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The Science

BioPharm International


Every protein produced through fermentation or cell culture needs to be purified before use. For mammalian cell culture, an important part of that purification will be the removal of parasitic viruses and other agents that could infect users of the drug. The problem of viral contamination of animal cells dates to long before the birth of biotech. When the polio vaccine was first produced in the 1950s, many latent viruses were present in the monkey cell cultures used. Some of those (even some lethal viruses) were transmitted to humans. In the 1940s, millions of doses of live yellow fever vaccine produced in eggs were contaminated by endogenous chicken retroviruses and hepatitis B. In the 1980s, human-sourced products like growth hormone and blood products were contaminated with hepatitis, HIV, and other agents.

Many pathogens that infect one kind of mammal will happily reproduce inside another. So cells intended for cell culture must undergo the process of viral characterization — that is, testing for any possible adventitious agents that may be present with the cells. If there are too many, or the viruses are too dangerous, a cell line may be scrapped and other cells selected.

Contamination by an adventitious or endogenous agent does not necessarily render cell lines useless. Virus screening was instituted to avoid such problems. Safety decisions are based on detailed risk-benefit analyses that have given mathematicians (particularly statisticians) a whole new career choice.

Insect cells. When insect cells are cultured to produce proteins, the process is very different from that for mammalian cells. Insect cells are eukaryotic, too, and capable of doing many of the same complex posttranslational modifications to proteins that mammalian cells do. But the cells themselves are not genetically modified directly. Instead, a system called baculovirus expression vector system (BEVS) is used. Viruses are even simpler forms of life than bacteria. They cannot replicate by themselves but require a host. A virus doesn't even have a cellular structure, existing only as a particle made up of a protein shell that protects a bit of DNA or RNA inside. That genetic material is injected into the insect host cell, where it takes over the ribosomes to build more viruses.

Baculovirus is a particular type of virus that replicates only in the cells of lepidopteran insects, an order that includes butterflies and moths. It is harmless to all other creatures. Normally, when the virus infects an insect cell, it injects the cell with DNA instructions that cause it to produce large amounts of a protein that coats and protects the virus. When baculovirus is used in biotechnology, molecular biologists replace the gene coding for the protective protein with a gene coding for a desirable protein. The baculovirus infects the insect cells, and the cells express the protein.

Just because the baculoviral vector does not infect mammals doesn't mean that insect cell lines are completely free of potentially dangerous viruses. They must be characterized just like CHO and other mammalian cells because other adventitious agents may be present even if they don't harm the cells. Insect cell expression is still a new technology. Mammalian cells (particularly CHO cells) are still the most commonly used nonbacterial system.

Cell lines and cell banks. Biopharmaceutical companies do not necessarily obtain animal cells from their original sources — hamsters, monkeys, insects, or humans. Instead, they work with vendors that maintain specific strains of the cell lines. In the United States the American Type Culture Collection (ATCC) maintains more than 4,000 different mammalian, insect, and other cell lines and hybridomas. ATCC's European equivalent is the European Collection of Cell Cultures (ECACC).

Certified cell lines come with comprehensive data files that provide their full history (species of origin, strain, and colony). Tissue donors are tested for viruses such as hepatitis, papillomavirus, and the human immunodeficiency virus. Immunization regimes are also recorded, and gene fusion, DNA cloning, and cell selection procedures are detailed. Cell lines are characterized by tests that ensure freedom from microbial contamination and that confirm the cells' specific function and identity. DNA "fingerprinting" is often used to verify identity.

Once a biopharmaceutical company obtains a beginning cell bank from a cell culture collection, its scientists will do the work of genetically engineering those cells for the company's particular use. They may not only add the gene of interest (often "amplified" or repeated copies of that gene so that each cell produces more protein), but they may also make other changes. For example, an animal cell line that naturally prefers to grow attached to a surface can be adapted to grow suspended in liquid like bacteria or yeasts. Cells can be modified so that they perform posttranslational modification of the protein in more desirable ways.


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