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Biologics vs. Small Molecules, What’s the Difference for Stability Testing?

Written by Mike Molloy, Protein Chemistry Director, Boston Analytical

There are similarities for stability testing of biologics vs. small molecules, but there are also significant differences. In this first of a three-part blog post, I’ll breakdown what is faced in stability testing of these two product types and how each are handled. The second post will focus on the difference in analytical testing and the final post will be on degradation. Hopefully you find this useful!

For any drug, an understanding of the length of time the drug can be expected to last before degrading to the point of being ineffective or toxic is critical.  For both small molecules and biologics, the theoretical process for proving the stable life-time of the drug is consistent.  Tests need to be done in controlled conditions of temperature and humidity that match the way the drug is anticipated to be stored.  For small molecules those conditions are often set for 25°C and 60% relative humidity. Biologics on the other hand, may be lyophilized or stored as a liquid, and the more common condition is refrigerated at 2-8°C

Samples of the drugs are stored for a pre-established period of time and tested at specified intervals to be sure of its purity, potency and safety.  From those tests, the shelf life can be estimated.  There are also accelerated storage conditions, which are harsher environments that can be used to intentionally degrade the drug more quickly and use that speeded-up data to estimate the true shelf life.  Accelerated testing can be used to get an initial approval for an estimated shelf-life, but testing under truer conditions must follow.

Like small molecules, proteins may be subject to stresses caused by exposure to light.  For proteins, the predictable impact of light as a degrading force changes some amino acid residues, primarily tryptophan, cysteine and tyrosine, all of which undergo largely understood and predictable chemical changes.  A photostability study will help to determine if storage under light exposure will cause damage to the protein.

Interactions with the packaging material need to be evaluated as they can also cause change in the product .  Proteins come into contact with the surface of the packaging.  In some cases, the packaging includes coatings on the surface with oils or silicon that can also affect the protein structure and function.  Aggregation can result, as well as increases in protein sticking to the glass surface with the addition of the coatings.  Thus the product must be tested for stability in the appropriate containers with all added elements present.

The container may also introduce contamination by leaching chemical compounds into the drug product.  A full extractables and leachables study will help to define the risk offered by the container system, and an appropriate choice of container can be influenced by these studies.

If the biological product is stored frozen, a study showing the effect of freeze thaw cycles must also be done.  Freezing provides a number of advantages including increased stability, but can also introduce difficulties.  The freezing process can change the environment the protein is in, such as pH changes introduced by freezing the buffers.  Proteins can be damaged and concentration of the protein in solution may be inconsistent if the freezing protocol is not optimal.  Likewise, the process of thawing frozen samples can cause damage, primarily by formation of crystals that can damage the proteins.  Because of these dangers, stability of the product during freeze/thaw cycles is a necessity.

The basic design of a stability study for biologics echoes that of small molecule studies.  In both cases a study is driven by a written, QA reviewed stability protocol.  A typical study will be two years in length (timing can vary depending on the nature of the drug) at a temperature and humidity specification commensurate with the intended storage of the marketed product.

For small molecules a typical condition is 25°C and 60% relative humidity.  For biologics it is typically 5°C or frozen (at those temperatures humidity control is not generally applied).  The samples will be tested at the beginning of the study to establish a baseline, then retested at 1 month, 3 months, 6 months, 9 months, 12 months, 18 months and 24 months.  For biologics there are added conditions applied such as orientation of the stored drug (upright or inverted).  Agitation may also be applied since biologics can be sensitive to physical forces.

The second post in this series will focus on analytical testing of protein drugs in stability studies.