Protein liquid formulation

The bacterial culture converts a portion of the supplied nutrient into vegetative cells, spores, crystalline protein toxin, soluble toxins, exoenzymes, and metabolic excretion products by the time of complete sporulation of the population. Although synchronous growth is not necessary, nearly simultaneous sporulation of the entire population is desired in order to obtain a uniform product. Depending on the manner of recovery of active material for the product, it will contain the insolubles including bacterial spores, crystals, cellular debris, and residual medium ingredients plus any soluble materials which may be carried with the fluid constituents. Diluents, vehicles, stickers, and chemical protectants, as the individual formulation procedure may dictate, are then added to the harvested fermentation products. The materials are used experimentally and commercially as dusts, wettable powders, and sprayable liquid formulations. Thus, a... 

Proteins must be stable during processing, storage, and reconstitution (if lyo-philized). Although liquid formulation is preferred for protein biopharmaceuticals, it may not always be the most stable presentation. The biopharmaceutical protein may need to be lyophilized to maintain stability. Lyophilization involves the removal of water from a frozen substance by sublimation and water vaporization under vacuum.17 But in some cases, this process may itself cause protein instability. 

A liquid formulation is usually comprised of a buffering agent, a stabilizer (which may also serve as a tonicity agent), a surfactant, and an anti-oxidant when protein oxidation is significant. Chelating agents are employed when metal ion catalyzed reactions predominate. A preservative may be included when a multi-dose formulation is desired. 

Different industries pose different challenges to the protein formulator. Many feed enzymes are sold as formulated liquid concentrates. In this case, the major requirements for a liquid formulation are enzymatic stability and preservation against microbial growth. It is sometimes not appreciated that the dominant factor affecting enzyme stability is the intrinsic stability of the enzyme itself formulation can do very little to correct for a structurally labile protein. Therefore, it is advisable to make stability an important criterion of the initial enzyme screening process. 

For liquid formulations, shaking the formulation increases the air/liquid interface in the formulation and often leads to protein denaturation. Several proteins are susceptible to denaturation by shaking, including human growth hormone (hGH) and recombinant factor XIII, both of which formed insoluble aggregates after shaking. 

The ability to achieve a higher concentration on reconstitution of the solid-state product also may allow the formulation scientist to side-step potential stability-limiting aggregation that may result in a liquid formulation of high-concentration protein. 

For liquid formulations, the choice of using either a salt or a carbohydrate to adjust the osmolality of the solution is made by the impact on protein stability. Sodium chloride is one of the most commonly used salts in the formulation of both traditional pharmaceutics as well as biological pharmaceutics. It is extremely safe, well tolerated, and inexpensive. However, the presence of sodium chloride in a formulation of rhuMAb HER2 was found to increase oxidation when the formulation was stored in stainless steel containers, presumably because the sodium chloride promoted corrosion of the stainless steel. Interactions of salts with the proteins must be investigated on an individual basis because the type and concentration of salt may lead to protein aggregation. 

In addition to the various physical techniques, a simple visual inspection of the formulation is conducted to determine if there are changes to the formulation. The human eye is extremely sensitive to many changes observed in protein formulations, and for this reason visual inspection is a valuable tool in the bag of techniques used to assess stability. For liquid formulations, the appearance of precipitates or a change in color of the formulation signifies trouble. For lyophilized formulations, the visual appearance of the lyophilized cake is an important characteristic of the formulation. Collapse or discoloration of the cake could indicate a compromised formulation. 

R.P. Scherersol system Liquid formulations for softgels, which incorporates the drug in a microemulsion preconcentrate or microemulsion form. Oral delivery of proteins and peptides enhanced oral bioavailability with reduced inter- and intraindividual variability in pharmacokinetics of certain drugs. Sandimmune Neoral (cyclosporine). 

Therapeutic proteins are usually prepared in liquid formulations or as freeze-dried powders that are to be reconstituted immediately before use. A number of the proteins have been found to be unstable when dried... 

For liquid formulations, stabiUzers such as sorbitol, glycerol, or propylene glycol are added to levels of 10% to 50% (w/w) to maintain the activity of the enzyme for up to several months at ambient conditions. Preservatives such as sodium benzoate, potassium sorbate, and sodium chloride are used to control microbial contamination. Buffers and surfactants such as Triton X-100 can also be added, although at lower levels than found in granulated cellulase enzymes due to the limits of tolerance of the proteins for these additives in Hquid solutions. 

In liquid formulation, optimizing the pH, in essence, optimizing the hydrolytic stability is a major issue. It is important to study the stability especially in the range from pH 3 to 10 early on in the formulation process. The solubility depends on the pH value, as does the physical and chemical stability. There is usually a close relationship between optimum solubility and stability. Minimum solubility is observed around the pi values of the proteins. Typically, one would determine the pH range for obtaining the proper solubility and concentration for dosage and then optimize the stability afterwards (18,67). 

Case Study Reference Standard Compared with a Lyophilized Formulated Dose In this case study, calibrators for an immunoassay were prepared from a lyophilized formulated dosage material for a therapeutic protein, and compared with the corresponding liquid formulation reference standard. Spiked control samples were likewise prepared from both sources of material and run in the... 

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