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Biopharmaceutical Analysis Overview

During development full characterization of the biopharmaceutical is required.  Due to their size, complexity, and heterogeneity analysis is typically more complex in comparison to the analysis of small molecules, therefore, a range of liquid chromatographic techniques alongside mass spectrometric detection are combined and utilized (Table 1).


Type of Chromatography


Ion exchange chromatography (IEX), chromatofocusing, capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF)


Size exclusion chromatography (SEC), capillary gel electrophoresis (CGE)


Reversed phase HPLC (RPLC), hydrophobic interaction chromatography (HIC), hydrophilic interaction chromatography (HILIC), micellar electrokinetic chromatography (MEKC)


Affinity chromatography

Table 1: Types of chromatography utilized in biopharmaceutical analysis (most highly used techniques shown in bold)

Amongst biopharmaceuticals monoclonal antibodies (mAbs) are the most promising class of therapeutic molecules, therefore, we will be focusing on the analysis of these types of molecules using IEX, SEC, RPLC, and HILIC [1].
The structure of a mAb is shown in Figure 1.  There are several characteristics inherent to mAbs which need to be considered to provide a detailed characterization of the compound (whilst this list is comprehensive it is by no means exhaustive): 

  • Amino acid sequence
  • Amino acid composition
  • Structural integrity
  • Higher order structures
  • Aggregation
  • Disulfide bridges
  • N- and O-glycosylation
  • N- and C-terminal sequence
  • Charge variants
  • Deamidation/isomerization
  • Oxidation
  • Clipping

Figure 1: Anatomy of a monoclonal antibody (mAb)

Many chemical modifications, which are generally unwanted and need to be characterized, are introduced during the manufacture and storage of mAbs.

The characteristics of biopharmaceuticals can be determined at different levels (Figure 2).  At the protein level the molecular weight (MW), structural integrity, charge variants, aggregation, and post-translational modifications can be determined.  Due to the size and complexity of the molecule at the protein level simplification through digestion or hydrolysis allows analysis of smaller fragments. For example, acid hydrolysis of the protein will yield the individual amino acids which can be analyzed using reversed phase liquid chromatography (RPLC) to give the amino acid composition.  A very common strategy in biopharmaceutical analysis is known as peptide mapping in which the protein is digested (i.e. trypsin digestion) to yield the corresponding peptides which are then analyzed to provide information on the amino acid sequence, modifications, modification sites, disulfide bridges etc.  When using an appropriate enzyme, such as peptide-N-glycosidase (PNGase F), the sugar moieties can be cut from the protein and analyzed to determine the glycosylation profile of the protein.  

Whilst UHPLC instruments are preferable, they are not essential. A standard 400 bar rated HPLC system can be used with long, narrow columns packed with small particles, as long as elevated temperatures and low volumetric flow rates are used to keep the operating back pressures below the 400 bar upper limit, especially at the start of the run when the mobile phase is predominately aqueous.

However, UHPLC is generally preferred; due to the increased efficiency, afforded by the narrower system capillary tubing and optimized extra column volumes, alongside the increased detector scanning speeds facilitating more accurate detection of the highly efficient and narrow peaks.

Acetonitrile (MeCN) is the organic modifier of choice and is employed nearly universally. Its reduced viscosity, lower polarity, and lower UV cut-off (imperative when detecting at <220 nm) provide numerous and sufficient benefits.


Figure 2: Biopharmaceutical characteristics determined at different levels 

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