With custom-designed oligopeptides becoming an increasingly popular option in
pharmaceuticals, it’s important to prevent spoilage of any kind.
Formulation of Four
Peptide Solutions in
15 Days
» by Fluence Analytics
Custom-designed oligopeptides are an increasingly accessible option in peptide-based technologies for pharmaceuticals. Following their synthesis, peptides are often subjected to HPLC purification and synthetic modification, which can
require the use of non-aqueous media or salts requiring later
removal. During the production of pharmaceutical peptides,
spoilage can occur in the form of aggregates, associates and other
means. Using ARGEN (Fluence Analytics), this app note demonstrates the rapid formulation of four peptide solutions that are
based on excipient effects correlated to increasing molecular
weight. The first peptide was formulated in five days for optimal
stability. The remaining three peptides were formulated in parallel
over 10 days.
Methodology
Four peptides of unknown primary structure were examined
against a panel of solution additives. Excipients included dimethyl
sulfoxide (DMSO), acetonitrile (MeCN), and guanidinium chloride (GnCl). Other conditions investigated were pH and peptide
concentration.
Each peptide was dissolved to 2 mg/mL in a 1:1 H2O:MeCN
cosolvent solution, which was adjusted to pH 7 before peptide dissolution, as prescribed by the peptide’s provider. This solution was
used to prepare 1 mg/mL solutions of the peptide with an excipient. These conditions included guanidine hydrochloride from 0 to
4. 3 M, DMSO from 0 to 20 percent by volume, and MeCN from
25 to 75 percent by volume. All solutions were held at 30 C for up
to 48 hours under constant monitoring by ARGEN.
Once the aforementioned assays were analyzed, a stock solution
with each additive at its optimal concentration was prepared for
each peptide. Each peptide was dissolved in its respective buffer
at 1 mg/mL, and pH was adjusted with dilute NaOH and HCl
solutions to identify the optimal pH between 2 and 10. This was
determined by characterizing the degree of aggregation for each
peptide at each pH level over a 48-hour period of aggregation
monitoring at 30 C. The formulated sample with the least aggre-
gation after 48 hours of monitoring correlates to the optimal stor-
age condition (variables included varied pH and varied concentra-
tions of additives). Solubility testing determined that all peptides
struggled to dissolve at concentrations greater than 10 mg/mL.
Results
Guanidinium chloride was shown to have stabilizing effects at
intermediary concentrations for the “A” group peptides. Stability
maxima were observed between 2 to 3 M of salt (Figure 1). The
light scattering signature of aggregation is steady and relatively
linear. This denotes a steady increase of normalized molecular
weight from its monomeric, unaggregated state, to that of a population of dimerized aggregates. Kinetic analysis showed that
peptides A-1 and A- 2 increased in stability by 250 and 350 times,
respectively, with the addition of the optimal concentration of
guanidinium chloride.
Both “B” group peptides, B-1 and B- 2, precipitated almost
immediately following the addition of saline solution. With the
understanding that the B group peptides are hydrophobic, this is
not unexpected. Though guanidinium is a chaotrope, its nature
as a salt increases the solution’s polarity, destabilizing the sparingly dissolved hydrophobic peptides. Further investigation would
greatly reduce the concentration of GnCl for these peptides to
analyze stability in a micromolar regime.
Across all four peptides studied, increasing concentration of
DMSO resulted in increased peptide stability. Three peptides
Peptide of
A.A.’s
Mass
(Daltons)
Hydrophilicity [Peptide] (mg/mL) pH [MeCN] (%) [DMSO] (%) [GnCl] (M)
A-1 7 850 Hydrophilic 2. 5 6 25 15.0 2.0
A- 2 7 850 Hydrophilic 1.0 6 25 20.0 3.0
B-1 36 4200 Hydrophobic 10.0 4 50 15.0 0.0
B- 2 15 1700 Hydrophobic -- 8 63.0 10.0 0.0
Table 1: Peptide characteristics and optimal conditions determined by rapid formulation with ARGEN.