University Animal Co-Products Research and Education Center (ACREC) team, is working to isolate proteins from rendering materials that have significant biomedical and veterinary applications with potential for great economic return. He also is studying ways to validate the efficacy of rendering processes for destroying the avian influenza virus.

Animal cell culture is a vital research tool for medical and veterinary studies. In the cell culture technique, living cells are grown in artificial media external to the whole organism. The success of this technique is dependent on carefully balancing the nutrients and growth conditions in the media. Currently, tissue culture growth matrices utilize bovine calf serum or fetal calf serum as a source of vital proteins and growth factors. Although some proteins and growth factors have been identified, many of the substances necessary for cell growth are unknown. Some of the proteins that have been identified are carriers for minerals, fatty acids, and hormones. Proteins necessary for cell growth include epidermal growth factor, fibroblast growth factor, insulin-like growth factor, platelet-derived growth factor, and interleukin-1. Other important proteins include fibronectin, which assists in the adhesion of cells, and albumin, which carries important cellular nutrients such as minerals, lipids, and globulins.

Early in the development of the cell culture technique, it was recognized that it was impossible to “go to the shelf” to add all required cell culture growth nutrients. Scientists realized they needed another approach. They experimented with mixtures of proteins and growth factors until they determined which worked best as media additives for cell culture. Bovine calf serum and fetal calf serum were selected based on cell culture growth enhancement and commercial availability. These sera have been used as the industry standard for decades. However, these media components are extremely expensive to the researcher – often costing $36 to $72 per 100 milliliter (equal to $1,362 to $2,725 per gallon).

Realizing the wealth of potential bioactive proteins and growth factors available in rendering materials, Scott set out to isolate a subcomponent from raw animal co-products that could be a more effective and yet less expensive source of cell culture growth factors and nutrients. After discussions with co-workers about bioactive proteins in bovine colostrum, and considering that those substances are produced by tissues which are deposited in the rendering stream, Scott decided to investigate fractions of proteins from bovine udders. He conducted extensive literature reviews and studied the bovine mammary structure. He eventually chose to focus on a particular site within the dairy and beef udder – the supramammary lymph node.

Scott and his team of graduate students are now investigating technologies to fractionate the proteins from the supramammary lymph node without damaging bioactivity. Initially, supramammary lymph nodes are dissected from bovine udders. These tissues are ground and processed through a series of steps that were developed in Scott’s lab with the assistance of Dr. Steve Ellis and graduate students Danelle Duffy and Alison Reed. The latest process developed has allowed a nine-fold improvement in protein recovery over earlier trials.

Scott chose to use MAC-T cells (mammary alveolar cells transfected with SV-40 large T-antigen) as a cell culture line for testing the isolated protein fractions for growth enhancement. MAC-T cells are epithelial cells derived from bovine mammary tissue. These cells are an excellent model for determining the effectiveness of protein fractions in enhancing cell growth.

Measuring the effectiveness of each protein fraction is a complicated process whereby cell division rates are determined via DNA copy analysis. Those fractions that manifest a bioactive response in the cell culture are further investigated by proteomics analysis. This process allows for identification of the actual protein or peptide sequence. Comparison of these sequences against known sequences in the database will determine if a new bioactive sequence has been isolated. The potential for identifying new bioactive peptides from rendered materials opens a new realm for research, diagnostic, and therapeutic activities.

The fractions isolated from supramammary lymph nodes produce far superior cell culture growth than the traditional bovine calf or fetal calf sera and, thus, have great economic potential. Worldwide, scientists routinely use cell culture techniques. On average, a laboratory will spend approximately $400 to $500 on fetal calf serum and/or bovine calf serum per cell line per year. Most small labs maintain three to four cell lines per year whereas large labs may grow hundreds of cell lines per year. The market potential for cell culture media additives could be millions of dollars.

Scott’s studies on a variety of biological and rendering tissue extracts have lead to identification of several other valuable peptides and factors that could be of value in human and animal therapies. Many of these extracts have the potential to be worth hundreds to thousands of dollars per microgram to milligram (equivalent to $10,000 to $1 million per ounce and up). Scott and his team are studying the use of these extracts for medical diagnostic testing, disease treatment, and for production of valuable products from cell culture such as hormones, growth factors, and monoclonal antibodies. Some biologically active proteins have been associated with potent anti-cancer and other activities.

Not only will this research potentially allow for greater profits from rendering materials, it will also add to the base of critical knowledge concerning bioactive interactions for cell growth, proliferation, and repair. This information will be helpful in furthering medical and veterinary medical knowledge that can lead to future advances in understanding cell processes as well as advances in diagnosis and treatment of diseases. Currently, there is very little scientific data reported about mammary gland extracts. Therefore, Scott is opening a new chapter in the understanding and use of these proteins.

Destroying Avian Influenza
In a separate project, Scott and co-workers are investigating ways to validate the thermal destruction of the avian influenza virus. Using sub-particles of two of the associated proteins (hemagglutinin and neuraminidase) as test peptides, the team is working to determine the least amount of heat necessary to destroy the pathogen. This information will be of utmost importance in avian influenza outbreaks. The rendering industry must have validation data ready to prove that the rendering process will destroy the virus. In the study, Scott has investigated ways to accurately measure peptide markers in the high fat environment of poultry rendering products. Initially, the fat interfered with the test methodology and gave false negatives. Scott tried a number of different procedures and eventually adapted a procedure that seems to be working well for high fat materials. Thermal trials are currently underway to determine the heat dose necessary to destroy the hemagglutinin and neuraminidase protein particles.

Scott’s research team includes master of science students Duffy, Reed, and Sara Garrett, as well as laboratory technicians Dr. Marcy Owens and Nancy Korn. The Clemson University ACREC is very proud to have Dr. Tom Scott and his team working on projects that are of such vital interest to not only the rendering industry, but to the biosecurity of the U.S. food supply and to the pool of knowledge in the biomedical fields.

ACREC Solutions – August 2007 RENDER | back