Liquid and Lyophilized Protein Formulations

In developing any formulation, excipients need to be selected only when their use is essential in imparting a desired pharmaceutical effect (i.e., stability or delivery). 

In fact, it is a regulatory expectation that an appropriate excipient be chosen and its level (amount) in a formulation be demonstrated and justified through formulation screening and development studies (29,30). The science of protein formulation development has become increasingly sophisticated over the past 20 years and its discussion is beyond the scope of this chapter. The interested reader is referred to excellent reviews on this subject for further study (31-34). 

While each formulation is unique, there are several general aspects with respect to excipient components in both liquid and lyophilized protein formulations. A comparison of the excipient components in liquid and lyophilized protein formulations is provided in Table 1. 

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. 

Buffer Maintain pH of formulation through product shelf life Maintain pH of formulation during lyophilization and upon reconstitution 

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Protein-based drugs have been formulated mainly as stable liquids or in cases where liquid stability is limiting as lyophilized dosage forms to be reconstituted with a suitable diluent prior to injection. This is because their delivery has been limited primarily to the parenteral routes of intravenous (IV), subcutaneous (SC), or intramuscular (IM) administration. There are a few drugs that have been developed for pulmonary delivery, such as rhDNase (Pulmozyme ) and an inhalable formulation of insulin (e.g., Exubra ). However, even such drugs have been formulated as either liquid or lyophilized or spray-dried powders. This chapter will focus only on excipients that are applicable to liquid and lyophilized protein formulations. 

We begin with a brief summary of the degradation pathways of proteins, followed by a discussion on the composition of liquid and lyophilized protein formulations and on various excipients in some detail. An important feature of this chapter is a comprehensive table (Appendix), which details the formulations of approved protein drugs through the year 2005. The table has been compiled with the help of several sources (1,10,11). 

Table 1 Excipient Components of Liquid and Lyophilized Protein Formulations... 

FT-IR can also be used to determine the tertiary structure of a protein. It does not require the protein to be in solution, and it can often be used to support early formulation development for either liquid or lyophilized proteins. 

The PEG could stabilize proteins by two different temperature-dependent mechanisms. At lower temperatures, it is preferentially excluded from the protein surface but has been shown to interact with the unfolded form of the protein at higher temperatures, given its amphipathic nature (57). Thus, at lower temperatures, it may protect proteins via the mechanism of preferential exclusion, but at higher temperatures possibly by reducing the number of productive collisions between unfolded molecules. PEG is also a cryoprotectant and has been employed in Recombinate, a lyophilized formulation of recombinant Antihemophilic Factor, which utilizes PEG 3350 at a concentration of 1.5mg/mL. The low-molecular weight liquid PEGs (PEG 300-600) can be contaminated with peroxides and cause protein oxidation. If used, the peroxide content in the raw material must be minimized and controlled throughout its shelf life. The same holds true for polysorbates (discussed below). 

Spray freeze drying also has been proposed as an alternative technology to produce light and porous particles for peptide and protein delivery. Liquid nitrogen is used as recipient agent, into which the formulation is sprayed. The formed microparticles are harvested and lyophilized eventually. DNase and monoclonal anti-IgE antibodies have been used to demonstrate the feasibility of this concept (Maa et al. 1999). Promaxx microspheres are manufactured in a phase-separation process between water-soluble polymers and therapeutically active protein that results in particles having a high protein payload of up to 90 percent (Brown et al. 1999). 

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. 

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. 

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