Chromatography


Ultrapure Water for HPLC–SEC Analysis


Katrin Töppner, Sartorius Stedim Biotech GmbH, 37079 Goettingen, Germany; Dr Dirk Hansen, Phenomenex Ltd. 63741 Aschaffenburg, Germany; Dr Elmar Herbig, Sartorius Lab Instruments GmbH & Co KG, 37075 Goettingen, Germany


High-performance liquid chromatography (HPLC) is an analytical procedure for the separation, identifi cation and quantifi cation of substances. Unlike other variants of biopolymer HPLC (ion-exchange, hydrophobic interaction and reversed phase chromatography) that in general are performed in a gradient mode, SEC (size exclusion chromatography) is generally an isocratic method [1].


Size Exclusion Chromatography


Size exclusion chromatography (SEC), which includes gel permeation chromatography (GPC) and gel fi ltration chromatography (a special SEC method performed under aqueous conditions), works with a porous matrix of spherical particles as a stationary phase. Small molecules are able to penetrate into the pores and are therefore retained. By contrast, very large molecules are excluded and pass through the column at linear fl ow velocity. Hence, molecules are separated based on their size, with the large molecules exiting the column fi rst and the small molecules later [2].


A constant composition of the eluant is used for this SEC method. To minimise enthalpic effects during SEC, a strong eluant with correspondingly high elution strength is employed. In SEC, this is usually a buffer with an adjusted pH and a salt additive. An SEC column suitable for solving the specifi c problem at hand must be chosen to successfully perform this analytical method. A successful approach then lies in the selection of a suitable solvent, an optimal fl ow rate, the appropriate injection volume and concentration, as well as in the treatment of the column, both during operation and ‘offl ine’ handling (regeneration and storage) [1].


SEC is a separation method frequently used for biopolymers. Its current areas of application are the following:


• Determination of molecular weight, e.g., that of antibodies (immunoglobulin G; (see Figure 1), peptides and proteins)


• SEC as a tool for studying conformational changes • SEC in downstream processing (subsequent processing and purifi cation of a product)


• A special application for SEC restricted-access columns for automated sample preparation of peptide and protein mixtures [1]


Over the past decades, biopharmaceuticals have gained an important position on the medical drug market. Among these biopharmaceuticals are therapeutic enzymes, coagulation factors, numerous hormones – such as insulin, epoetin or growth hormones – monoclonal antibodies (mAbs) and biotech engineered vaccines [3].


SEC analysis is a standard method in downstream processing for determining the purity and aggregation states of the target molecule during biomanufacturing by recombinant production of therapeutic proteins and antibodies.


Determination of differences in molecular weights to detect contaminants and aggregates is based on the use of highly pure eluants that do not interact at all with the stationary phase.


Water of the special quality required for SEC can be purchased from various manufacturers or prepared cost-effi ciently on demand directly on site using a lab water purifi cation system, such as the arium®


pro VF.


The impurities and contaminants resulting during upstream processing (preparation of solutions) from the fermentation medium (host cell DNA, host cell proteins, components of the medium, such as albumin, insulin and transferrin), as well as microorganisms and endotoxins, must be reduced during downstream processing. This process is also a source of impurities (addition of proteases, leakage of protein A). The mAb product itself also forms impurities, such as split, aggregated antibodies, deamidated or oxidised forms of the mAb or incorrectly folded molecules [4] that must be detected.


For quality control of the purity of the target protein, SEC analysis, among other methods, is used to detect aggregated antibodies based on their molecular weight separation.


Figure 2. Current arium® pro


VF ultrapure water system (photo courtesy of Sartorius)


Preliminary trials were conducted to test whether the ultrapure water employed in these runs was suitable for use in SEC analysis. Ultrapure water was produced as described in [5] and used both for these preliminary trials and for further tests to determine the aggregation states of monoclonal antibodies.


The preliminary trials were designed to clarify whether the arium® pro VF utilised


(a predecessor model with the same technical specifi cations for production of ultrapure water as those of the current system shown in Figure 2) can be used for SEC analysis to detect aggregates and monomers of a monoclonal antibody. Furthermore, the effect of various buffer substances was tested.


The following materials were used in the test series (see Table 1):


Table 1. Materials HPLC


Column Dionex (pump/sampler/column oven/UV detector) Phenomenex Yarra SEC 3000 (OOH-4513-KO 300 x 7.8 mm) Precolumn Phenomenex SecurityGuard GFC 3000 (4 x 3mm, article no. AJO-4488)


Mobile phase 0.1 M sodium phosphate /0.1 M sodium sulphate, pH 6.6, conductivity 23 mS/cm in arium®


pro VF ultrapure water


The column used was a Phenomenex Yarra SEC 3000 (3 µm), which is an SEC column packed with modifi ed silica. Modifi cation of the new-generation ultrapure silica gel ensures minimal protein adsorption, which is essential for excellent recovery and reliable quantitation. The packing in the Yarra column employed has a pore size of 290 Å. This permits a linear range for native proteins between 5 kDa and 700 kDa [6].


Table 2. SEC-HPLC Method


Figure 1. Immunoglobulin G (IgG antibody; photo detail courtesy of Sartorius)


Experimental Evaluation of the Water


This paper describes the qualitative study of a recombinantly produced monoclonal antibody as an example of the use of SEC analysis. The solution employed stems from our in-house manufacture.


Parameters/Settings Flow rate Time


Maximum pressure


[ml/min] [min] [bar]


Temperature of column oven [°C] Injection volume UV detector


[µl] [nm] 1 20


180 25 5


220, 260, 280


LAB ASIA - NOVEMBER/DECEMBER 2016


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