As mentioned in part I of this series, there are four major applications areas of ultrahigh-pressure liquid chromatography (UHPLC) for biotechnology: peptide mapping, amino acid analysis (AAA), intact protein and antibody analysis, and glycan analysis or glycoprofiling. The first two of these areas were extensively covered in Part I. This installment will emphasize intact protein–antibody analysis and glycan analysis or glycoprofiling and why they are used.
In part I of this two-part series on the current usage of ultrahigh-pressure liquid chromatography (UHPLC) in biotechnology, we introduced the fundamentals of performing UHPLC and discussed specific applications for peptide mapping and amino acid analysis (AAA) (1). Readers are encouraged to read part I before part II. There are four major applications areas where UHPLC has become important for biotechnology: peptide mapping, amino acid analysis, intact protein characterization, and glycan analysis or glycoprofiling. These applications are essential analytical challenges in biopharmaceutical development, in which UHPLC has proven valuable (2–6). The first two topics were discussed in part I; here, we will focus on the latter two (6,7).
Using much smaller particle diameter packing materials, and shorter or narrower columns, has improved virtually all chromatography for larger proteins or antibodies, as well as for their smaller cousins. Such trends will, of course, continue into the future. When using UHPLC for biotechnology applications, perhaps the very first areas of emphasis have been intact proteins, especially mixtures of protein variants in a drug substance (DS), or antibody variants, isoforms, or glycoforms.
The structure of intact proteins presents a difficult analytical problem because the pharmacological activity of these large molecules is altered by small chemical changes to the protein. The modifications affect a tiny fraction of the chemical properties, so it is necessary to use multiple modes of separation to detect and measure them. The common approaches of reversed-phase chromatography, size-exclusion chromatography (SEC), and ion-exchange chromatography (IEC) are now available in UHPLC.
The reversed-phase high performance liquid chromatography (HPLC) of intact proteins, especially large molecules such as antibodies, is usually characterized by broad, diffuse, and poorly resolved peaks, with low plate counts and often large asymmetry values. These molecules are the "bad actors" of HPLC because their high molecular weights, slow mass transfer, and low diffusion coefficients lead to large peak volumes. Specific chemical interactions also degrade the analysis through mixed modes of separation (hydrophobic and hydrophilic patches and ionic binding), as well as poor solubility in most HPLC solvents. When UHPLC materials were being developed for proteins, it was efficient to consider both implementation of small particles with shorter diffusion distances and optimized particle chemistry for reduced chemical interactions. As illustrated in some of the figures in part I, this combination has facilitated using UHPLC for proteins or antibodies. For example, Figure 1 in this installment compares two different columns with the same base particle, bonded phase, and bonding chemistry, operated under identical conditions in two different particle sizes: 3.5 µm and 1.7 µm. It is a controlled comparison between conventional HPLC (3.5-µm particles) and UHPLC (1.7-µm particles). The relative retentions for all of the peaks are the same in the two chromatograms, but more resolution is apparent with the smaller particles. The sample consists of light chains (LC) and heavy chains (HC) of an antibody (immunoglobulin, or IgG) with the heavy chains having different degrees of glycosylation or modifications (post-translational modifications, or PTMs). In addition, the sample was reduced and intentionally alkylated only partially to further increase the sample heterogeneity as a test of chromatographic resolving power. The improved resolution with the UHPLC packing material and instrumentation is apparent. It also should be noted that the run time could be reduced by using different dimensions of the columns. Thus, the area of intact proteins remains one of the four most important applications of UHPLC in use today. It will surely remain so in the future.
Figure 1: UHPLC separation of light and heavy chains of a reduced and partially alkylated monoclonal antibody (IgG). (Reprinted with permission from reference 8.)