Oxidation. Certain amino acids (tryptophan, methionine, cysteine, histidine, and tyrosine) are susceptible to oxidation. Metal ions
(copper, iron) can accelerate the process. Higher pH values, fluorescent lights, and hydrogen peroxide can all cause or contribute
to oxidation, but atmospheric O2 is obviously the main culprit. If the amino acids along a polypeptide chain are deformed by oxidation, the molecule can be
irreversibly altered, and the new molecule created may not perform the necessary drug action.
Hydrolysis. Hydrolysis of a side-chain amide on a polypeptide's glutamine or asparagine residues can yield a carboxylic acid. The process,
called deamidation, is facilitated by elevated temperature and pH, and its effect is to destroy the activity of most proteins.
The peptide bonds that hold amino acids together in the chain can also be severed by hydrolysis — particularly where aspartic
acid residues are located. This effect is usually due to heat or to pH levels that are too low.
Crystalline structure of a sugar, often used as a pharmaceutical excipient
Disulfide exchange. Cysteine residues form disulfide bonds, which are important to protein structural integrity. Shuffling of these bonds, where
two sulfur atoms from two different amino acid molecules link up, often changes the three-dimensional structure, causing a
loss of activity.
The Ingredients and the Recipe
Proteins are most stable in a "glassy," amorphous, and highly viscous matrix (freeze-dried or spray-dried). Technically speaking,
glass is just a very hard gel. Liquid solutions must be kept very cold. Maximum stability occurs when conditions restrict
molecular interactions and conformational changes. Various technologies offer several options to formulators, and more are
being developed all the time. By far the most widely accepted methods of protein stabilization are additives and excipients,
cryopreservation, and freeze-drying. Spray-drying, cryogranulation, and undercooling are less proven options.
Additives and excipients. Salts and nonelectrolytes (such as ammonium sulfate and glycerol) help stabilize proteins in high temperatures and low pH
when freezing is not an option, but they still require low-temperature storage. Sometimes they must be removed before the
drug is used, which can be inconvenient, time-consuming, and expensive. Also, the active ingredient must be diluted, allowing
further waste and variability in the final product just as in reconstituting freeze-dried products.
Cryopreservation (freezing) can extend the shelf life of unstable products and improve containment if the freeze-thaw process is consistent.
Frozen product can be transported safely for final formulation elsewhere and stockpiled to optimize the fill and finish process,
which can reduce processing costs. More detail on this process begins in Chapter 4.
Many formulation scientists believe that proteins cannot be frozen and thawed without damage because no practical methods
exist for doing so on a large scale. The usual method, which is both slow and nonreproducible, involves small volumes in bags,
bottles, or vials freezing at —20°C or below. A slow freeze alters the physical properties (pH, diffusion, reaction rates)
of the aqueous solvent medium or mixture, which can denature proteins, particularly over extended time. The solution is then
thawed at room temperature, with some components thawing before others. Ice recrystallization in the thawing process creates
mechanical stress. Processes for freezing and thawing protein solutions must, therefore, be well controlled.
Cryogranulation. One answer to the control problem might be cryogranulation. "Standard practice for finished product manufacturing of parenteral
dosage forms begins with the bulk drug substance in the solid state, at which point it is dissolved in a suitable solvent
during the formulation or compounding stage before filtration. However, some drug substances, particularly proteins, are very
difficult, if not impossible, to crystallize," D.J. Schmidt and M.J. Akers wrote in BioPharm (April 1997). Cryogranulation uses a stream of liquid nitrogen to quickly create frozen discrete pellets. The main challenge
is controlling thawing and reformulation on the clinical or final product manufacturing side.
Lyophilization (freeze-drying) doesn't always have an "either-or" relationship with cryopreservation; sometimes both are used. Because lyophilization
is so often used, there is an expanded discussion later.