Dosage Formulations

Proteins, peptides, antibodies and other biopharmaceuticals, like small-molecule drugs, must be characterized before and as a part of the formulation process with respect to physical-chemical stability and biological activity. While tests specific for each drug vary, some of the key analyses are the same. Common analyses, using a number of established and validated methods, concern amino-acid composition, amino-acid sequence, peptide mapping, disulfide linkages, molecular weight, endotoxin levels, residual host cell factors (proteins, DNA, and growth reagents), and inactive and degraded protein products. These methods include mass spectroscopy, high-pressure liquid chromatography (HPLC), sodium dodecyl sulfate polyacry-lamide gel electrophoresis (SDS-PAGE), isoelectric focusing, and immunoassays.

With respect to endotoxin content, the FDA requires that each dose have less than 0.5 endotoxin units (EU) for each ml of drug solution with a maximum tolerated limit of 5 EU for each kg of body weight. Additional tests, such as analyses to detect the degree of posttranslational modification (e.g., glycosylation, sulfation, phosphorylation, and formylation) may be required for every batch of protein. Relevant in vitro and in vivo biological testing also is carried out to verify that a given lot of protein product exhibits potency that falls within predetermined standards.

The physiochemically and biologically characterized proteins and peptides are further formulated and subject to stability studies. The goal of these studies is to develop a unique combination of excipients, solution pH, buffer, and container that will produce an optimum dosage form. Biopharmaceutical formulations should be stable in storage and in vivo. Protein and peptide formulations contain excipients to stabilize protein activity and reduce inactivation or loss due to adsorption to the container, oxidation, or hydrolysis. In some cases—insulin, for example—divalent cations such as Zn++ are added to increase the duration of insulin effect. Additional drug delivery considerations are discussed in Chapter 13.

While formulation of proteins in solution or suspension are less costly to produce, not all therapeutic proteins can be stably stored in solution or suspension, even when refrigerated (4°C) or frozen (-20°C). In those cases freeze-dried formulations of proteins may be used as an alternative. Freeze-drying or lyophilization typically produces an amorphous form of

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




Enhancement Effects

Human epidermal

Triton X-100


~2 fold increased stability at 60°C

growth factor

(0.02% w/v)


~1.5 fold stability increase at 60°C

(0.05% w/v)



Pluronic F-127


~3 fold increased stability at 4°C


(10% w/w)



Recombinant human



~50 fold increased stability at 37°C

keratinocyte growth

(0.5% w/v)



Human growth



375-1500 fold increased stability at 50°C


trehalose, or




Recombinant human

Sucrose or


4-12 fold increased stability at 50°C




receptor antagonist

(1% w/v)




~2 fold increased stability at 45°C

(0.5% w/v)





~2 fold increased stability at 35°C

(0.5% w/v)


Immunoglobulin E



Dramatic increase in stability at 5°C


(6:4 w/w)

and 30°C [26]

protein that can be readily rehydrated or resuspended in water just prior to use.

Lyophilization is a two-step process— freezing the protein solution and drying the frozen solid under vacuum. The drying step can be further divided into the primary event, removal of frozen water, and the secondary event, removal of nonfrozen protein-bound water, estimated to be 0.30 to 0.35 g/g protein—slightly less than surface-bound water (protein hydration shell) [12]. Protein lyophilization does not always yield increased stability compared with frozen liquid formulations. For example, the oxidation rate of lyophilized IL-2 is not slower than that of a frozen liquid formulation containing proper excipients or stabilizers [13]. Many of the factors affecting protein instability and stabilization in the freeze-drying process have been elucidated and recently reviewed [14-16]. The stability of freeze-dried protein formulations can be improved by controlling moisture content and pH, and adding cryostabilizers or protectants such as sucrose, polyols, surfactants, and polymers (Table 5.8).

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