BioPharmaSpec provides a mass spectrometry service for the structural and physicochemical characterization of your biopharmaceutical. From our globally located labs in the US and Europe, BioPharmaSpec scientists apply high resolution mass spectrometry expertise to biopharmaceuticals such as recombinant proteins, antibodies, antibody drug conjugates (ADCs), fusion proteins, bispecifics and biosimilars to characterize them according to the ICH guidelines.
What is Mass Spectrometry?
MS works by accurately identifying the masses of components passing through the system optics. Certain types of mass spectrometers (e.g. quadrupole orthogonal time-of-flight [Q-TOF] instruments) have the capacity to generate fragment ions from peptides either through isolation of specific components (MS/ MS analysis) or through the use of energy switching within the instrument (MSe ).
High resolution mass spectrometry has significant advantages over more simple chromatographic techniques, particularly during the development of biopharmaceuticals such as proteins.
Protein Mass Spectrometry
Proteins are a class of molecule that are amenable to mass spectrometric fragmentation at the peptide backbone. These fragmentation pathways result in a series of ions differing in mass by one amino acid. The sequence of the peptide can then be determined by looking at the mass differences between fragment ions derived from the same fragmentation pathway.
Since most amino acids have different chemical structures and therefore different masses, the identity of the amino acid can be determined by the mass difference between associated fragment ions. The limiting factor in the effectiveness of this as an approach is the size of the peptides that can be readily fragmented. Energy considerations and the need to limit dissipation of energy through the ionized molecule to promote fragmentation mean that the maximum size for fragment ion generation is about 2-3kDa, which translates to a molecule of approximately 18-28 amino acids in length. Clearly, this is much smaller than most proteins of therapeutic value (e.g. IgG monoclonal antibodies, which are composed of two light chains of approximately 214 amino acids each and two heavy chains of approximately 450 amino acids each). Therefore, these proteins need to be reduced down to a mixture of constituent peptides to generate meaningful fragmentation data.
This peptide generation is achieved through the use of specific protease digests in conjunction with on-line liquid chromatographic (LC) separation of the digestion products and UV and mass spectrometric analysis of the separated components. The most useful form of ionization for mass spectrometric analysis of peptides is electrospray, which produces peptide ions with multiple degrees of charging that are amenable to fragmentation. The whole procedure is therefore described as on-line LC/ES-MS analysis.
Figure 1: Peptide mapping data obtained from online LC/ES-MS analysis of a digest of a monoclonal antibody. The low- and high-energy mass spectra acquired during elution of the peak highlighted in red are shown. These data can be used to confirm the molecular weight and provide sequence information for each observed peptide
All mass spectrometers have the following components:
- an ionization source e.g. Matrix Assisted Laser Desorption Ionisation (MALDI) and Electrospray ionisation (ESI). Both of these create quasimolecular ions from the component of interest in the gaseous phase,
- a mass analyzer, which accurately measures the m/z ratios of the ionized species and
- a detector to provide the response.
BioPharmaSpec has a large range of mass spectrometers that are applied to different aspects of protein characterization. These include:
- Waters Xevo G2-XS Q-TOF Mass Spectrometers with ES and Nanospray
- Waters Xevo G2 Q-TOF Mass Spectrometers with ES and Nanospray
- Waters Xevo TQ-S Mass Spectrometers with ES AB
- Sciex 5800 MALDI TOF/TOF Mass Spectrometers
- Sciex TripleTOF 6600 QTOF Mass Spectrometers
Uses of Mass Spectrometry
BioPharmaSpec uses mass spectrometry to provide high-quality protein characterization information such as:
- intact molecular weight analysis,
- primary (de novo) amino acid sequence
- peptide mapping
- including analysis of Post Translational Modifications (PTMs)
- disulfide bridge analysis
- carbohydrate structure
- including monosaccharide composition,
- monosaccharide linkage and
- N and O oligosaccharide profiling.
The above methods for part of the ICH guidelines that should be followed when a protein is to be characterized according to globally accepted regulations. For a full description of the protein characterization methods required under these guidelines, please visit our protein characterization pages.
The ICH guidelines also state that an assessment must be made to show the absence of any contaminants, such as process related impurities. Such impurities include reagents such as inducers and antibiotics or downstream derived contaminants such as solvents or enzymes.
BioPharmaSpec uses Mass Spectrometry to develop (where necessary), optimize methods for the detection of process related impurities.
Detection of Host Cell Proteins
Host Cell Proteins (HCPs) are proteins from originating from the production cell line which are contaminating the drug product. HCPs are removed from the final product using purification techniques but it is very likely a small amount of HCP material will still be in the final product.
HCP is usually detected using an ELISA based method, initially with an off-the-shelf kit and latterly with a developed ELISA specific to your product. The regulators also recommend the use of Mass Spectrometry as an orthogonal technique for the accurate detection of HCPs.
Mass Spectrometry has the advantage of detecting a wider population of HCPs in a de novo manner without specialized polyclonal antibody reagents or product and process-specific method development.
BioPharmaSpec can provide a mass spectrometry service for HCP analysis including accurate detection of HCPs of interest i.e. those present in amounts >100ng per mg of product or those that are immunogenic.