Phu Duong Ph.D. Senior Application Chemist Agilent Technologies

In recent years, monoclonal antibodies (mAbs) have become one of the major biopharma products in response to the need to treat various diseases. These antibodies have been engineered with a specific genetic makeup for better targeting of disease agents. During the development of these antibodies, Protein A and G analytical affinity columns are used for determining their titer or concentration from various cell culture supernatants, to select the high-yield clone. An inert polymeric monolith is used as the support for both the Protein A and Protein G columns. Both columns have high affinity for antibodies, and so, they bind only to antibodies in cell-culture supernatants. However, they have different selectivity, as indicated in Table 1.

This application note introduces the Agilent Bio-Monolith Protein G column. The column is designed for high speed and high loading capacity. Data are presented to show the linearity with high specificity. Linearity analysis reveals the column’s capability for accurate quantitation analysis of mAbs in cell-culture supernatant. In addition, lifetime analysis data indicate that the column can be highly reproducible, and has a long lifetime with stable and low backpressure. The Bio-Monolith Protein G column is complementary to the Bio-Monolith Protein A column, to provide more options for titer determination of monoclonal antibodies.

Specificity and Selectivity

As indicated in Table 1, the Protein G column has higher binding affinity for different humanized monoclonal antibody subclasses than the Protein A column, and only Protein G has affinity for the IgG3 subclass. Data in Figure 1A demonstrate the specificity of the Bio-Monolith Protein G column and its ability to monitor titer, that is, the presence and concentration of antibodies in the supernatant. The column was injected with the sample containing purified recombinant humanized mAb, IgG3, which was spiked in the supernatant of CHO cells. This mAb was expressed in the CHO cell line. The data show that IgG3 was the only protein captured and eluted from the column, at about 1.6 minutes at 1.0 mL/min, whereas all host-cell proteins were not captured by the column, and eluted at the flow-through peak.

To show the different selectivity between Protein A and Protein G columns, both were separately injected with monoclonal antibodies IgG1, IgG2, and IgG3. Both IgG1 and IgG2 were captured by the two columns (data not shown) but IgG3 was not captured by Bio-Monolith Protein A. IgG3 was eluted as a flow-through peak, and IgG3 was only captured and eluted by the Bio-Monolith Protein G column (compare Figures 1A and 1B).

A more rugged test was performed to confirm that the specificity of the Bio-Monolith Protein G column was such that it had no binding affinity for host-cell proteins. Host-cell protein samples from E. coli cell lysate, CHO-cell lysate, supernatant, and insect-cell lysate were used. These lysates contained host-cell proteins that were extracted by lysing buffers that contained sodium dodecyl sulfate, a chemical that greatly influences nonspecific binding to the column. These samples did not contain antibody, only host-cell proteins. The test was run because rugged samples give nonspecific results if the column is not well designed.

Figure 2 shows no evidence of any protein from any host-cell supernatant being absorbed by the column. All host-cell proteins were eluted as flow-through peaks. The data suggest that the Bio-Monolith Protein G column did not have any affinity for host-cell proteins.

Accurate Quantitation

Accurate quantitation of mAb titer is essential during the early stages of development when the cell line is selected, and also during manufacture when the amount of mAb in the cell-culture supernatant determines the optimum harvest time. To demonstrate the ability of the Bio-Monolith Protein G column in the accurate quantitation of mAbs, different amounts (μg) of purified IgGs were injected onto the column. Data of peak areas versus amounts of IgGs generated were used to construct the linearity lines, to determine the accuracy of the analysis. Figure 3 shows the linearity of peak areas from the Protein G column, showing that the column can be used for quantitation of mAb in harvest cell-culture media with different concentration ranges. The column was injected as low as 2 μg IgG. The signal-to-noise ratio was no higher than 1:1 for 2 μg (data not shown). The maximum loading capacity for this column is approximately 400 to 500 μg IgG (data not shown), which covers the range of concentrations achieved during cell-line selection and production.

Wider Loading Range

Figure 4A shows the linearity comparison for a wide loading range of IgG3 between the Bio-Monolith Protein G column and another vendor’s 2.1× 30 mm, 4,000Å, protein G column. The Agilent column generated a linear loading range from 25 to 200 μg IgG3, whereas the other column could only generate linear data between 25 and 100 μg IgG3, as recommended by the other vendor’s literature. The reason for not having a linear line at a higher loading range was because the column was not able to retain all material at the higher loading range. In fact, at a 200 μg loading range, the other vendor’s protein G column had a break-through peak for the mAb (some IgG3 was not retained on the column and eluted as a flow-through peak), as shown in Figure 4B.


The Agilent Bio-Monolith Protein G column has a high affinity for monoclonal antibody subclasses. It is evident that the column can capture and accurately quantitate across a wide loading linearity range for mAb from supernatants. The columns can be used effectively to quantitate the amount of monoclonal antibody with various flow rates, without sacrificing data. The operating backpressure was significantly low, illustrating that the Bio-Monolith Protein G column can be operated with <600 bar HPLC instruments. The flexibility of the column with different acidic eluents enables easy and straightforward experimental design. Agilent Bio-Monolith Protein A and G columns are thus complementary, thus Protein G has affinity for mAbs that do not bind to Protein A, and vice versa. These columns provide more options for rapid titer determination for a wider range of mAb variants.

Phu Duong Ph.D., is a senior application chemist at Agilent Technologies.

Read the full application note, including materials and methods, method, conditions and more here. Learn about Agilent’s Protein G columns here.

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