Understanding lyophilization formulation development: the author covers the fundamentals of lyophilization and provides case studies about the development of lyophilized biopharmaceutical products and the importance of biophysical characterization in form

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Understanding lyophilization formulation development: the author covers the fundamentals of lyophilization and provides case studies about the development of lyophilized biopharmaceutical products and the importance of biophysical characterization in formulation and the lyophilization process
Feb, 2004
by Frank Kofi Bedu-Addo
Pharmaceutical Technology
The author covers the fundamentals of lyophilization and provides case studies about the development of lyophilized biopharmaceutical products and the importance of biophysical characterization in formulation and the lyophilization process.
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Lyophilization or freeze-drying is often used to stabilize various pharmaceutical products, including virus vaccines, protein and peptide formulations, liposome, and small-chemical drug formulations (1-4).
Often a pharmaceutical product may be susceptible to physical and chemical degradation when stored as a ready-to-use solution. The goal of the formulations scientist is to identify the right formulation conditions, the right excipients in optimal quantities, and the right dosage form to maximize stability, biological activity, safety, and marketability of a particular product.
Desired characteristics of a lyophilized product
A lyophilized product should possess certain desirable characteristics, including
* long-term stability
* short reconstitution time
* elegant cake appearance
* maintenance of the characteristics of the original dosage form upon reconstitution, including solution properties; structure or conformation of proteins; and particle-size distribution of suspensions
* isotonicity upon reconstitution (in some cases, also for bulk solution).
The lyophilization process
The lyophilization process consists of three stages: freezing, primary drying, and secondary drying.
Freezing. During this stage the formulation is cooled. Pure crystalline ice forms from the liquid, thereby resulting in a freeze concentration of the remainder of the liquid to a more viscous state that inhibits further crystallization. Ultimately, this highly concentrated and viscous solution solidifies, yielding an amorphous, crystalline, or combined amorphous-crystalline phase.
Primary drying. The ice formed during freezing is removed by sublimation at subambient temperatures under vacuum. This step traditionally is carried out at chamber pressures of 40-400 Torr and shelf temperatures ranging from -30 [degrees]C to + 10 [degrees]C. Throughout this stage, the product is maintained in the solid state below the collapse temperature of the product in order to dry the product with retention of the structure established in the freezing step. The collapse temperature is the glass-transition temperature ([T.sub.g]) in the case of amorphous products or the eutectic temperature ([T.sub.e]) for crystalline products.
Secondary drying. The relatively small amount of bound water remaining in the matrix is removed by desorption. During this stage, the temperature of the shelf and product are increased to promote adequate desorption rates and achieve the desired residual moisture.
Possible destabilizing effects of the lyophilization process
Freezing. Freezing damage can occur with labile products such as liposomes, proteins, and viruses (5,6). initial ice-crystal size depends on the relative contributions of nucleation and crystal growth of ice. A rapid nucleation and growth rate resulting from a large degree of supercooling leads to a larger number of small ice crystals, which in turn presents a large ice-water interface (7). Exposure of proteins to this ice-water interface can lead to denaturation. Freezing stresses also can disrupt the liposome bilayer and emulsion structure.
The freezing step will determine the structure of the final dried cake as well as the drying rate. Small ice crystals produce pores with lower volume-surface area, thus resulting in lower diffusive flux and slower sublimation rates (7).
Drying. Removal of the hydration shell from proteins and products such as liposomes during drying in the absence of the appropriate stabilizers can cause destabilization of the protein structure and fusion of liposomes (8,9). Extremely low water content in the final product can result in destabilization, and optimal water content should be determined (10). The desired residual moisture must be correlated to stability during long-term storage as part of development studies.
Excipients in a lyophilized formulation
The design of a lyophilized formulation is dependent on the requirements of the active pharmaceutical ingredient (API) and intended route of administration. A formulation may consist of one or more excipients that perform one or more functions. Excipients may be characterized as buffers and pH adjusters, bulking agents, stabilizers, and tonicity modifiers.
Buffers. Buffers are required in pharmaceutical formulations to stabilize pH. In the development of lyophilized formulations, the choice of buffer can be critical. Phosphate buffers, especially sodium phosphate, undergo drastic pH changes during freezing (6,11,12). A good approach is to use low concentrations of a buffer that undergoes minimal pH change during freezing such as Tris, citrate, and histidine buffers (13).
Bulking agents. The purpose of the bulking agent is to provide bulk to the formulation. This is important in cases in which very low concentrations of the active ingredient are used. Crystalline bulking agents produce an elegant cake structure with good mechanical properties. However, these materials often are ineffective in stabilizing products such as emulsions, proteins, and liposomes but may be suitable for small-chemical drugs and some peptides (14,15). If a crystalline phase is suitable, mannitol can be used. Sucrose or one of the other disaccharides can be used in a protein or liposome product.
Stabilizers. In addition to being bulking agents, disaccharides form an amorphous sugar glass and have proven to be most effective in stabilizing products such as liposomes and proteins during lyophilization (1,8,9,16). Sucrose and trehalose are inert and have been used in stabilizing liposome, protein, and virus formulations. Glucose, lactose, and maltose are reducing sugars and can reduce proteins by means of the mailard reaction (17-19).
Two hypotheses have been postulated to explain the stabilizing effects of the disaccharides.
* The water replacement hypothesis: Disaccharides have been found to interact with these products by hydrogen bonding similarly to the replaced water. a The vitrification hypothesis: Disaccharides form sugar glasses of extremely high viscosity. The drug and water molecules are immobilized ill the viscous glass, leading to extremely high activation energies required for any reactions to occur (8,9,16,20,21).
Tonicity adjusters. In several cases, an isotonic formulation might be required. The need for such a formulation may be dictated by either the stability requirements of the bulk solution or those for the route of administration. Excipients such as mannitol, sucrose, glycine, glycerol, and sodium chloride are good tonicity adjusters. Glycine can lower the glass-transition temperature if it is maintained in the amorphous phase. Tonicity modifiers also can be included in the diluent rather than the formulation.
Glass-transition temperature and its significance
When heated, sugar glasses undergo a second-order transition from a rigid state to a viscoelastic rubbery state. The temperature at which the vitreous transformation occurs is the [T.sub.g]. When a product exceeds the [T.sub.g] value, the rigid glass softens to become a highly viscous rubbery material and collapses. The [T.sub.g] value of a formulation can be determined by differential scanning calorimetry (DSC), and the collapse temperature is measured by freeze-drying microscopy (22-24). Primary drying is always performed at the highest possible temperature while maintaining the product below the collapse temperature. A 5 [degrees]C increase in product temperature can lead to a decrease in drying time by a factor of two (13).
The dried amorphous product material also has a [T.sub.g] value. As water is removed during secondary drying, [T.sub.g] increases. Storage below [T.sub.g] is important for several products to maintain the rigid-glass structure and hence stability of the product (25,26).
Formulation example 1: development of a lyophilized liposome formulation
Preformulation studies were performed to select optimal pH, ionic strength, and excipients to optimize stability of the drug and lipids (F.K. Bedu-Addo, R. Coe, S. Bhamadipatti, and J. Pawelchak, The Liposome Co., 1997). A diacyl phosphatidylcholine was used as the matrix lipid, cholesterol was included in the formulation to improve rigidity of the bilayer, and a negatively charged lipid was included to improve blood circulation time. Mannitol was selected as the bulking agent, and maltose was a stabilizer. Dynamic light scattering and microscopy were used to monitor liposome fusion and disintegration. Lipid and drug stability were monitored by reverse-phase high-performance liquid chomatography (RP-HPLC). Water content was evaluated by Karl Fisher titration.