Best Practices for Formulation and Manufacturing of Biotech Drug Products - How to maintain product stability and prevent particulates. - BioPharm International
The freeze and thaw behavior of proteins has been studied extensively, but primarily in small or microscopic volumes and often
in conjunction with lyophilization. The use of these small volumes in literature studies makes the process aspects difficult
to relate to the freezing and storage of bulk proteins. A few studies have, however, elucidated fundamental aspects of the
impact of freezing on protein structure and interaction with ice and are reviewed by Bhatnagar, et al.15
Figure 1
An unavoidable feature of freezing is cryoconcentration as water converts to ice and excludes the solutes (and protein), ultimately
creating a viscous glassy matrix (Figure 1). This can affect the embedded protein in a number of ways. If the buffer salts
are prone to crystallization because of saturation, significant pH shifts can occur. Among the common buffers used for biologics,
the sodium phosphate buffer mixture is particularly susceptible, and the pH can change from seven to near four on precipitation
of the dibasic salt; the actual value is dependent on strength and rate.15 Even if the salts do not precipitate, buffer pH is sensitive to temperature, and therefore, pH shifts will occur during
freezing and in the frozen state. Other excipients in the formulation can also cryoconcentrate. Although there is a complex
dependence on factors such as the rate of cooling and composition, phase and state diagrams provide some insight into the
cryoconcentrated system. If sodium chloride (NaCl) is present, a eutectic is formed at –21.2 °C which has a concentration
of 23.3% w/w, i.e., an approximately 25-fold increase from 0.9% w/w normal saline. For most carbohydrates (including disaccharides),
the concentration of solute in a maximal freeze-concentrated glass is around 80% w/w.16 Reactions that could lead to incompatibilities in the matrix are slowed down because of the low temperature, but the cryoconcentration
of solutes can counteract this effect. Reactions such as oxidation can be enhanced, especially because the solubility of oxygen
increases as temperature drops, while ice formation also excludes gases. Other potential incompatibilities among the solutes,
including the protein, can be exacerbated. Proteins also interact with the ice surface with a consequent perturbation of their
native structure. Proteins can partially denature at the ice interface through weakening of hydrophobic bonds as well as adsorption
on the ice surface.17 This phenomenon is largely reversible after thawing, although some fraction of the protein may become irreversibly damaged.
More importantly, depending on the storage temperature (in relation to the glass transition temperature of the cryoconcentrated
mass), this loss of protein structure can result in aggregate formation because the partially unfolded molecules interact
with other species around them. Storage above the glass transition temperature (Tg') of the matrix will allow greater mobility
for this to occur. Similarly, other solutes (e.g., NaCl, glycine, mannitol, sorbitol) can phase separate, crystallize, or
undergo phase transitions over time if frozen into nonequilibrium states during the freezing process, leading to protein destabilization.18 Maximally freeze-concentrated carbohydrate solutions relevant to biologics formulation tend to have a Tg' below –30 °C.19 Less than maximally freeze-concentrated systems have even lower Tg' levels. Proteins themselves have Tg' levels in the range
of –10 to –15 °C, but freezing without cryoprotectants is generally not viable.19–20 Practical storage areas always have a degree of temperature variability within which they are controlled. Temperature fluctuations,
especially in the vicinity of and above the Tg', can be especially detrimental because the rates of the processes described
above will increase significantly more than would be expected based on the nominal storage temperature.
Anurag S. Rathore, PhD, is a consultant, Biotech CMC Issues, and a member of the faculty in the department of chemical engineering at the Indian Institute of Technology. Rathore is also a member of BioPharm International's Editorial Advisory Board.
Articles by Anurag S. Rathore, PhD
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