Lyophilization excipient

T. W. Randolph, Phase separation of excipients during lyophilization Effects on protein stability, J. Pharm. Sci., 86(11), 1198 (1997). 

The product is presented in lyophilized format and contains sodium phosphate, sucrose and polysorbate as excipients. It is usually administered as once-weekly s.c. injections, typically for periods of 6 months. 

After initial cell fermentation and product extraction from the producer cells, the crude preparation is subject to multiple chromatographic steps, including ion-exchange, hydrophobic interaction chromatography and gel-filtration chromatography. The purified product is presented in lyophilized form in vials (1 mg active/vial) and excipients include a phosphate buffer, sodium chloride and serum albumin. 

Neorecormon (tradename, also known as epoetin beta) is a recombinant human EPO first approved for medical use in the EU in 1997. It is indicated for the treatment of anaemia associated with various medical conditions, most commonly chronic renal failure and cancer patients receiving chemotherapy. Neorecormon is produced by recombinant DNA technology in a CHO cell line and is manufactured as outlined in Figure 10.5. It is presented in lyophilized format at various strengths (500-10 000 IU/vial) and contains phosphate buffer, sodium chloride, calcium chloride, urea, polysorbate and various amino acids as excipients. 

Keratinocyte growth factor is a 140 amino acid, 16.3 kDa member of the FGF family. It differs from native keratinocyte growth factor in that the first 23 N-terminal amino acids have been deleted, which improves its stability. After cell growth the product is recovered and purified by a multistep chromatographic protocol. It is presented in lyophilized format in single-use vials and containing mannitol, sucrose, polysorbate 20 and histidine as excipients. It is administered by daily i.v. injection, usually for several days. 

Once the protein is purified, it will be formulated to produce the drug product. This could be as simple as diluting the protein in phosphate-buffered saline, or as complex as addition of excipients and lyophilization. The mark of... 

A variety of analytical methods, such as ELISA and HPLC, can be used to evaluate the effect of excipients or lyophilization on the stability of the biophar-maceutical product. Some parameters the analytical methods should evaluate are degradation, chemical and physical changes, aggregation, adsorption, and loss of biological activity. 

Inclusion of a cryoprotectant in the formulation can stabilize the protein during the freezing and drying stages of lyophilization.17 Excipients frequently... 

The finished form is a sterile, lyophilized powder formulated with tri-methamine, mannitol, and sucrose as excipients. 

TABLE 5.8. Examples of excipients used to enhance protein stability in solution and lyophilized formulations... 

Dosage form Xigris is supplied as a sterile, lyophilized powder in vials containing 5 and 20 mg drotrecogin aha (activated) and excipients. 

As a result, various excipients (from small molecules to polymers) have been added to the aqueous solution prior to lyophilization. Some of the most common are carbohydrates [86], polymers [87], organic buffer [81], competitive inhibitors [42], or nonbuffer salts [88]. 

Cyclodextrins (sometimes called cycloamyloses) make up a family of cyclic oligosaccharides composed of 5 or more a-D-glucopyranoside units linked 1 —> 4, as in amylose (a fragment of starch), and have been used as activating macrocyclic excipients in nonaqueous biocatalysis. Griebenow et al. observed that lyophilization of methyl-[3-cyclodextrin with subtilisin Carlsberg in a 6 1 weight ratio resulted in a 164-fold rate enhancement in THF for the transesterification rate of sec-phenethyl alcohol with vinyl butyrate (Scheme 3.3) [98]. 

A particularly interesting, and extremely simple, method of activating enzymes for use in nonaqueous media is lyophilization in the presence of nonbuffer salts. In the process, a great deal about excipient-enzyme-water interactions has been learned along with an appreciation of how enzymes adjust to novel microenvironments while retaining their intrinsic catalytic properties. 

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). 

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. 

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

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