By Maxime Laugier
The development of injectable biopharmaceutical products is based on the ability of the formulation expert to overcome three main technical hurdles. First, you must preserve the activity of the native molecule, without damaging it or changing the active structure. Second, you must conserve or improve the bioavailability of the molecule in order to determine a targeted therapeutic activity. Finally, you have to ensure an acceptable product expiry date while considering the volunteer recruitment program of the clinical study, and you must adjust storage conditions which facilitate the distribution of the products and their convenient medical use. This article will focus on the strategy required for the development of such biotech products and on the main critical product and process variables involved in the stabilization of biomolecules and parenteral drug products in particular.
First Step: Characterization Of The Biomolecule And Drug Product Requirements
At the beginning of the development program, it is important to focus on the structural characteristics of the biomolecule and to define carefully the needs and specifications of the parenteral drug product. Nowadays, the characterization of most chemical compounds is easy, considering the large choice of conventional equipment available, such as infrared (IR), differential scanning calorimetry (DSC), X-ray diffraction, and mass spectrometry.
The specifications of the related parenteral product must be defined according to clinical purpose. The therapeutic indication targeted, the patient typology, the administration route (intravascular, intramuscular or subcutaneous), the therapeutic dose, and the posology required (simple or multiple dose) must all be suitably defined.
Second Step: Preformulation And Formulation Studies
Based on these preliminary requirements, the next step is to assess properties of the biomolecule under solid state and solution, throughout the preformulation studies. This approach aims to define the most appropriate conditions for the solubilization of the active ingredient, for its stabilization and administration. The biomolecule is tested according to several temperature, time, and atmospheric conditions, in combination with a wide range of agents such as solvents, buffers, stabilizers, solubilizers, and tonicity modifiers. In order to test which of the candidate components and conditions are best-suited for the upcoming drug product formulation, the effects on the structure of the protein are verified. In fact, the denaturing of a protein is commonly associated with a change of the spatial structure without dislocation of the peptides linkages. The main consequences of such change are a decrease in biological activity, a modification of optical properties, and a loss of solubility, which leads to product precipitation. Additional preformulation trials are implemented according to different active product concentrations, pH, buffer, and ionic conditions. Some inactive ingredients such as surfactants, tonicity modifiers, cryo- and lyoprotectants, and bulking agents are also tested in combination with the biomolecule in order to identify the most appropriate candidate for the drug product formulation. These excipients are generally recognized as safe (GRAS) chemicals and are sourced for their low endotoxin levels. Structural changes of the protein are influenced by environmental conditions. The stability of the biomolecule is therefore assessed according to negative, ambient, or elevated temperatures. The structural changes of the molecule are also measured after exposure to stressed conditions such as thawing and freezing cycles, air oxidation, and light. Compatibility, leachable, and adsorption studies are also performed between the biomolecule and the equipment used throughout the manufacturing process, such as vessels, sterilization filters, and tubing.
Compatibility testing with the packaging components is also performed to choose suitable vials and closure. In order to define the most appropriate administration conditions, the biomolecule’s stability and solubility is tested in conventional infusion solutions (such as glucose, sodium chloride, and Ringer solutions). Moreover, the compatibility study of the biomolecule with the components of the infusion device is required if some of the materials are different from those involved in the manufacturing.
After identifying the key factors for the stabilization of the biomolecule, it is necessary to assess the quality of the solution stored for six weeks or three or six months, in cold (2 to 8 degrees Celsius) and accelerated (40°C/75% RH) conditions. If the liquid formulation is not stable enough, the lyophilization of the product represents one of the best approaches to keep it safe for one or two years.
Third Step: Freeze-Drying Process Development
Lyophilization is a method that contributes to keeping a product stable in a dry state by dehydration. This method is based on sublimation, which determines the transfer of a solvent from a solid state to a gaseous one without any transition throughout the liquid state. This method is also based on desorption, which is an extreme sublimation improved by the increase of the product temperature in order to achieve the desired residual moisture. The lyophilization process consists of three stages: freezing, primary drying, and secondary drying. During the freezing stage, the formulation is cooled, and crystalline ice forms from the liquid. The solution becomes viscous and solidifies, yielding an amorphous, crystalline, or amorphous-crystalline phase. During the primary drying, the ice formed is removed by sublimation at subambient temperatures under chamber-negative pressure. The product is maintained in solid state below its collapse temperature throughout this step. At the end of this phase, secondary drying occurs, which aims to remove the bound water remaining in the matrix, by desorption. During this phase, the temperature of the product is increased under maximum vacuum, to promote desorption and achieve the targeted residual moisture. The most important phase of the freeze-drying process is the freezing step. Freezing must preserve the structure and integrity of components and avoid any residual interstitial liquid phase. Freezing temperature and kinetics influence crystal growth and, as a result, have an impact on the solvent sublimation speed during the primary drying. To assess such parameters, freeze-drying microscopy and thermal analysis represent reliable solutions to determine critical temperatures of the product like glass transition temperature in the case of amorphous products or the eutectic temperature for crystalline products.
For the freeze-drying of proteins, it is essential to pay attention to cryoprotective and lyoprotective agents, which are able to protect the biomolecule throughout freezing and drying steps. A cryoprotective agent is able to substitute water molecules by the polar groups and hydrogen bonds and to keep the three-dimensional structure of the protein. Glucose, glycine, dextrans, and polyvidone are appropriate excipients in such an approach. A lyoprotective agent provides an amorphous structure to the freeze-dried product which protects the active molecule. These kinds of excipients are able to limit the configuration changes of proteins during sublimation and desorption phases.
The relatively small volumes of bound water which remain after primary drying are removed by desorption during secondary drying. Biopharmaceutical lyophilized products may have a range of residual moisture, in the final product, of between 0.5% and 2.5%. In order to ensure the stability of the biopharmaceutical product, especially if the active molecule is sensitive to an oxidizing environment, it is advisable to backfill protein formulations with nitrogen gas to maintain a nonreactive headspace.
The development of a stable and reliable biopharmaceutical drug product must take into account a number of considerations such as therapeutic requirements, properties of the biomolecule, and product and process variables. Considering it is difficult to take into account all of the possible contingencies that one may encounter, a rational approach must be implemented in order to achieve the development of a biopharmaceutical product in a quick and effective manner.
About The Author
Maxime Laugier joined CREAPHARM PARENTERALS in early 2007 as the pharmaceutical development and projects director. He has over 14 years of experience in pharmaceutical development, investigational medicinal products manufacturing, and industrial scaling up with European CMOs.