Advantages of Isopropanol for the Reverse
Phase Analysis of Peptide Pharmaceuticals
Daniel R. Studelska & Claire E. Brook
Peptides Analytical R&D[ Background ]Cadenza column

Why would using Isopropanol be beneficial?

Gradient:
10-40%B(10min)
40-60%(20min)
10%(30min)

TFA/acetonitrile/water
YMC Pro C18 3μ

There is currently a worldwide acetonitrile shortage; as a result of the shortage, acetonitrile
costs have risen dramatically.
What makes Isopropanol a suitable replacement for acetonitrile?
Blank

The major reason isopropanol is avoided is its higher viscosity. As we show, with the rightcolumns, this is not an issue.

TFA/isopropanol/water
Cadenza CD- C18 3μBlank

Acetonitrile can produce unwanted artifacts during gradient elution. While isopropanol has its
own unwanted gradient behaviors, they often do not overlap with acetonitrile, making
isopropanol the preferred solvent in the examples presented here.

Fig 2: Analysis of peptide reaction mixture.
Fig 1: Less baseline noise with isopropanol.

[ Summary ]For a recent product, extant analytical methods were all release methods of marginal use for inprocess analysis. The peptide contains cysteines, and is synthesized in a totally reduced form. The
active peptide is folded into a compact form via the formation of –S-S- bridges, thus the “linear”
reduced peptide must be oxidized to produce the correct product. The first two columns employed
were not able to resolve the reduced from the oxidized form. A release method column could resolvethese species, but the gradient was not optimal, and we altered mobile phase composition and thegradient to maximize resolution, and to mitigate a very noisy baseline. With particles smaller than 5
microns (3.5 or 3 µ), the baseline noise with acetonitrile was particularly bad where the producteluted.
To address this, we developed a method employing isopropanol/water gradients instead of theacetonitrile/water. We employed Cadenza CD-C18 reverse-phase columns; 3 µ columns notable forhigh resolution (200,000 plates/m) and low backpressure. The low backpressure, combined with thegreater eluent strength of isopropanol, compensated for its increased viscosity. With a 4.6 x 150 mm
column we were able to run at 1 ml/min and stay within the recommended pressure limits of thecolumn. More importantly, we obtained excellent separation of reduced and oxidized peptide. Inview of the current supply issues and higher expense of acetonitrile, we think this approach deservesconsideration.

[ Materials & Methods ]Figure 3: Separation of reduced from oxidized peptide
during disulfide bridge formation.

Figure 4: Purification of correctly folded product.

We employed either a Cadenza CD-C18 column or an Intrada WP-RP. The Cadenza is 4.6 x 150 mm
with 3 µm particles containing 12 nm pores; the Intrada is 4.6 x 250 mm with 3 µm particles containing
30 nm pores. The mobile phases were the same for both columns:
Mobile Phase A : 0.1% TFA in 8.6% IPA/water
Mobile Phase B : 0.1% TFA in 96% IPA/water
For the Cadenza:
Flow Rate: 1.0 mL/min
Temperature : 40 °C
Wavelength: 220 nm
Injection Volume: 10 µL
Gradient %B (interval in min): 0-2 (5), 2-8(15), 8-12(25), 12-100(30),100(33), 0(50)Vydac Protein & Peptide C18 5μ0.1% TFA acetonitrile/water

Intrada WP-RP 3μ0.1% TFA isopropanol/water
Method 1

For the Intrada:
Flow rate mL/min (interval in min): 0.6-1.0 (30), 1.0 (42), 0.6 (48)Temperature : 55 °C
Wavelength : 214 nm
Injection Volume : 10 µL
Gradient %B (interval in min) : 9-15 (30), 15-100 (35), 100 (38), 9 (48)Intrada WP-RP 3μ0.1% TFA isopropanol/water
Method 1
Fig 5: Improved separation of a 37-mer peptide.

The Cadenza was employed for the chromatographs presented in figures 1-4; the Intrada wasemployed for the chromatographs presented in figure 5. Figures 1, 3 and 4 are analyses of the cysteine
peptide mentioned in the introduction. Figure 2 is an analysis of a synthetic step for a different peptide,and figure 5 shows the development of an analytical method for a third peptide.

Intrada WP-RP 3μ0.1% TFA isopropanol/water
Method 2