Perspective on LC-MS(/MS) for biotherapeutic and biomarker proteins in research and regulated Bioanalysis: a consolidation of more than a decade of experience across the European Bioanalysis Forum community (Part 1: “The What”)

ABSTRACT

Following up on our most recent discussion paper focusing on the continued regulatory challenges for bioanalysis of biotherapeutic and biomarker proteins with LC-MS/MS, the European Bioanalysis Forum reports back on their internal discussions on and experience with method development for biotherapeutic and biomarker proteins in research and regulated bioanalysis. Due to the broad array of topics discussed, this information is spread over two research papers, where one focusses on the fundamental principles on which the technology is built (i.e., the what?) and another on the practical considerations (i.e., the how). In this paper, we discuss ‘the what’. Both papers should be helpful for the bioanalytical community to better understand the challenges and provide an insight on why bioanalysis of biotherapeutic and biomarker proteins with LC-MS/MS should not be compared with the more traditional LC-MS/MS assay for small molecules or ligand binding assays for biotherapeutics.

Plain language summary

Article highlights.

• Since 2011, the European Bioanalysis Forum (EBF) has been a key player in large molecule analysis via LC-MS(/MS), focusing on science, challenges, advances and regulatory landscapes

• In 2020, the EBF formed a team to share experiences in large molecule analysis, leading to two manuscripts: one addressing “what” to measure and the other focusing on “how” to measure.

• This first paper, focusing on the What?, covers fundamental principles of quantitative protein bioanalysis, discussing critical aspects like whether to measure intact or partial proteins, selecting signature peptides, reference materials and internal standards.

• A second paper, focusing in the How?, explores practical methodologies such as sample treatment, automation and data processing.

• In the paper, we emphasize the importance of accurate protein measurement, because proteins often exist in various forms with different chemical structures and isoforms.

• In the paper, we compare top-down (intact protein analysis) and bottom-up (peptide-based) approaches, highlighting the strengths and limitations of mass spectrometry. Special attention is given to the selection of signature peptides to ensure sensitivity, stability and relevance. In addition, we emphasize the importance of stable isotope-labeled internal standards, which is deemed crucial for ensuring consistency and reliability in quantification.

• The EBF aims to foster discussion on the complexities of bioanalysis for biotherapeutic and biomarker proteins, which we feel to be different from small molecule LC-MS/MS assays.

• While our paper references the ICH M10 guideline on assay requirements, it does not aim to re-write or challenge it. Instead, the focus is on the scientific requirements for hybrid or immunocapture LC-MS(/MS) assays, which are not fully clarified by ICH M10.

• The EBF has previously discussed these regulatory challenges and aims to provide insights to help the bioanalytical community and regulatory authorities better understand the complexities of LC-MS(/MS) bioanalysis for large molecules compared with traditional methods for small molecules.

1. Introduction

Since 2011, the European Bioanalysis Forum (EBF) has played a key role in the area of large molecule analysis by LC-MS(/MS). This role has been broad and has covered discussions and recommendations on science, challenges, advances, interpretation, strategies and the regulatory landscape, including workshops or sessions at the Open Symposium or discussion or recommendation papers [1–4]. Continuing on this path, a team was formed in the EBF in 2020 to bring together the experiences of large molecule analysis by LC-MS(/MS) and identify areas of opportunity or concerns and share science and processes between our members. One of the deliverables of this team is to share our experience and learnings with the broader community on technology and process aspects. We decided to write two manuscripts, one discussing ‘the what’ and a second one discussing ‘the how’. In these papers, we will not go into the details of the continued regulatory challenges our community observes, i.e., no additional clarity given by the recent ICH M10 [5] on regulatory expectations. These have been discussed in our recent discussion paper [6]. Some of the EBF discussion go hand in hand with other discussions in industry on the subject [7].

Our feedback sets out to draw upon the scientific experience and learnings. Initially we planned to cover the “what” and the “how” in one paper, but because of the magnitude and wealth of information, it has resulted in two separate papers. This paper covers the fundamental principles of quantitative protein bioanalysis, the “what” – what to measure (intact or partial protein), what signature peptide to select, what reference materials to use and what internal standards to select. Our second paper [8] explores in-depth discussions on practical methodologies, the “how” - sample treatment, automation, instrumental analysis, data processing and analysis and method development and validation.

In several paragraphs, there is a potential link to assay requirements referred to in the ICH M10 guideline. Our paper does not intend to re-write or challenge the ICH M10 guideline on those aspects per se, but focusses on the scientific requirements we feel are paramount and which are not necessarily clearly articulated for the complex world of hybrid or immunocapture LCMS(/MS) assays for biotherapeutic and biomarker proteins.

For decades, ligand binding assays (LBA) have been the gold standard for quantification of proteins and large molecules. Even though this technique shows high sensitivity and selectivity, there are some and prerequisites and limitations to use LBA such as availability of detection antibodies, anti-drug antibodies /neutralizing antibodies (ADA/Nab) binding, shed target. Unlike small molecules and smaller linear peptides, proteins and other macromolecules are structurally complex and their quantification via LC-MS(/MS) is therefore less straightforward. Analysis of large molecules by LC-MS(/MS) has made major progress over the last decade and has developed to an orthogonal method to LBA which can help to overcome some of the beforehand mentioned problems.

In the field of protein bioanalysis, accurate measurement of proteins is essential for understanding their functions and pharmacological activities. Proteins can exist in various forms, either free or bound to endogenous binding partners, such as target molecules or anti-drug antibodies. Moreover, proteins often have multiple isoforms with distinct chemical and three-dimensional structures that can undergo further modifications during their presence in the body. The ability of a specific protein form to exhibit pharmacological activity depends on its binding extent, structural modifications and location.

Measuring proteins accurately is a complex task due to their large size and structural heterogeneity. Different analytical methods, such as LC-MS(/MS) and binding assays, target specific structural parts of these multifaceted protein molecules, resulting in the measurement of total, free, or bound protein concentrations, as well as the detection of various isoforms or modified forms. Therefore, it is crucial to clearly define the scientific question and the research context to design an optimal analytical method that yields meaningful and unambiguous results.

This text discusses the use of mass spectrometry (MS) analyzers for protein bioanalysis, including the triple-quadrupole (QqQ) technology and high-resolution mass spectrometry (HRMS). The authors explore the advantages and limitations of a top-down (intact protein analysis) versus bottom-up (peptide-based analysis) approach in MS. They emphasize the importance of selecting appropriate signature peptides for quantification, considering uniqueness, sensitivity and relevance to the protein's active form. Additionally, the use of reference materials and internal standards is discussed to ensure the accuracy and reliability of protein measurement.

2. What needs to be measured?

Typically, proteins are present both in their free form and bound to one or more endogenous binding partners, such as a circulating target molecule and/or ADAs. In addition, a protein usually is not homogeneous and inert, but instead often exists as a set of isoforms with different chemical and three-dimensional structures, which may all undergo additional structural changes during their residence in the body. Whether or not a specific protein form is capable of showing pharmacological activity, depends on the extent and the location of the binding of a protein and on the nature and the location of the structural modifications.

The large size of most protein analytes adds another level of complexity because any analytical method, be it LC-MS, a binding assay or a hybrid method, is directed to only a small structural part of these multiple protein molecules. As a result, it depends on the analytical principle of the method and on the exact part of the protein to which it is directed whether a total, free or bound protein concentration is obtained, whether one, several or all isoforms are targeted, whether the unchanged or modified protein form or the sum of both is picked up and finally whether the concentration found is actually representative for the active form of the protein.

The exact scientific question that needs to be answered should therefore be clearly defined with the respective project teams and it should be well understood which kind of concentration result offers most value in the given research context. Only then can an optimal analytical method be designed that will provide meaningful and unambiguous results. LC-MS offers many tools for specifically targeting a desired concentration and the relevance of these will be discussed in more detail in the next sections.

3. Measurement of intact or partial protein structure

There are two types of mass spectrometric analyzers to perform protein bioanalysis: the QqQ technology and HRMS. QqQ mass spectrometry using a bottom-up approach, whereby intact proteins are digested into peptides prior to introduction into the mass spectrometer, remains the gold standard for quantitative bioanalysis as it offers high sensitivity, a wide dynamic range and sufficient selectivity when operated in the selected reaction monitoring (SRM) mode. The emergence of HRMS by means of fast-scanning systems such as Qq time of flight (TOF) or Q-Orbitrap systems provides a higher resolving power, a wider mass range and has opened new possibilities in integrating qualitative and quantitative workflows both when intact protein analysis is required or for bottom-up workflows.

One important decision is whether to perform a top-down or a bottom-up analytical approach. The bottom-up approach using QqQ technology has the advantage of being more sensitive and the synthesis strategies for the peptide reference materials have been optimized for mass production and are therefore more easily sourced (see reference material). Using the SRM mode, specific transitions (combination of a specific product ion originating from a specific precursor mass after fragmentation) allow for high specificity and sensitivity of the quantification. Hence, the bottom-up approach using a proteolytic digest has been the mainly used approach in bioanalytic protein analysis and continues to be very popular and suitable. However, due to the low resolving power and the limited range of mass-to-charge ratio (m/z) of quadrupoles, a proteolytic cleavage step needs to be performed to convert proteins into peptides. As a result, the bottom-up approach can provide amino acid sequence information only for the targeted peptides and the processing of the sample results in a loss of labile post translational modifications. Therefore the identification of so-called “proteoforms” becomes more challenging.

In the top-down approach, the whole molecule is measured in its intact form and modifications (such as post translational modifications, sequence variations and splice isoforms) can be directly assigned to a specific proteoform. Thus, in contrast to LBA and LC-MS/MS technologies that target selected regions of a complex drug molecule, intact protein analysis studies define these complex molecules as a whole entity and are capable to discriminate important high-level structure or biotransformations which often occur with biotherapeutics, such as glycosylation, clipping, degradation and mass additions/losses. In addition, this allows to quantify total drug related material. If a generic antihuman immunoglobulin (IgG) or anti human-fragment crystallizable (Fc) is used for immunocapture, interferences can be reduced. Moreover, intact protein or antibody subunit sample preparation is often simpler than for the bottom-up approach. No digestion step is required, and protein mass detection is achieved over a wide and expected MS scan range without the need for surrogate peptide identification, synthesis and peptide MS optimization. However, the intact protein analysis approach is less established and sensitivity is typically lower. The most sensitive intact or subunit HRMS methods published so far for nonclinical pharmacokinetic (PK) studies can measure monoclonal antibodies (mAb) down to approximately 50 ng/ml. Thus, the recent improvements in sensitivity and mass resolution of HRMS systems enable its application in quantitative bioanalysis, especially when monitoring shorter PK profiles in high dose nonclinical toxicology studies, but might not be appropriate yet for longer duration PK characterization or a low-dose human clinical trial.

Furthermore, a middle-down approach using an HRMS system, may be employed for antibody subunit analysis by disulfide reduction and/or partial digestion, specifically, when top-down analysis is not sufficiently sensitive or for the analysis of mass variants localized in the subunits of large molecules such as mAb, antibody-drug conjugates or fusion proteins. These complex biotherapeutics can undergo biotransformation or catabolism and generate modifications such as glycoform changes, clipping products, or payload conjugation, among many others. If so, monitoring these in-vivo mass variants also called critical quality attributes (CQA) may be required for nonclinical and clinical studies to better understand how these biotransformations can affect toxicity and therapeutic efficacy. The middle down approach can detect very small mass variants like deamidation, acetylation, hydrolysis or oxidation. Generic detection using intact or subunit analysis has another advantage as it can also support analysis and quantification of co-dosed mAbs in a single and generic assay, assuming each therapeutic binds to the capture protein. In addition, for the analysis of some clinical monoclonal antibodies' biomarkers, which are produced in certain gammopathies and are unique for each patient, developing an LC-MS/MS method could be challenging and time-consuming as it requires a long sample preparation time and above all the need to find a specific surrogate peptide for each mAb from each patient. To this purpose, the use of LC-HRMS technology by the middle-down analysis of the monoclonal antibody light chains allows to obtain isotopically resolved mass spectra of these light chains which is mandatory for a robust deconvolution of the LC-MS/MS signal, and consequently for a robust quantification of these subunits.

Moreover, HRMS is an excellent alternative to QqQ using SRM methods for compounds that don't fragment well using collision-induced dissociation, for example for disulfide-rich cyclic peptides or for glycopeptides. The low intensity of the product ions observed in the MS/MS spectra of these compounds could not allow to achieve a good sensitivity using an SRM mode with a QqQ. HRMS operated in MS mode (using a full scan or a more sensitive targeted-SIM mode) can be an attractive platform for the quantitative analysis of these compounds.

4. Signature peptide selection

The choice of a peptide for protein bioanalysis by LC-MS/MS using the bottom-up approach must be made very carefully: the peptide must be unique for the targeted protein or the targeted isoform, with sufficient sensitivity, selectivity and stability.

First and foremost, the uniqueness of the chosen peptide is essential to obtain a selective, and in continuation, a robust and sensitive bioanalytical method, which is suitable for use in all nonclinical and/or clinical studies in a development program of a biotherapeutic drug. It must be ensured that the amino acid sequence of the signature peptide does not occur as part of any other endogenous protein in the biological matrix of interest, to avoid a contribution from non-analyte proteins to the detection signal.

In practice, the first step of the LC-MS workflow is to find the amino-acid sequence of the protein using internal or external databases and to perform a theoretical, in-silico digestion of the protein by the enzyme of choice, to predict the peptides formed after digestion. Uniqueness is then established by comparison to a database containing all peptides of the proteome of the species of interest. To ensure sufficient sensitivity and stability, there are some further empirical rules for selecting good signature peptides: the ideal size should be 8 to 18 amino acids and it is recommended to avoid peptides with structural elements that may lead to missed cleavages and peptides with glutamate or glutamine at the N-terminus which are prone to form pyroglutamate. In addition, it is best not to select peptides containing methionine which are subject to oxidation, and asparagine and glutamine which may be deamidated and, if possible, favor peptides with proline or histidine near the N-terminus, because these can generate very intense fragment ions.

Generally, for quantification of total protein, peptides containing amino-acid residues with potential post-translational modifications should also be avoided. Proteomic databases can not only help to predict the uniqueness of the peptide, but also its sensitivity and the most abundant fragment ions, provided the peptide was seen in an LC-MS/MS experiment.

After the in-silico analysis, a confirmation must be performed by an experimental digestion of spiked protein in matrix and blank matrix to select the most selective and sensitive peptides which will be used for further method development.

Next to these analytical considerations, it is important that sufficient attention should be paid to the region of interest in the protein structure for selecting the proper signature peptide, and to the objective of any given nonclinical or clinical study. For example, if correlation to the activity of a therapeutic protein is required, it is desirable to select a peptide from the structural area of the protein that is involved in its target binding or activity, such as one of the complementarity-determining regions (CDRs) in a mAb or the active site of an enzyme. If assessing exposure is the main goal, this is less important and a unique peptide from any part of the protein will usually do. For quantification of humanized or fully human mAbs, it may often be possible to select a human-specific signature peptide from the constant part of the protein to allow analysis in a variety of non-clinical matrices. It is also increasingly recognized that therapeutic proteins undergo all sorts of in vivo biotransformation reactions, which can have a dramatic effect on their pharmacological activity, e.g., when an amino acid in the CDR of a mAb is deamidated, oxidized or isomerized. Whenever assessment of an active concentration is desired, the signature peptide should contain such a biotransformation-susceptible amino acid in order to represent the active form of the protein. Conversely, if biotransformation does not impact activity, it is best not to include the biotransformation site in the signature peptide to avoid underestimation of the active concentration.

Also, when it comes to the number of signature peptides to be selected, a good understanding of the structure of the protein, its working mechanism and biotransformation and the objective of the trial are essential. In straightforward situations, such as for single-unit or stable proteins, just one peptide often provides sufficient information. However, the capability of LC-MS/MS for multiplexing allows monitoring multiple peptides relatively easily and including additional peptides can be helpful for building up knowledge of a bioanalytical assay, especially in the early parts of its lifecycle. Consistent concentration results for different peptides adds to the confidence in method performance, and monitoring a single peptide is typically adequate in later phases. In some situations, including multiple signature peptides may be of interest, e.g., when a protein has more than one structurally important region, such as a bi-specific antibody or an antibody-drug conjugate, or when it is important to monitor both active and total concentrations, each by a separate peptide. In addition, LC-MS permits simultaneous quantification of multiple analytes, which can be needed in many different situations. Examples include the measurement of different isoforms of biomarkers and the pharmacokinetic assessment of multiple co-dosed proteins or of a protein and one or more degradation products, such as truncated forms, again each by its own unique signature peptide.

One practical, but quite important final remark is that the options to select proper signature peptides are not endless. Especially for human proteins in human matrix, there may be just a few peptides whose amino acid sequences do not occur in the background proteome. In that respect, it is important that different digestion enzymes be compared, to increase the chance of finding a unique peptide sequence with the right analytical and conceptual properties.

5. Reference materials

Any lot of material may be used as a reference standard if the quality specification is suitable. However, it is preferable that the batch of the reference standard used for preparation of calibration samples and quality control samples is the same as that used for dosing in the non-clinical and clinical studies and where possible the same applies to the choice for method development.

Smaller proteins and peptides are often quantified intact and therefore an intact standard is required. Larger proteins however (>10–20 kDa) are often analyzed using digestion with surrogate peptide monitoring. It is greatly preferable to use the intact protein as an analytical standard for analysis and method development and only if one is not available the surrogate peptide could be used as an alternative with the caveat that steps should be taken to ensure that the analytical method truly reflects the concentration of the desired intact analyte, for example by analysis of characterized control standard samples.

Irrespective of requirements for certificates of analysis for validated assays as per ICH M10, best practice for documentation of identity and quality of reference standards should consider that reference standards should have appropriate levels of detail to assure the bioanalytical scientist is in receipt of appropriate material. A well-characterized reference standard is required that is representative of the material being measured in the biological samples. In addition to the typical certificate of analysis requirements, such as name, source, batch / lot number, storage conditions, expiration or retest date and protein content, it is also important to understand properties such as the protein identity (e.g., amino acid sequence) and the potential for the standard to contain different isoforms. Full scan or product mass spectra to confirm molecular weight and sequence can be helpful and any information concerning, for example propensity to form aggregates and impurity content are useful when embarking on method development.

As well as several commercial suppliers, the European and US Pharmacopeia can be useful suppliers of certain protein and peptide materials and these come provided with certificates of analysis.

Peptides and proteins frequently demonstrate greater adsorption to container materials than small molecules, either through hydrophobic interactions for neutral compounds or electrostatic interactions for charged peptides. Non-linear behavior of analytical standards following dilution is a classic symptom of non-specific binding of the analyte to the container. This should be assessed during method development by testing solutions of the reference standards at concentrations that are relevant to your proposed storage and handling during bioanalysis e.g., primary reference standard and working stock solutions. Stock solutions should be prepared at concentrations above which adsorption is found to be problematic and may be more concentrated than is routine for small molecule solutions. Different containers such as polypropylene versus glass containers, lo-bind tubes and formulation conditions may need to be tested and it is highly recommended to avoid low concentration matrix free aqueous solutions. Adjusting the pH of the storage solvent relevant to the isoelectric point of the protein can change the charge state and affect the electrostatic adsorption. Addition of suitable anti-binding compounds, including surfactants such as BRIJ-35 or TWEEN-20, or a carrier peptide such as leucine enkephalin may also be considered. These compounds, added at a concentration in excess of the analyte, act as displacement agents to compete with the analyte for adsorption to binding sites on the surface of the labware.

Stock solution stability should be assessed during development, usually this would be at refrigerated and/or frozen conditions. As peptides and proteins can precipitate from solution and matrix following freeze-thaw, this should also be assessed.

6. Internal standards

The selection of an appropriate internal standard (IS) is crucial in the development of robust, accurate protein assays with a mass spectrometric end point. The purpose of the internal standard is to compensate for the variability in the extraction, sample processing or acquisition. To achieve that, the ideal internal standard needs to display physical and chemical properties that are almost identical to the target analyte. This is most commonly achieved using stable isotope labelled (SIL) analogues as internal standards. Owing to their near identical properties, these molecules are extracted consistently from assays optimized for the target analyte. They also co-elute chromatographically and are simultaneously measured by mass spectrometry detectors. The most common internal standards employed in mass spectrometric bioanalysis of proteins are SIL intact protein (SIL-protein), SIL peptides (SIL-peptide) and SIL extended peptide (SIL-ex peptide). Each class of internal standard has inherent advantages within the context of proteolytic digestion assays, the most common means of protein quantification by mass spectrometry.

SIL-protein internal standards are typically considered the gold standard approach for proteolytic digestion assays. As an isotopologe of the protein analyte, a SIL-protein IS is added at the beginning of the assay and will compensate for all sample preparation steps undertaken during the assay. This includes immunocapture steps, protein enrichment steps and importantly the proteolytic digestion step. Furthermore, the proteolytic peptide resulting from digestion of the internal standard will be the SIL-peptide version of the digested analyte protein. Thus, variability during downstream sample handling, chromatography and detection steps will also be compensated. While SIL-proteins are the ideal internal standard for protein workflows, the availability of recombinant SIL-proteins is typically very limited owing to the complexity, time and cost involved in their production. For example, for a protein containing cysteine bonds, glycosylations or other post-translational modifications, these need to be represented in the same manner. Otherwise, the extraction or immunocapture can behave differently introducing a bias. Consequently, structural analogue proteins or SIL-peptides are commonly employed as internal standard candidates in protein bioanalysis

Analogue protein internal standards are proteins that share partial homology with the target protein analyte. These proteins have the advantage of being commercially available at low cost. However, a structural protein analogue is unlikely to adequately compensate for proteolysis effects, will yield a structurally different surrogate peptide(s) and will not co-chromatograph with the target analyte. A structural analogue can only compensate effectively for volumetric effects. This approach would typically not be recommended for protein workflows involving enrichment strategies or extensive post-digestion clean up at the surrogate peptide level owing to the divergent physical and chemical properties of the analyte and internal standard. However, an adequate analogue protein IS may be selected for simple protein assays with reference to the physical properties of the analyte protein such as hydrophobicity and protein size. Often the stage your molecule is in will determine which internal standard is available.

In the case where a SIL-protein is unavailable it is common to use a stable isotope-labelled version of the surrogate peptide as an internal standard. SIL-peptides are often added to the assay before or immediately post enzymatic digestion. SIL-peptides have the advantage of being easily synthesized, relatively inexpensive and cover a broad spectrum of analytical steps involved in a quantitative protein assay. SIL-peptide internal standards typically incorporate 13C, 15N or 2H in the same peptide structure as the target peptide to maintain the same physicochemical properties while introducing the mass difference that is required for detection by mass spectrometry. SIL-peptides are good internal standard candidates as they effectively compensate for extract recovery, peptide degradation, matrix effects and mass spectrometry ionization efficiency. However, they cannot compensate for variation in immunocapture or proteolytic digestion. When planning a protein method development, it is recommended that any SIL-peptide internal standards synthesized incorporate a mass differential of at least +6 Da relative to the target surrogate peptide to maintain mass separation at the higher order peptide charge states. In addition to this, it is recommended that immunocapture, enrichment and digestion steps are carefully optimised in method development to limit any introduced variability that cannot be tracked by a SIL-peptide IS.

Stable isotope-labelled extended peptides (SIL-ex peptides) provide a good compromise between SIL-protein and SIL-peptide candidates. SIL-ex peptides, occasionally referred to as winged-, extended-, or cleavable-peptides are SIL-peptides that have typically been extended by 3 to 6 additional amino acid moieties at both the C- and N- terminals. This extended SIL-ex peptide is added to the sample's pre-digestion such that the analyte and IS are digested in parallel. This approach enables the normalization and control of assay variability resulting from the digestion step in the workflow. In this case, the IS compensates for any inter-sample variability in digestion efficiency and peptide degradation in addition to all downstream sample processing and analysis.

SIL-ex peptides offer a good compromise between the practicalities of availability, cost and adequate compensation coverage across the spectrum of steps that may be employed in a protein assay. However, the kinetics associated with digestion of a simple, small SIL-ex peptide are likely to be different that the kinetics associated with complete digestion of the target protein. These phenomena may warrant investigation during method development to ensure an appropriately robust bioanalytical assay is provided.

7. Conclusion

In conclusion, accurate protein measurement is essential to gain valuable insights into biological functions and therapeutic efficacy. The use of LC-MS(/MS) is an important part of the bioanalysis toolkit and understanding the “what” before embarking on an assay is critical. This paper has laid the groundwork by addressing the fundamental principles and theoretical underpinnings essential for understanding quantitative protein bioanalysis. As we transition from the conceptual to the practical, paper 2 takes you through the specifics of the “how” and covers sample treatment, automation, instrumental analysis, data processing and analysis, method development and validation. Drawing from the extensive expertise of the European Bioanalysis Forum, it offers a practical blueprint for translating theoretical foundations into effective methodologies. We hope that both papers, together with earlier recommendations from the EBF can help for the bioanalytical community and the regulatory authorities to better appreciate the challenges and provide an insight and food for discussion on why bioanalysis of biotherapeutic and biomarker proteins with LC-MS/MS should not be compared with the more traditional LC-MS/MS assay for small molecules or LBA for biotherapeutics. As ever, the EBF is ready to be a partner in those discussion.

Disclaimer

The views and conclusion presented in this paper are those of the European Bioanalysis Forum and do not necessarily reflect the representative affiliation or company's position on the subject.

Financial disclosure

The authors have no 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.

Competing interests disclosure

The 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.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.