Preformulation studies

Product development of a peptide or protein drug starts with preformulation studies on the bulk material. These studies include physicochemical characterization of the bulk material and evaluation of its solubility and stability.5 The primary sequence and carbohydrate profile of the protein will be established, and its extinction coefficient will be determined. The solubility profile of the protein, its stability, and its isoelectric point (pi) will determine which pH should be used for formulation development. For example, a pH range of 3.0 to 8.0 may be investigated at a constant ionic strength of 100 to 150 mM. Then, using the pH of maximum stability, effect of ionic strength on stability may be investigated using an NaCl concentration of 0 to 1 M. Using optimized pH and ionic strength, effects of various buffer species on stability can be screened. Using the optimized buffer system, the effect of protein concentration itself on protein stability can be screened. Protein:excipients screening studies will also be run using the optimized buffer system, and other studies such as freeze-thaw stability, agitation stability, and ultraviolet (UV) light stability will be run.6 The peptide content of the bulk powder should also be determined and stated for other users, such as for the formulation scientist who will take over after the preformulation studies are completed. Most chloride and acetate salts of peptides are hygroscopic and may contain a nonstoichiometric amount of acid; thus, obtaining required amounts by just weighing without adjustment for peptide content can lead to error.7

Relevant stability-indicating assays are developed, and any potential stability issues or formulation challenges are identified. Preformulation studies should also characterize the impurities in the bulk protein as these will affect the stability of the product. For example, presence of a protease, even at very low concentrations, can destabilize the product.8 However, variants (which may result from the manufacturing process) with similar biological activity as the product are considered to be product-related substances rather than impurities. Impurities, when present, may be product related or process related. Process-related impurities may include host cell proteins, culture media components, lipids, polysaccharides, and viruses. These impurities can affect the immunogenicity of the protein as discussed in Chapter 1. Trace levels of deoxyribonucleic acid (DNA) from host cells may also be present and can be quantitated using a DNA hybridization test. The process history and purification history of a protein, such as its exposure to different pH, buffers, and contaminating proteases, may also affect its potency, stability, safety, pharmacokinetics, or immunogenicity and thus should be consid-ered.9 However, a purified protein is not necessarily stable. Although the advances in biotechnology can now provide us with highly purified products, the purified protein may actually be more susceptible to processes such

Table 4.3 Analytical Methods for Degradation/Purity Tests Degradation reaction Analytical methods

Purity testing SDS-PAGE (reducing and nonreducing), UV assay,

SEC-HPLC, CEX-HPLC, LAL test, ECP enzyme-linked immunosorbent assay (ELISA), Western blot, DNA hybridization

Deamidation Isoelectric focusing, ion exchange chromatography

Oxidation Reverse-phase HPLC, peptide analysis

Aggregation Size exclusion chromatography, SDS-PAGE, light scattering, analytical ultracentrifugation as shear, agitation, and the like. This is because the purified protein does not have the natural environment that normally contributes to its stability. This environment may sometimes include other proteins, carbohydrates, lipids, or salts that help to stabilize the structure.10

The analytical methods most useful for degradation or purity tests are listed in Table 4.3. Based on analytical testing and all the preformulation work, specifications will be developed for the bulk protein. These will typically include appearance, color, bioassay (potency), and purity specifications based on UV, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reverse-phase high-performance liquid chromatography (RP-HPLC), or cation exchange-HPLC (CEX-HPLC) assays. Limits for specific degradation products can be based on the analytical methods listed in Table 4.3. Limits for endotoxin and residual DNA can also be determined by the Limulus amoebocyte lysate (LAL) test or DNA hybridization test, respectively. Generally, the final product should contain no more than 100 pg of cellular DNA per dose. The rabbit pyrogen test will detect all pyrogens and is subject to false positives and high variability. Thus, the LAL test for pyrogenic lipopolysaccharides from Gram-negative bacteria such as Escherichia coli (endotoxins) is also used.11 The identification test can include analytical methods such as amino acid analysis, tryptic mapping, circular dichro-ism, N- and C-terminal sequencing, isoelectric focusing, or mass spectrometry. For a detailed discussion of these analytical methods, refer to Chapter 2. As would be clear from this discussion, preformulation is a broad term, and many of the topics discussed elsewhere in this book would also be relevant for an understanding of preformulation activities. Once a final formula is developed, additional specifications such as those relating to pH, bioburden, osmolarity, and particulate matter will be developed, and sterility testing will be performed.

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