In Vitro Often Preferred Over of In Vivo for Adventitious Agent Testing

The biopharmaceutical industry favors replacing in vivo testing with a panel of in vitro and molecular based methods to detect a wide range of viral contaminants.

^{This article is posted on our Science Snippets Blog} 

Biopharmaceutical manufacturers know that a viral contamination during production of vaccines is one of the biggest threats to the safety and efficacy of the final product. Since it was first introduced in the 1950s, in vivo testing has been the standard process for detecting virus contamination. But in recent years, manufacturers’ preference is now in vitro testing combined with molecular based methods such as polymerase chain reaction and Next Generation Sequencing (NGS). These are more sensitive and can detect a breadth of viral contaminants.  

The Risk of Adventitious Agents in Vaccine Production

There are multiple points where a virus can enter the production stream. Some come from the raw materials used for production, while others enter from the process or product itself. The most common contamination risks are:

• Raw materials, particularly those derived from animals
• Cell substrates, which are the starting point for all biologics products
• Viral substrates used in viral vaccine and viral vector manufacturing
• Faulty cleaning procedures that fail to eliminate contaminants
• Manufacturing equipment that is not properly maintained or cleaned
• Inadequate production facilities with limited or no contamination controls
• Direct or indirect human contact with the product

The most common known contamination risks come from:

• Bacteria
• Chlamydia
• Mycoplasma
• Prion
• Protozoa
• Rickettsia
• Viruses (environmental, endogenous, replication competence)
• Yeast

Because there are so many opportunities for contamination, adventitious agent testing is an essential part of any manufacturing process to ensure product safety.

Key Differences Between In Vivo and In Vitro Testing

To better understand why in vitro testing is preferred over in vivo testing in many biopharmaceutical manufacturing processes, it is important to understand the differences between the two methods.

In Vivo Testing

In vivo testing of drugs involves injecting animals—suckling mice, nude mice, or guinea pigs—and embryonated eggs with the product. Testers make clinical observations over a specific time period, based on the viruses for which they are testing, to look for signs of a viral infection. Next, they conduct hemagglutination tests or pathology exams to determine whether there is a viral infection risk. If more than 80 percent of test subjects survive, the test is valid.

This type of tests meets all regulatory requirements and can provide an additional level of safety to prevent viral contamination. However, one of the key drawbacks to this type of test is how long it takes. In vivo assays require 42 days for the experiment, then another two to six months to finish the study.
 

In Vitro Testing

Rather than using small animal subjects for the tests, in vitro testing of drugs uses indicator cell lines with a demonstrated capability for viral growth. Compared to in vivo testing, in vitro tests make it much easier to validate for Limit of Detection (LOD) and generate a GMP assay. Since industry ethics dictate that companies should reduce, refine, or replace the amount of animal testing required (the three Rs), manufacturers can perform more studies with in vitro than they would be able to with the in vivo method.

In vitro assays are more sensitive and detect more viruses when compared with in vivo options. In one study, in vitro testing correctly identified 100 percent (16 of 16) viruses, while in vivo only identified 55 percent (6 of 11). Additionally, in vitro testing requires less time—typically 14 to 28 days—and manufacturers can easily validate the test.
 

Is There Ever a Use for In Vivo Testing?

While in vitro testing is clearly the most effective method for detecting all types of adventitious agents, there are still some situations when manufacturers continue to use in vivo testing. Regulations still require testing on cell substates and viral seed stocks with potential consideration for batch intermediates and End of Production Cell Banks.

For decades there was a persistent belief that you could not grow certain viruses in cell cultures after they are isolated from clinical samples. However, that is changing as companies can demonstrate viral safety with a combination of in vitro assays, sequencing, and polymerase chain reaction (PCR) based testing methods that show a product is free from most adventitious agents.

The Future of Adventitious Agent Testing

Most biopharmaceutical manufacturers need guidelines for adventitious agent testing that provide the necessary flexibility to assess statistically significant samples within the context of their own product and manufacturing systems. The guidelines have been around since before the introduction of current high-potency and low batch size manufacturing for gene therapy. These guidelines make it hard to minimize product loss while also assuring that the testing methods offer the highest level of sensitivity to detect adventitious agents.

There are even more exciting innovations coming in the future as we move away from in vivo testing options. Some of the most promising innovations include:

• 3D bioassays
• Next generation sequencing (NGS)
• High-throughput sequencing (HTS)
• PCR species-specific panels

These methods are more effective at detecting viruses quickly and are more sensitive than current testing methods. NGS and HTS testing offer the promise of detecting both infectious and non-infectious viruses—something in vivo and in vitro testing cannot do.

Find Out More About Adventitious Agent Testing with Sartorius

Sartorius offers a wide range of in vitro and in vivo assays for detection of adventitious agents and supports manufacturers in selecting the optimal combination of tests for their safety strategy. Learn more about the options available and talk to an expert about your needs.