Lyophilization process

In addition to the additives used in a formulation to help stabilize the protein to freezing, the residual moisture content of the lyophilized powder needs to be considered. Not only is moisture capable of affecting the physicochemical stability of the protein itself, equally important is the ability of moisture to affect the Tg of the formulation. Water acts as a plasticizer and depresses the Tg of amorphous solids [124,137,138]. During primary drying, as water is gradually removed from the product, the Tg increases accordingly. The duration and temperature of the secondary drying step of the lyophilization process determines how much moisture remains bound to the powder. Usually lower residual moisture in the finished biopharmaceutical product leads to enhanced stability. Typically, moisture content in lyophilized formulations should not exceed 2% [139]. The optimal moisture level for maximum stability of a particular product must be demonstrated on a case-by-case basis. 

Depending on the processing variables, including the additives, the end product of a lyophilization process may be amorphous or crystalline or of intermediate crystallinity. As explained below the amorphous product has a higher free energy and therefore dissolves faster and tends to be less stable and more hygroscopic than the crystalline product. The choice between the crystalline and amorphous material may depend on whether an improved solubility or improved stability is required. 

Chase, D., R. Monitoring and control of the lyophilization process using a mass flow controller. Pharmaceutical engineering, p. 92-98, Jan.lFeb. 1998... 

During the lyophilizing process, the sample has to get external heat because sublimation of ice withdraws heat resulting in cooling the probe, but avoid melting of the sample. 

During simulated lyophilization processes, the pressure inside the freeze drier should be measured and compared with the set values. 

Media-hlled vials (soybean casein digest broth or fluid thioglycollate medium) are hlled on the hlling line, transported to the freeze drier, loaded into the chamber, subjected to a simulated lyophilization process, stoppered, sealed, and incubated for 14 days at 25°C. The minimum number of vials to be used is 5000. 

The method employed for lyophilization process simulation testing generally is similar to those used for solution fills with the addition of the transport and freeze-drying steps. However, it should focus on loading and sealing activities, which are presumed to be the greatest source of potential contamination. 

Where anaerobic organisms have not been detected in the environmental monitoring or sterility testing, lyophilizer process simulation tests should utilize TSB and air. 

Alternatively, an appropriate number of glass vials are filled to the proper level with sterilized WFI, following which the filled bottles are subjected to the lyophilization process. The processed bottles are then filled with a known volume of a sterile liquid medium, sealed, and incubated as described above. 

The second station is the stoppering station. The filled vials are partially stoppered, keeping enough opening required for the lyophilization process. For liquid products, the vials are fully stoppered. The vibratory bowels and stoppering assembly are easily disassembled for cleaning and sterilization before use. 

The need for two stages in the lyophilization process is that the free water, frozen to crystals, is readily removed under low pressure by sublimation. However, water bound by hydrogen-bonding to the crystalline lattice or molecular matrix of the product, especially if it is proteinaceous, is more difficult to remove and requires a much longer time and increased heat exposure. 

Allison, S.D., Molina, M.C., Anchordoquy, TJ, (2000). Stabilization of lipid/DNA complexes during the freezing step of the lyophilization process the particle isolation hypothesis. Biochim. Biophys. Acta, 1468, 127-138. 

Surfactant Prevent/control aggregation, particle formation and surface adsorption of drug Examples include polysorbate 20 and 80 Employed if aggregation during the lyophilization process is an issue May serve to reduce reconstitution times Examples include polysorbate 20 and 80... 

Buffers for lyophilized formulations need additional consideration. Some buffers like sodium phosphate can crystallize out of the protein amorphous phase during freezing resulting in rather large shifts in pH. Other common buffers such as acetate and imidazole should be avoided since they may sublime or evaporate during the lyophilization process, thereby shifting the pH of formulation during lyophilization or after reconstitution. 

Samples of Concentrates. Concentrates or dried organic residues from extraction or lyophilization processes should be stored at —70 °C or lower. Little information is available regarding the stability of samples stored cryogenically. Thus, research should be conducted in this area to determine the best method for the storage of such samples to prevent the degradation and development of artifacts. 

Development studies, summarized within a distinct report on the physiochemical aspects, drug substance attributes, and finished product characteristics, become critical parts of the validation package. Such data is also valuable for future integration into a manufacturing operation. This includes the scientific rationale for formulating and bulk-handling procedures, lyophilization processing parameters, finished product analysis, and stability requirements. 

Development activities encompass drug substance characterization, formulation design, packaging selection, and process development for manufacturing. Each of these aspects influence the lyophilization process. For a drug substance, upstream processing and quality aspects of the starting material need to be quantified. These quality aspects include both assay and purity. In particular, levels of... 

Lyophilization processes consist of the manipulation of environmental conditions of subambient temperatures and subatmospheric pressures. These extraordinary conditions are created by the lyophilization equipment. The success of the process, therefore, relies heavily on the operating performance of the lyophilizer. Confidence in the ability of the equipment to create these necessary environmental conditions is achieved through the successful completion of a comprehensive IQ and OQ. Without the proper performance of the equipment, there is limited opportunity for successful processing of materials. 

Trappier, E. Scale-up Strategy for a lyophilized process. American Pharmaceutical Review Fall 2001. 

Bruells, M. Folestad, S. Sparen, A. etal., In-situ near-infrared spectroscopy monitoring of the lyophilization process Pharm. Res. 2003, 20, 494-499. 

Genin, N., Schott, G., Rene, F. Control of operation cycles and final product quality in the vacuum lyophilization process. Recents Prog. Genie Proc. 9, No. 39, Strategic d Acces au Produit Industrie , 73-78,1995... 

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