Sugars (or saccharides) raise Tg and act as stabilizers. Dextran, lactose, maltose, sucrose, and trehalose are used, but the
latter two are preferred. Acidic amino acids (glutamic acid, glycine, histidine, threonine) are used to adjust pH in one direction,
and alkaline amino acids (arginine, lysine) are used to adjust it the other way. Other pH and buffering agents include HCl,
NaOH, PO4, and acetic acid.
Antioxidants help protect against oxidation by scavenging oxygen for themselves — just as they do in the body. Ascorbic acid
is used, but citric acid is preferred, and it can be used as a pH adjuster as well.
A nonionic surfactant (usually a polysorbate) is often added as a finishing touch to inhibit protein aggregation. It is often
needed during earlier processing steps anyway, so it may be easier to keep it in the solution than to filter it out (unlike
processing contaminants such as peroxides, which must be removed). Surfactants and bulking agents such as mannitol and certain
biodegradable polymers are often needed for low-concentration formulations. Clinicians and patients may not feel confident
about a vial with hardly anything in it, and tiny amounts of drug substance are difficult to handle.
Lyophilization
A lyophilized biopharmaceutical protein requires protection during both the acute stages (the freezing and drying) and during
storage, when excipients must form an amorphous solid (glass) with the protein. Freeze-drying may cause certain stresses on
proteins that can result in unfolding, so specific conditions must be determined and stabilizing additives must be appropriate
for use during both the freezing and drying stages. Part of a formulation plan is to stabilize the product during each stage:
freezing, drying, and dry storage. Some proteins unfold during lyophilization but refold if immediately rehydrated. However,
the goals are long-term storage (one or two years), acceptable cake morphology, and adequate dissolution properties.
Eutectic Point (Te) and Glass Transition Temperature (Tg)
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The process. There are four steps to the freeze-drying process. Freezing forms an amorphous solid of the protein and excipients, with
associated water in crystalline form. Annealing, an optional step, increases ice-crystal size and allows crystallization of
bulking agents (such as glycine or mannitol), removing them from the amorphous portion and increasing Tg. Primary drying sublimes
water ice at temperatures lower than Tg (to avoid collapse of the cake). The higher the Tg, the higher the mixture's temperature
and sublimation rate can be. Increasing the protein to excipient ratio will increase Tg. Secondary drying removes water from
the amorphous phase by an increase in sample temperature, which still may not exceed Tg. Luckily, as water leaves the amorphous
phase, Tg increases. Sample temperatures play a larger role than the duration of secondary drying for determining final water
content of the lyophilized cake. Low moisture increases Tg and thus increases the temperature at which the product can be
stored.
A drug solution is first frozen at atmospheric pressure, and then water is removed by a reduction of pressure in the lyophilizer
chamber, collecting the water as ice on a condenser. Samples are placed in glass vials and frozen, either before being put
in the lyophilizer or on the lyophilizer shelves. The samples contain ice crystals, unfrozen water, amorphous solids (including
the therapeutic protein), and crystalline additives. Pressure is reduced, and the ice crystals sublime. That constitutes the
primary drying.
It is harder to remove the unfrozen water trapped in an amorphous solid. So after primary drying, a secondary drying stage
removes that water by increasing the temperature. The final temperature of this secondary process is the key factor in determining
residual moisture in the dried cake. The pressure is kept the same for secondary and primary drying to avoid protein collapse.
The ideal result is a porous cake with little residual moisture. Porosity is important in later reconstituting the product.
Sometimes an annealing step (in which the product is kept at a set temperature) is added before the primary drying or near
the end of secondary drying to crystallize excipients. This assures that crystallization and moisture release don't happen
in an uncontrolled fashion later on during shipping and storage. Phase changes (crystallization of formulation sugars, for
example) during shipping or storage can be disastrous. Moisture can even transfer from rubber vial-stoppers during storage
unless savvy formulators plan for and prevent it.
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