LC–MS/MS of large molecules in a regulated bioanalytical environment – which acceptance criteria to apply?

Abstract

BioanalysisVol. 5, No. 18 EditorialFree AccessLC–MS/MS of large molecules in a regulated bioanalytical environment – which acceptance criteria to apply?Magnus Knutsson, Ronald Schmidt & Philip TimmermanMagnus KnutssonFerring Pharmaceuticals A/S, Copenhagen, DenmarkSearch for more papers by this author, Ronald SchmidtSanofi-Aventis Deutschland GmbH, Frankfurt, GermanySearch for more papers by this author & Philip Timmerman* Author for correspondenceBioanalysis Department, Janssen R&D NV, Turnhoutseweg 30, Beerse, Belgium. Search for more papers by this authorEmail the corresponding author at ptimmerm@its.jnj.comPublished Online:23 Sep 2013https://doi.org/10.4155/bio.13.193AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: acceptance criteriaEuropean Bioanalysis ForumLC–MSpeptideproteinregulated bioanalysisAnalysis of large molecules has become the talk of the day in the bioanalytical community. The increasing importance of peptides and proteins as therapeutic agents, together with the enormous possibilities offered by new MS-based technology, has opened a new world for the bioanalytical scientist. The European Bioanalysis Forum (EBF) has been following these new developments closely and have dedicated a Topic Team (TT) to discuss and share experiences on the bioanalysis of large molecules with LC–MS-based technologies. In this Editorial, the EBF wants to share their perspective on how to integrate LC–MS of peptides and proteins in regulated bioanalysis.With larger molecules, that is, peptides, proteins and oligonucleotides, becoming increasingly important as therapeutic agents in the future, the scientific community is building their experience in analyzing these molecules within the framework of regulated bioanalysis. Traditionally, these types of molecules are analyzed using ligand-binding assays (LBA) and to date LBA remains the working horse, especially for proteins.However, a lot of progress has been made by the MS/TOF instrument vendors to improve the performance of these systems. And with success: the mass ranges are increasing and overall sensitivity, especially in the higher mass ranges, has improved tremendously. At the same time, advances in software and IT hardware have also enabled faster scanning and more flexible approaches to data mining. Another feature of MS-based assays is their selectivity. As a consequence, bioanalytical approaches based on MS for peptide and protein analysis are gaining increased interest [1–3].In addition, MS methods can have a complementary selectivity to LBA methods. Where the latter methods detect molecules based on binding affinity and 3D conformational structure, they may not be able to distinguish between a protein and its metabolites. MS-based methods do have that ability and will be able to generate more accurate data on unchanged peptide/protein concentrations, compared with LBA, in cases where metabolism hampers accurate data from LBA. MS methods normally give total drug concentrations. For LBA methods, this may depend on the type of assay, and they can give either total or free drug concentration. Depending on the project in development, the crossreactivity of LBAs towards analyte-like compounds (potentially active metabolites) can be an advantage to better describe the PD. However, in case we need to document the accurate PK behavior of the compound, MS-based methods may be the better choice.Because of the increased interest within the industry to discuss the bioanalysis of large molecules, the EBF hosted a Focus Meeting in June 2011 ‘Large meets Small‘[4] to discuss the topic. As a follow-up to this workshop, the EBF decided time at their annual strategy meeting in spring 2012 to continue the discussion. A TT devoted to the analysis of large molecules using LC–MS/MS was established. The TT was further divided into two sub-teams: one focusing on company bioanalytical strategies when developing peptides/proteins, another focusing on the regulatory aspects of large molecule bioanalysis using LC–MS/MS. The initial work from the latter team was presented and discussed at the 5th EBF Open Symposium in Barcelona [101]. In this Editorial, the current thinking of the EBF on which acceptance criteria to apply for LC–MS-based bioanalysis of large molecules in regulated bioanalysis will be discussed.LC–MS(/MS) approaches for bioanalysis of large moleculesWhen using LC–MS based technology for bioanalysis of large molecules two main approaches are frequently used: either the analyte is measured as intact analyte or as digested analyte.▪ Intact analyte LC–MS(/MS) approachThis approach normally follows a traditional LC–MS(/MS) bioanalytical protocol, that is, it involves conventional sample preparation steps (e.g., protein precipitation or SPE) prior to the LC–MS(/MS) quantification [5–7]. Using current state-of-the-art LC–MS(/MS) instrumentation, this approach is predominantly used for peptides, small proteins and oligonucleotides with a molecular weight typically below 4–8 kDa. As an alternative, hybrid LC–MS/MS platforms have been employed, that is, by combining an immunoaffinity sample preparation step with the LC–MS/MS quantification [8,9].▪ LC–MS/MS approach using a digestion stepThis approach is more complex and mainly used for proteins or larger peptides. In addition to the intact analyte approach, this approach involves a (enzymatic) digestion step, where the protein/peptide is digested into smaller peptides [10,11]. One or several of the resulting smaller peptides can be identified as signature peptides, in other words, smaller peptides being selective and quantitatively representative for the intact molecule, and quantified in the subsequent LC–MS(/MS) quantification. There are several protocols for analysing proteins with the digestion approach, but they generally all involve a sample preparation step that could either be based on conventional techniques [12,13] or on an immunoaffinity extraction step [14,15]. Dependent upon the nature of the analytical problem (e.g., size of analyte and required sensitivity) the sample preparation step for the digestion LC–MS(/MS) approach can either be employed on the protein level, peptide level or on both levels.LC–MS platformsFor both the intact and the digested analyte approaches, today it is most common to use conventional LC–MS/MS triple quadrupole instruments for quantification.However, the usage of TOF/HRMS instruments for LC–MS bioanalysis of intact proteins have recently been reported [16]. So far, the majority of these publications are used in drug discovery, but certainly this approach will gradually find its way in regulated bioanalysis.Needless to say that also the increased separation power offered by miniaturization of LC, again resulting in increased sensitivity and selectivity, has contributed to the success of peptide/protein analysis using LC–MS-based methods.ISAs always when dealing with LC–MS/MS assays, the choice of IS is crucial. For large molecules, the availability of a suitable IS might also be an obstacle. Whenever possible, a stable labeled IS is recommended in regulated bioanalytical environments. In the context of peptides and proteins, this is not different and implies we need to have a labeled peptide (for peptide analysis), or either a labeled intact protein (resulting in labeled signature peptides after digestion) or a labeled signature peptide (that will not compensate for the digestion step) in the case of protein analysis.Acceptance criteria for LC–MS/MS bioanalysis of large moleculesWhen moving from an existing analytical platform, that is, LBA, for the large molecule class of compounds to a new analytical platform, in other words, LC–MS/MS, the obvious question arises: which acceptance criteria should be used? Should it be driven by the class of compounds, the analytical technique acceptance criteria history or something else?The current guidelines on bioanalysis make a distinction for the acceptance criteria: for LBA the 4–6–20 rule applies (four out of six QC samples should be within 20% of the nominal value), whereas 4–6–15 (four out of six QC samples should be within 15% of the nominal value) is applied for LC–MS/MS assays [17,18]. Although debatable from a scientific decision-making perspective, this distinction was largely driven by the higher variability of a LBA method compared with a LC–MS method, and has been endorsed by industry and regulators. At the time these expectations were defined, there was a clear separation on which molecules were analyzed with which technique (LC–MS/MS for small new chemical entities [NCEs] and LBA for peptides/proteins). In the last years, and because of the aforementioned technology improvements, the need for this separation is fading and molecules historically analyzed using the 4–6–20 paradigm are now analyzed using a technology requiring 4–6–15 based on small-molecule insights.The acceptance criteria for LC–MS/MS (4–6–15) in the current regulations is primarily based on the experience for analysis of one class of compounds (small NCEs), where the analytical challenges are normally less demanding. However, just because the analytical technique is capable of reaching certain acceptance criteria for one class of compounds, it may not be appropriate to apply the same acceptance criteria of another class of compound (peptides/proteins) where the analytical challenges are of another dimension.In order to be able to set proper acceptance criteria, it is important to consider all the factors influencing the assay performance. The use of LC–MS(/MS) for large molecules is generally much more complex compared with applying the technique for small NCEs; for example, the behavior in the mass spectrometer (fragmentation and different charge states), extraction recovery, carryover, the availability of a suitable and representative IS, and the added complexity of using an immunoaffinity sample preparation step or enzymatic digestion.The topic of appropriate acceptance criteria has been intensively discussed within the EBF TT, and the different member companies have shared their experiences and views.The current thinking from the EBF TT is to start with a conservative approach when defining acceptance criteria and not to propose acceptance criteria that are still too demanding for the technology/analytical approach, which is still under development. Our position is not only driven by the fact that analyzing peptides and proteins is a developing science; we also try to consider the lack of added value for the patient by setting the bar too high. By aiming at these higher standards (i.e., 15 instead of 20%), the time and cost for establishing an assay may increase. Also, from a patient-safety perspective, the industry has been comfortable in accepting 20% inaccuracy for a LBA-based peptide/protein assay. Changing the technology should not trigger changing the acceptance criteria if there is no compelling safety or PK need. As we move forward, if and when the technology/analytical approach matures, our proposed acceptance criteria could be subjected to revision, based on scientific needs.Thus, the EBF propose the following when it comes to acceptance criteria for large molecules using LC–MS/MS: for smaller intact analytes (e.g., peptides and oligonucleotides) it is normally recommended to apply 4–6–15 acceptance criteria. For larger intact analytes, especially if a hybrid LC–MS/MS approach is employed, 4–6–20 acceptance criteria are recommended. As normal practice within regulated bioanalysis, these acceptance criteria should be defined prior to the method validation (i.e., method validation study plan) or sample analysis (analytical plan). Experience from method development is a good source to define the acceptance criteria prior to validating the assay for a compound.As always, there might be situations where alternative acceptance criteria can be defendable to achieve valid results for a given analytical challenge without jeopardising the safety of the patient [19]. In most cases, 4–6–20 acceptance criteria will be achievable for digested analyte assays, but taking the increased complexity of these assays into consideration there might be situations and assays where a widening of the acceptance criteria beyond 4–6–20 based on method establishment data should be allowed. An example could be the analysis of digested analyte (e.g., proteins or large peptides) using LC–MS/MS.ConclusionThe EBF is pleased with the increased possibilities offered by LC–MS(/MS) to the bioanalytical scientist for the analysis of peptides and proteins. As part of their current ongoing discussions, it is the EBF‘s current thinking not to copy regulated requirements for small-molecule bioanalysis for peptides and proteins when analyzing them using LC–MS(/MS), with the exception of small intact peptides. At the same time, we want to focus the scientists‘ attention on the potential complementary information generated by LC–MS in addition to LBA data on a specific large molecule as an important strategic opportunity to increase the PK/PD knowledge. Hence, the use of both technologies should be considered and LC–MS should not necessarily replace LBA for peptides and proteins.AcknowledgementsThe authors would like to thank D Schmidt (Sanofi), leader of the European Bioanalysis Forum topic team, and the rest of the European Bioanalysis Forum Topic Team members, A Guenzi (F. 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Hoffmann – La Roche), G Krantz (Novartis), C Siethoff (Swiss BioQuant), G Rohde (Bayer), M Blackburn (Covance), MP Andersen (Novo Nordisk), P Lassahn (SwissBioanalytics), R Kay (Quotient Bio Analytical Sciences) and S Wood (Celerion) for their contribution to the discussions within the Topic Team.DisclaimerThe views expressed in this editorial are the ones of the European Bioanalysis Forum and do not necessarily represent the views of the individual members companies.Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download