Intact Protein Analysis by RPLC Using Hand-Portable HPLC

Introduction

Biopharmaceuticals are rapidly becoming more popular and in recent years have increased from just one, to currently seven of the top 10 drugs. Biopharmaceuticals are proteins and peptides, such as monoclonal antibodies, that are genetically engineered from living cells. The size of these molecules typically fall in the range of 2 to 2,000 kDa. Compared to small molecule pharmaceuticals, biopharmaceuticals have more complex structures and more reactive groups which often lead to lower stability and chemical properties that are easily modified. As a result, the quantitation identification, heterogeneity, impurity content and activity of each batch of target biopharmaceuticals must be thoroughly investigated before release. Due to the complexities of proteins, there is a need for powerful analytical techniques for their characterization, among which capillary liquid chromatography (LC) is one of the most important.

Transferring from established LC methods to micro- and nano-flow capillary LC is a simple procedure
if you follow these guidelines. The mobile phase flow rate must be significantly reduced; other separation conditions (i.e., stationary phase, temperature, mobile phase components and gradient program) remain nearly the same. If the ratio between column length (L) and particle size (dp) is kept within +50% and -25% (as referenced in the United States Pharmacopeia guidelines), then transferring the method should start with adjusting the mobile phase flow rate to keep the linear velocity unchanged. More specifically, flow rate should be scaled according to the change in cross- sectional area of the column, which is proportional to the square of the ratio of the column diameters.

Overview

This brief application note demonstrates the use of hand-portable capillary HPLC by the Axcend
Focus LC for intact protein analysis. Specifically, the separation of five test proteins in the range of 5.8 to 45 kDa is shown using reversed phase (RP) conditions. Other protein samples can be analyzed using the Axcend Focus LC merely by adapting established conventional high performance LC methods to the capillary column format.

5 Protein Test Compounds

Run Conditions

Instrumentation: Axcend Focus LC w/UV Detector at 275 nm wavelength

Column: CoAnn C4, 1.7 μm FPP, 300 A, 100mm

Mobile Phase A:  0.1% TFA in water

Mobile Phase B:  0.1% in acetonitrile

Flow Rate: 1.25 uL/min

Injection Detail: 0.2 uL

Sample concentration: 0.25 mg/mL carbonic anhydrase, myoglobin, human insulin, ovalbumin each and 0.1 mg/mL cytochrome C mixture in water

UV detection wavelength: 275 nm

Column temperature: 22° C

Chromatographic Results

Summary

• The Axcend Focus LC is well suited for the separation of biomolecules, such as peptides and proteins.
• Transferring methods from conventional to capillary LC should involve scaling the mobile phase flow rate according to the change in column cross section.
• Chromatographic results obtained using the Axcend Focus LC demonstrate reproducible separation of a mixture of five test proteins in the size range of 5.7 to 45 kDa within 14 min.
• Good peak shapes comparable to conventional HPLC are observed for all proteins.
• Improved resolution and greater peak capacity can be obtained by using longer capillary columns.