Peptide-Based Drug Design

Serum Stability of Peptides 183
� Conjugation of the peptide with polyethylene glycol (PEGylation) is quite
powerful (23), increasing the peptides’ water solubility and reducing their
immunogenecity (24).
� Conjugation of the peptide to albumin (25) or the Fc domain of, for instance,
human gamma immunoglobulin (26) may extend the half-life of the peptide.
2. Human blood samples can rather easy be converted to human serum with the
use of BD Vacutainer R○ SST centrifugation tubes (Becton Dickinson Diagnostics,
Franklin Lakes, NJ). If serum purification is considered over custom orders, the
reader should consider pooling serum from several individuals. The serum should
also be heat inactivated at 56◦C for 30 min. Inactivation may cause precipitation;
thus sterile filtration of the serum is recommended.
3. Obviously, tissue homogenates, or even fractionated tissue homogenates (27),
other than human blood serum can be tested using the described method, but for
pharmacological testing, human blood serum is usually the most relevant medium
and is therefore used as the example in this chapter.
4. Several factors are known to inflict on the peptides serum stability, causing
misleading results:
� Nonhuman plasma is known to have different levels of certain peptidases
compared to human serum and may in turn give misleading (too high or too
low) stability results (28,29).
� Plasma from patients with pancreatitis or other disease states may change the
plasma peptidase activity—for example, certain viral diseases (30).
� Serum from subjects/animals of different ages may also affect the level of
certain peptidases (31).
� Plasma prepared with strong chelators such as ethylene diamine tetraacetic acid
(EDTA) may act as peptidase inhibitors for metalloproteases and peptidases
using divalent cations as cofactors (28).
� Immunoassay methods may not be stability specific (32).
� Radiotracer methods where the probe degradation occurs through a labelspecific
pathway are not suitable (33).
5. Measurement of the proteolytic serum activity should ideally be done at different
time intervals. However, studies have demonstrated that an adequate time point
for detection of peptide degradation in sterile serum is 1 h. This time point gave
almost full degradation of the unstable control peptide, with comparable levels of
the degradation products with both RP-HPLC and mass spectroscopy (15).
6. A higher concentration of TCA will push the equilibrium of charged or polar
substances with some size, even peptides, further from soluble toward precipitation,
thus reducing the amount of detectable peptide in the sample (34).
7. Amphipathic peptides may stick to amphipathic surfaces, i.e., cells, media components,
or chemical instruments, thus making their quantitative analysis rather
difficult (35).
8. Retro-orbital bleeding is suggested as blood samples for measuring peptides
in vivo stability/pharmacokinetics in mice should be as sterile as possible to