Product & Process Considerations In the Development of Lyophilized
Product & Process Considerations In the Development of Lyophilized
A review of how to identify the critical product and process variables that need to be optimized to formulate a successful lyophilized biopharmaceutical drug product.
Gerald R. Magneson, Karen K. Jette and Thomas R. Kovalcik
Pharma & Bio Ingredients
The development of a parenteral drug product (DP) typically involves stabilizing an active pharmaceutical ingredient (API) that either is unable to meet the desired DP shelflife as a liquid formulation, or is not readily marketed and distributed as a frozen liquid. The API biomolecules that we have encountered range from high molecular weight proteins and peptides to DNA and siRNA. In addition, we have extensive experience in lyophilized synthetic molecules, such as antibiotics and oncology DPs, as well as nuclear imaging agents. While we have employed different drug delivery technologies in developing DPs, this article will focus on the critical product and process variables involved in stabilizing parenteral drug products by lyophilization.
Lyophilization is a process where an aqueous solution is frozen and its water content is largely removed by sublimation of water vapor under reduced pressure and temperature. This technology was developed for large scale commercial manufacturing in the 1930s and demonstrated its therapeutic utility during the production of lyophilized human plasma to treat wounded servicemen during the Second World War.1 The equipment and processing methodology has advanced and matured in its pharmaceutical applications. Although lyophilization technology has been employed to produce stable protein pharmaceuticals for decades, the potential deleterious effect of dehydrating proteins is well documented as a significant problem.2 Since a number of labile biomolecules fail to recover their functional activity upon rehydration, it is imperative to maintain the native state of the biomolecule during the lyophilization process.3
Additionally, there is an increased risk of aggregate formation caused by non-native protein interactions within the lyophilized cake matrix. Hence, the key to successfully developing lyophilized biopharmaceuticals is, first, to identify those variables that may harm the biomolecule's structure. Then, in order to circumvent biomolecular damage, one may formulate with stabilizing excipients or optimize process parameters (e.g., by increasing the freezing rate to decrease mannitol crystallization during the lyophilization cycle).
While alternative technologies such as spray-drying,4 vacuum drying5 and supercritical fluid technology6 have been developed, in part, in an attempt to decrease the extended processing times and expense involved in large-scale manufacturing, lyophilization is a popular choice as a proven technology, because of the difficulties inherent in verifying the safety and equivalence of an alternative process for manufacturing a protein pharmaceutical. Some of the beneficial properties of lyophilized products include:
Ease-of-use for handling, shipment and storage
Short reconstitution time
In this article, we will review how to identify the critical product and process variables that need to be optimized to formulate a successful lyophilized biopharmaceutical drug product.
Customer-Specific Development Questions
As soon as a customer's confidentiality needs are met, several questions need to be answered to ensure that product development efforts will satisfy their needs and specifications. Usually, the first question is, what are the structural characteristics of a customer's API? If the API is a synthetic molecule, the customer will likely possess its structure via conventional instrumentation; e.g., NMR, IR and mass spectroscopy. However, for biomolecules such as high molecular weight proteins, one must utilize a multitude of analytical methods that obtain overlapping and complementary information to gain insight about the protein's structural characteristics. When very little structural or stability information on a biomolecule is known, a good place to start is the amino acid sequence, which can be used to explore the fundamental properties of the protein or peptide, such as its molecular weight, theoretical isoelectric point and extinction coefficient. Furthermore, a survey of the scientific literature may provide structural and stability information on this API, or a similar biomolecule purified from different tissues or organisms.
Certain customer information is critical to develop a successful lyophilized dosage form. The clinical indication for this potential drug product is useful in order to know where the DP will be administered (e.g., self-administered at home, in the physician's office or in the hospital) as well as the route of administration (e.g., IV, SC, IM, topical or inhalant). The patient population factors, such as age, disease state and sensitivity to excipients, may significantly influence formulation strategy. Dose requirements for the DP (e.g., single injection, multidose, dosage frequency, fixed versus variable dosing) also influence formulation development. Finally, customers may have specifications for the container/closure system (e.g., vial/stopper) to best accommodate their dosage form in the marketplace *(see Table 1).
Once the customer's needs are clear, the next step is to ascertain basic physiochemical properties of a biomolecule. Such information is obtained under aqueous and solid state conditions by conducting screening or pre-formulation studies. Variables in biomolecule formulation that are commonly investigated include: buffers/pH, stabilizers, solubilizers and tonicity modifiers. In the case of a multi-dose (multiple injections) drug product used over several days, one may include preservatives in the variables list.7
In order to test which of the candidate components is best suited for drug product formulation, these variables must be evaluated for their effect on the native structure and function of the API by employing stability-indicating methods. These stability-indicating methods may be acquired from the customer or developed to support formulation studies. These test methods should measure changes in secondary, tertiary and quaternary protein, or peptide structure (e.g., far-UV Circular Dichroism (CD) and Fourier Transform-Infrared Radiation (FT-IR) spectroscopy for secondary structural analyses; near-UV CD and intrinsic protein fluorescence for tertiary structural analyses and static, or dynamic light scattering, with or without Size Exclusion (SEC), or Reversed Phase (RP-HPLC) chromatography for quaternary structural analyses). In addition, one should evaluate the effect of formulation variables on the therapeutic function of the biomolecule by performing a bioassay (e.g., measuring enzymatic activity, determining immunoreactivity of a monoclonal antibody against its epitope, etc.
Pre-formulation studies help to identify those variables that appear most critical for stabilizing the biomolecule at the desired storage temperature as well as at least one elevated temperature; e.g., 40?C. These typically include the following screening studies:
pH, buffer and ionic strength compatibility studies (physiological buffers such as histidine, phosphate and acetate at wide pH range and salt concentration)
Different biomolecule concentrations
Effect of excipients (cryoprotectants, lyoprotectants, surfactants, bulking agents and tonicity modifiers
Initial container compatibility (glass vial and rubber stopper)
Environmental stressors (air oxidation, metal ions, light)
The nature of these screening studies entails using pre-existing stability information, such as observations
Figure 1: Design-Expert Plot made during protein or peptide process development, or literature results, to make educated formulation assumptions that are tested by multiple methods in an attempt to identify those components most likely to enhance the stability of the biomolecule. Furthermore, these formulation components and excipients usually are GRAS (Generally Recognized as Safe) chemicals that are sourced for their high purity and low endotoxin levels.
One must be careful not to place too much emphasis on the effect of components on the aqueous solubility of the biomolecule. For example, a protein may be more stable to thermal stress in a high ionic strength formulation in solution, but less stable following multiple freeze-thaw cycles relative to a low ionic strength formulation. Freeze-thaw instability is likely caused by the concentration effect on electrolytes and protein in the interstitial matrix between ice crystals and their interactions in the matrix (i.e., a 25-fold concentration of salt occurs in a 4% (w/v) solids formulation). To better assess these components in a model drug product format, it is preferable to test biomolecule formulations that are freeze-dried using a very mild lyophilization cycle in a research tray dryer.
In evaluating biomolecule formulations that are stressed at elevated temperatures, it is important to note that some proteins may retain nearly all of their functional activity, while experiencing extensive structural perturbation. Since there are obvious health risks inherent with the presence of protein aggregates in a parenteral biopharmaceutical, one must be aware of the appearance of non-native structure in the biomolecule as observed by stability-indicating assays. Hence, one must identify those beneficial product variables and their effective concentration range to stabilize biomolecules in lyophilized screening formulations against suitable response factors (e.g., bioassay activity, alterations in secondary structures and percentage soluble aggregates by SEC-HPLC).
After identifying the key product variables for stabilizing the API, the optimizat