January 1, 1970 (Vol. , No. )
Alexander Schwahn European Support Expert Thermo Fisher Scientific
This is the first article in a series of four, describing the characterization of protein-based bio-therapeutics by bottom-up analysis at peptide level (Peptide Mapping Analysis). This article summarizes the characterization attributes that are commonly addressed by this approach and looks at the potential challenges that need to be overcome to ensure optimal results.
30 years after the first market introduction of an antibody-based biologic drug product, the momentum of the biopharma market remains unbroken. Today, about 20% of the total pharma market is based on biologics and the biopharma industry continues to grow with an annual rate of more than 8%. Since 1996, biologic drug substances, especially the antibody-based ones, have seen a steep evolution and more and more refined and sophisticated products have become available. Starting from the engineering of fully human monoclonal antibodies (mAbs) that blend perfectly with the body’s own antibody repertoire to avoid an unwanted immune response, over antibody-drug-conjugates that allow the directed delivery of a small molecule drug to a limited population of carefully defined target cells (e. g., cancer cells) or the combination of different target structures in antibody-fusion proteins or bi-specific antibodies to harness a combinatorial effect, resulting in higher selectivity and efficacy.
One thing that all “new” and “old” bio-therapeutics have in common is that a rigorous analytical characterization is required to ensure efficacy and safety for the patient. Analytical requirements and challenges have risen with each new product class and will probably see a new high with the advent of the biosimilars, in terms of depth of analytical characterization needed. Biological drug substances inevitably display a degree of product heterogeneity as a result of the biotechnological manufacturing process applied and their tremendous structural complexity in comparison to conventional small molecule drugs. Adding to this complexity are posttranslational modifications (PTMs) of the “bare” protein such as glycosylation or the specific formation of disulfide-bridges that are crucial requirements for the functionality of the protein. This complexity however can give rise to a virtually unlimited number of potential combinations of modifications. Some of them are introduced by the production cell itself (e.g., alteration of the proteins amino acid sequence or the aforementioned PTMs). Others are introduced chemically or mechanically (e.g., modification of amino acid side chains such as deamidation and oxidation or the formation of aggregates) during the production process, product storage, or even during the analysis of the finished drug product. While the correct modification of specific amino acid side chains is an absolute requirement for product functionality, incorrect modifications may alter the product efficacy or cause adverse effects and thus pose a potential risk to product safety. Modifications in this category are considered as critical quality attributes (CQAs) and require careful monitoring throughout the production process.
Peptide Mapping: Protein Analysis from the Bottom up.
Among the different methods advised by the global regulatory agencies for the testing of identity, purity, and heterogeneity of biological drug products, bottom-up analysis of a protein on the peptide level by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is among the most versatile and powerful. The method can be used to ensure the identity of a protein-based drug by verifying the correct amino acid sequence. The presence of necessary as well as unwanted and potentially harmful modifications can be assessed and quantified simultaneously, often allowing to “pin-down” the precise location of the modification within the protein’s primary structure. A potential draw-back however is that any combinatorial aspect is lost during the process as the protein is digested into characteristic peptides prior to the analysis. Generally, sequence-specific endopeptidase such as Trypsin, or Lys-C, are used to digest the target protein into its characteristic peptides (the peptide map or fingerprint in the original context). The generated peptides are then separated by liquid chromatography ([U]HPLC), predominantly using the reversed phase principle, and introduced into an accurate mass tandem mass spectrometer. The precise masses of the intact peptides are recorded (MS1) followed by fragmentation analysis in MS2. Both the accurate mass of the intact peptides as well as their pattern of fragmentation in MS2, even in cases where a modification is present, can be precisely predicted from the known amino acid sequence of the bio-therapeutic. A systematic comparison of the experimental and predicted data using sophisticated computer algorithms then allows researchers to verify the amino acid sequence of the peptide or to detect sequence deviations and modifications. Accessible modifications comprise the protein glycosylation, the annotation of disulfide bridges and the detection of free cysteins, together with any modification of the amino acid side chain that results in a mass shift, such as deamidation (Asn, Gln) or oxidation (Met, Trp, Cys) events or the detection of modifications of the protein termini (e.g., N-terminal pyroglutamte formation or C-terminal Lysine truncations).
The quality of the sample preparation often pre-determines the quality and reproducibility of the final results. As such the actual proteolysis or digestion method applied has a high impact on the overall outcome. Current protocols tend to be lengthy, often conducted overnight, and labour intensive, requiring steps to facilitate protein denaturation that can introduce artefacts or compromise digest efficiency. An example is the carbamylation of arginine and lysine side chains by urea, a chaotrope commonly used to facilitate protein unfolding prior to its digestion. These modified residues not only complicate the peptide map and make accurate quantification more difficult, they also can prevent the trypsin cleavage at these sites thereby diminishing the digestion efficiency. The slightly alkaline conditions that allow for optimal trypsin cleavage harbour an additional risk to introduce artificial modifications, with deamidation of asparagine and glutamine being among the most prominent events followed by pyroglutamate formation at the proteins N-terminus.
The next article in this series will focus on a novel digestion approach featuring a heat-stabile immobilized trypsin, the Thermo Scientific™ SMART Digest™ Kit that overcomes most of the limitations above and allows for reliable protein digestion in less than an hour, using a simple and automation friendly 3-step protocol. Subsequent articles will discuss the advantages of separation and detection of peptides, with advanced UHPLC and mass spectrometry solutions, e.g., Thermo Scientific™ Vanquish™ UHPLC and Thermo Scientific™ Q Exactive™ Mass Spectrometry systems.
Dr. Alexander Schwahn (email@example.com) is the European Support Expert for the BioPharma Industry at Thermo Fisher Scientific.