Objective Ultrafiltration and diafiltration (UF/DF) operations have been demonstrated to clear leachables from drug substance, however there is limited data available. Consequently, comprehensive and systematic characterization of leachables clearance during UF/DF is required and essential. Methods To achieve this, the reduction capacity for 28 selected organic compounds spiked into 3 different proteins during UF/DF processes was investigated using liquid chromatography high-resolution mass spectrometry. Selection of compounds was based on their presence in representative biomanufacturing processes. Results Most compounds (24) showed clearance over 98% across the process for the 3 protein materials. The specific protein characteristics and process parameters for each protein had a minimal impact on clearance, with sieving coefficients essentially the same for each one of the 3 protein processes. The sieving coefficient is a parameter that characterizes clearance of compounds during UF/DF. Physicochemical properties of the compounds under study significantly influenced their clearance, with the octanol-water coefficient (Log P) being the most crucial factor. Compounds with Log P < 4 had sieving coefficients close to ideal clearance, and compounds with Log P > 7 showed lower but still significant clearance (> 93%). Other important parameters were established to be molecular weight, polarizability and solvent accessible surface area. Modelling tools based on Orthogonal Partial Least Squares (OPLS) regression were created to predict sieving coefficients. Conclusions The present work has created a strong background to describe the ability of UF/DF to remove potential organic leachables. Application of these modelling approaches becomes critical to support product safety assessments. Demonstration of significant removal along UF/DF operations confirms risk reduction of leachables coming mostly from upstream stages.

Comprehensive characterization of monoclonal antibody (mAb) charge heterogeneity is essential to ensure product quality, maintain batch consistency and support biosimilar development. Charge variant analysis (CVA) is widely used to separate acidic and basic proteoforms from the main species. However, cation-exchange chromatography coupled to mass spectrometry provides limited information and cannot localize the post-translational modifications (PTMs) responsible for mAb heterogeneity. Here, the coupling of pH-gradient CVA with native top-down mass spectrometry (TD-MS) for proteoform-specific analysis of trastuzumab is presented. Individual charge variants were chromatographically separated under native conditions and directly fragmented on the chromatographic time scale using higher-energy collision dissociation (HCD), electron-transfer dissociation (ETD) and ultraviolet photodissociation (UVPD). The addition of proton-transfer charge reduction (PTCR) helped reduce spectral congestion and enhanced the detection of high-mass fragment ions, resulting in improved sequence coverage. This workflow enabled the complete sequencing of the complementarity-determining region (CDR) 3 and the direct identification and insights into the location of key PTMs at the intact-protein level, including deamidation, succinimide and N-terminal pyroGlu for individual proteoforms. Comparison of five trastuzumab samples (originator and biosimilars) demonstrated high reproducibility in fragmentation patterns, sequence coverage and variant assignment, highlighting the robustness of the method. Although limitations remain due to the challenges of fragmenting intact mAbs under native conditions, this work establishes a proof of concept for CVA native TD-MS characterization of mAbs to complement bottom-up and middle-down analyses, and has potential for broad applicability for antibody-based biopharmaceuticals.

During early-stage biotherapeutic development, analytical methods play an important role in candidate screening, process development, formulation screening and stability determination. However, developing sensitive and robust analytical methods is challenging in the early stages as there is often insufficient product knowledge and limited sample amount. Here we present a high-sensitivity multi-attribute method (MAM) centred on nano-flow LC-MS/MS for the characterisation of mAb-based molecules. Using NISTmAb as a reference, run-to-run variation was examined, in terms of peak area, peak width, peak asymmetry, retention time precision and mass accuracy. The ability to detect and quantify commonly observed product quality attributes (PQAs) was also evaluated for a wide range of injected digest amounts from 1 ng to 100 ng. LOD and LOQ were between 0.6 to 7.0 ng of injected protein amount (R² > 0.99), for a selection of low abundant PQAs including deamidation, oxidation, succinimide and lysine content, following regulatory considerations on the implementation of MAM in QC. The nano-flow MAM approach was then applied as an in-process test to study PQAs and impurities of a chimeric IgG1 mAb. Low amounts of samples were digested followed by high resolution nano-LC-MS/MS, Orbitrap-based mass detection, operated within a Code of Federal Regulations (CFR) Part 11 compliant data system for data acquisition and data analysis. Finally, the new peak detection (NPD) aspect of the method was also evaluated, as an in-process test for detection of low-level impurities. The results highlight the potential of nano-flow MAM for early-stage process development and its suitability for QC applications.

Abstract Monoclonal antibody (mAb) glycosylation is a critical quality attribute that is difficult to rationally engineer and rapidly assess during cell line development. Here, we investigate whether cell-surface glycosylation can serve as a predictive indicator of mAb product glycosylation following targeted glycogene engineering in CHO cells. Five key glycogenes (COSMC, FUT8, B4GALT1, ST3GAL4, ST6GAL1) were investigated in two mAb-producing CHO cell lines. Product glycan analysis revealed consistent, gene-specific effects across hosts, including loss of core fucosylation, and tuneable galactosylation and sialylation. Lectin-based surface profiling reliably reflected product outcomes for COSMC and FUT8 modifications but showed limited predictive power for galactosylation and α2,3-sialylation, highlighting glycosylation pathway redundancy and context dependence. This study provides the first systematic, cross-cell line evaluation of lectin-based cell-surface glycan profiling as a predictor of mAb product glycosylation, establishing its practical utility and inherent limitations for CHO glycoengineering workflows. Graphical abstract

β4-galactosylation is a critical quality attribute of therapeutic monoclonal antibodies (mAbs), enhancing complement-dependent cytotoxicity, antibody-dependent cytotoxicity, and antibody-dependent cellular phagocytosis. Despite its therapeutic importance, galactosylation remains the most variable glycosylation motif due to its sensitivity to cell culture conditions. Here, we describe a dual genetic engineering strategy applied to two mAb-producing CHO cell lines, DP12 and VRC01, to simultaneously overcome the cellular machinery and metabolic bottlenecks that limit β4-galactosylation. The first engineering event knocks out COSMC, the chaperone required for core 1 β-1,3-galactosyltransferase 1 activity, to redirect UDP-Gal consumption from O-linked β3-galactosylation towards mAb Fc N-linked β4-galactosylation. The second event overexpresses β-1,4-galactosyltransferase 1 (β4GalT1) to augment cellular galactosylation machinery. Each modification was characterised individually (COSMC- and GalT+) and in combination (C-/GT+) across both cell lines in batch and fed-batch cultures. The combined C-/GT+ strategy consistently achieved greater than 90% mAb Fc β4-galactosylation, irrespective of host cell line or culture mode. Metabolic characterisation confirmed that both engineering events alleviate their respective bottlenecks: COSMC knockout redirects UDP-Gal flux and β4GalT1 overexpression increases N-galactosylation capacity. The C-/GT+ strategy also reduced production of Man5 glycans, which accelerate serum clearance and pose immunogenicity risks. Metabolic profiling suggests that the COSMC knockout may attenuate UTP consumption and contributes to reduced Man5 production. C-/GT+ glycoengineering had no negative impact on mAb titre. Our results establish the C-/GT+ dual glycoengineering strategy as a robust approach for consistently achieving high mAb galactosylation across diverse cell culture conditions, with the additional benefit of reduced Man5 glycans.

Untargeted analysis using liquid chromatography high-resolution mass spectrometry (LC-HRMS) provides a comprehensive approach to determine process equipment-related leachables (PERLs) in biomanufacturing processes. A shortcoming of current analytical methods is their inability to provide quantitative information, due to limited availability of reference standards, constituting an important obstacle in understanding the fate of PERLs and their human exposure levels if present in the final drug product. This study presents a semiquantitative workflow based on ionization efficiency (LogIE) measurement of PERLs analyzed by LC-HRMS. A partial least squares (PLS) model based on physicochemical parameters of 31 compounds was built and validated to predict LogIE of unknown compounds and to facilitate their quantitation. The semi-quantitative analysis was internally validated by calculation and comparison of residuals at 5 concentration levels within the linear dynamic range. The workflow was applied and tested to investigate leachable clearance during ultrafiltration/diafiltration (UF/DF) processes. Three monoclonal antibodies were spiked with leachables generated from polymeric materials under selected conditions to simulate realistic leachables concentrations. Determined leachable concentrations across UF/DF allowed calculation of clearance percentages (> 90%) and sieving coefficients (S). S values were compared with experimental values from previous studies, determining an average error of 2.7%, indicating acceptable performance of the semiquantitative approach. Results from this wider study under realistic leachable concentrations confirmed Log P as the dominant property in explaining clearance. Lowest clearance is anticipated for compounds with Log P > 7 (highly hydrophobic) while the highest clearance is expected for compounds with Log P < 5 (highly and moderately polar).

Monoclonal antibody (mAb) glycosylation is a critical quality attribute that is difficult to rationally engineer and rapidly assess during cell line development. Here, we investigate whether cell-surface glycosylation can serve as a surrogate readout of mAb product glycosylation following targeted glycogene engineering in CHO cells. Five key glycogenes (COSMC, FUT8, β4GALT1, ST3GAL4, and ST6GAL1) were investigated in two mAb-producing CHO cell lines. Product glycan analysis revealed consistent, gene-specific effects across hosts, including loss of core fucosylation, and tunable galactosylation and sialylation. Lectin-based surface profiling reliably reflected product outcomes for COSMC and FUT8 modifications but showed limited correspondence with product galactosylation and α2,3-sialylation phenotypes, highlighting glycosylation pathway redundancy and context dependence. These findings demonstrate that lectin-based profiling is most effective for identifying glycoengineering outcomes associated with nonredundant pathways rather than as a universal predictor of product glycosylation.

Host cell proteins (HCPs) are endogenous proteins generated in cellular production systems alongside the biotherapeutic of interest. Removal of HCPs is crucial as they can be detrimental to product efficacy and patient safety. Due to its ability to determine individual HCP concentrations, liquid chromatography tandem mass spectrometry is increasingly utilized as an orthogonal method to ELISA for HCP monitoring. For protein biotherapeutics like monoclonal antibodies, their dynamic range makes detection of low-level HCPs difficult. The Orbitrap Astral MS has the potential to overcome such challenges, offering improvements in protein identifications in complex sample matrices while simultaneously reducing analysis times. Here, we utilize the Orbitrap Astral MS to perform HCP analysis on 36 protein biotherapeutics. Our workflow used a short 60 samples-per-day separation method and was initially benchmarked against four previously published studies, demonstrating comparable levels of HCP identifications. 236 HCPs were detected across the cohort and 55% of those found to be quantifiable in at least one product using label free quantitation. Functional analysis revealed that most detected HCPs had functions related to catalysis or binding, predominately catalytic activity (46%, 97 gene IDs) or protein binding (44%, 91 gene IDs). Nearly 80% of quantifiable HCPs were detected at concentrations below 10 ng/mg, with 8% detected below concentrations of 1 ng/mg. These included HCPs considered as "high-risk" by the Biophorum Development Group. This study shows how new generation mass spectrometry instruments can enable detection of low-level HCPs while allowing for a rapid and more informed understanding of a product's HCP content.

Protein biotherapeutics such as monoclonal antibodies (mAbs) are generally produced using cell-based production systems that generate endogenous proteins, commonly referred to as host cell proteins (HCPs), along with the desired drug products. A critical challenge is the removal of HCPs from the final drug product as they can be detrimental to product efficacy and patient safety, even at low levels. HCP clearance is monitored during process optimisation and assessed before product release. Tandem liquid chromatography-mass spectrometry (LC-MS/MS) is increasingly utilized as an orthogonal method to enzyme-linked immunosorbent assays (ELISA) for HCP monitoring during downstream processing due to its untargeted nature and ability to determine total HCP concentrations. The dynamic range of the recombinant proteins makes detection of low-level HCPs difficult. However, the recently developed Orbitrap Astral MS has the potential to overcome such challenges, having previously demonstrated significant improvements in protein identifications in complex sample matrices while simultaneously reducing analysis times. Here we utilise the Orbitrap Astral MS to perform HCP analysis on a cohort of 37 protein biotherapeutics. Its speed and sensitivity enable the use of a short 60 samples-per-day (SPD; 24-minute injection to injection) separation method, dramatically reducing analysis time compared to standard LC-MS methods without sacrificing HCP identifications. 544 HCPs were detected across the cohort and 63.60% of those found to be quantifiable in at least one product using Hi3 quantitation. Over 80% of quantifiable HCPs were detected at concentrations below 10 ppm, including approximately 9% below concentrations of 1 ppm. These included HCPs considered as “high-risk” by the Biophorum Development Group. Excluding an outlier, on average 20 HCPs were identified per product. Overall, this study shows how the Orbitrap Astral MS can improve detection of low-level HCPs while significantly reducing analysis times, allowing for a rapid and more informed understanding of a products HCP content.

Cell therapies (CT) have demonstrated life-changing benefits and curative options to patients with unmet medical needs. Recent commercial successes have strengthened support for the industry; strong clinical responses are propelling additional CT products toward commercialisation. However, manufacturing of CT products continues to create challenges. Extractables and leachables (E&Ls) are a significant concern for the CT industry, which relies exclusively on single-use systems (SUSs). Investigation of the impact of SUS materials that encounter the cell-based product is a new field and the generation of more information is critical. Here, a proof-of-principle study is presented, demonstrating evidence of effects of leachates on T cells. Jurkat cells, a prototypical T cell line, were cultivated in media previously incubated in single-used bags (SUBs) utilised during incubation/expansion stages. Leachables present in the media were identified by high resolution mass spectrometry (HRAM). The physiological condition of T-cells was assessed using biological assays. Media components and metabolites were analysed over time using a direct infusion-mass spectrometry (DI-MS) method. Media containing leachables resulted in cell growth inhibition and early onset of the apoptosis/necrosis pathways. Changes in mitochondrial membrane potential suggested that leachables are cytotoxic via ΔΨm depolarisation, involving the intrinsic apoptotic pathway in the initiation of cell death. Key metabolic pathways were also significantly affected, producing accumulation of toxic metabolites and degradation of nucleic acids and lipids.

Oligonucleotides offer a powerful class of new therapeutic modalities, which requires the support of robust and sensitive analytical methods. The primary structure of RNA-based therapeutic drugs is considered a critical quality attribute by regulatory agencies, and must be empirically confirmed to ensure quality, safety and efficacy, together with the analysis of the 5' and 3' termini, and any site-specific modifications. This study highlights the use of amine-based ion pair reversed-phase LC coupled to a high-resolution MS and MS/MS method (HRMS) for the characterisation of small RNA-based molecules, single stranded antisense oligonucleotides (ss-ASOs), designed with different chemical modifications from second and third generation categories, including backbone modifications, sugar modifications at the 2' position and base modifications. The study evaluates the applicability of an alternative ion pairing reagent, with moderate hydrophobicity, and the use of a mild ESI source, with the aim to reduce the formation of ion pair-adducts and in-source induced impurities. The developed method allows for sequence verification and confident identification of low abundant impurities with high sensitivity and high mass accuracy at the intact and sequencing level. Intact analysis allowed for the detection and quantification of full-length products with different sizes and purity values, and the detection of multiple impurities and degradants, even without being fully chromatographically resolved, with high sensitivity. Most common impurity found in analysed RNA-based ss-ASOs was the presence of the phosphodiester (PO) conversion, with fractional abundances from 9.2 % to 1.2 %, followed by failure sequence shortmers.

The introduction of trace elemental impurities through the use of single use technologies (SUTs) during biopharmaceutical manufacturing is a key concern as the presence of process equipment related leachables (PERLs) such as process related elemental impurities has potential harmful implications on product quality and patient safety. ICH Q3A provides a regulatory road map for PERLs but excludes biologic drugs like monoclonal antibody (mAb) therapies. Consequently, the industry lacks a synchronized, risk-based testing strategy for PERLs based on the likelihood of their presence in the final drug product. Ultrafiltration and diafiltration (UF/DF) operations have been demonstrated to clear leachables from the drug product during downstream purification. Hence, it is attractive to characterize PERL behavior during UF/DF to inform subsequent extractable and leachable (E&L) evaluation of the formulated biologic. The reduction capacity of twenty-two elements spiked into concentrated protein samples during UF/DF processes was investigated, using an inductively coupled plasma-mass spectrometry (ICP-MS) method, which was developed and validated according to ICH Q2 (R1) guidelines. Most elements (18) were efficiently cleared (>97 %), representing a 100-fold (2-log) reduction or higher after 10 diavolumes. Clearance was linked to UF/DF process parameters, most notably pH, as well as to the physicochemical properties of the studied elements. Mathematical models based on Orthogonal Partial Least Squares (OPLS) regression were developed and validated using the sieving coefficient to characterize and predict the clearance behavior of elemental impurities during UF/DF. Results from this study lay a solid foundation for the understanding and prediction of UF/DF capacity to remove elemental leachables. PERL clearance modelling emerges as a valuable platform to support industry and regulatory bodies in developing sophisticated risk-based PERL testing strategies, ultimately ensuring patient safety. The data presented herein demonstrate the significant risk reduction that the UF/DF process provides for processing steps upstream in mAbs and other biologics processes.

Fc-fusion proteins are medicines developed for the treatment of complex diseases which feature enhanced pharmacokinetic properties compared to other therapeutic protein formats. A commonly used strategy for the extension of protein half-life in circulation is the introduction of negative charges on the protein, preferably through N- and O-glycans equipped with negatively charged sialic acid moieties. While enhancing pharmacokinetics, the presence of many glycosylation sites can coincide with a considerable protein heterogeneity, which renders analytical characterisation increasingly difficult. In this work, we demonstrate that the complexity of a highly glycosylated intact Fc-fusion protein can be well resolved using mass spectrometry (MS)-friendly anion exchange chromatography (AEX) with pH gradient elution. The application of the developed method allowed for the separation of 9 clear chromatographic peaks and the acquisition of high-quality protein spectra and thus, MS detection of intact protein isoforms. The resulting data enabled the identification of 268 protein features which represents a ∼ 25-fold increase in detected forms compared to output that could be obtained by simple size exclusion chromatography-mass spectrometry. The underlying cause for the separation selectivity observed in AEX was found to be differential protein sialylation while varying numbers of hexose and N-acetylglucosamine units on glycans were identified as other major contributors to the high heterogeneity observed.

Adeno-associated virus (AAV) viral vector-based gene therapy is advancing rapidly, offering potential treatments for rare and severe diseases. The AAV capsid consists of a combination of three viral proteins (VPs), VP1, VP2, and VP3, ranging from 59 to 81 kDa and present at a theoretical bulk ratio of 1:1:10. This study employed hydrophilic interaction liquid chromatography (HILIC) and mass spectrometry (MS) to achieve robust separation and detailed characterisation of AAV9 capsid proteins. Advanced top-down MS approaches combining multiple fragmentation techniques (HCD, ETD, EThcD, and UVPD) were successfully applied, increasing the sequence coverage up to 40% for VP3 and confirming N-terminal acetylation on VP1 and VP3. The workflow demonstrated high reproducibility between injection duplicates and was subsequently applied to the characterisation of in-house produced biological replicates of AAV9 samples from HEK293 cells, showing consistent results across them. Analysis of AAV9 derived from Sf9 insect cells, a more complex sample due to higher levels of modification of the capsid VPs, further evidenced method versatility. Overall, this study highlights the potential of HILIC-MS and advanced top-down MS approaches for detailed characterisation of AAV capsid proteins.

Over the past few years, the implementation of mass spectrometry (MS) in QC laboratories has become a more common occurrence. The multi-attribute method (MAM), and emerging intact multi-attribute method (iMAM), are powerful analytical tools utilising liquid chromatography−mass spectrometry (LC-MS) methods that enable the monitoring of critical quality attributes (CQAs) in biotherapeutic proteins in compliant settings. Both MAM and iMAM are intended to replace or supplement several conventional assays with a single LC-MS method utilising MS data in combination with robust, semi-automated data processing workflows. MAM and iMAM workflows can also be implemented into current Good Manufacturing Practices environments due to the availability of CFR 11 compliant chromatography data system software. In this study, MAM and iMAM are employed for the analysis of 4 batches of a glucagon-like peptide-Fc fusion protein. MAM approach involved a first the discovery phase for the identification of CQAs and second, the target monitoring phase of the selected CQAs in other samples. New peak detection was performed on the data set to determine the appearance, absence or change of any peak. For native iMAM workflow both size exclusion and strong cation exchange chromatography were optimized for the identification and monitoring of CQAs at the intact level.

Os autores externos submeteram sua publicação para apresentação de trabalho no evento "International Symposium on Immunobiologicals", que foi coordenado e organizado pelo Instituto de Tecnologia em Imunobiológicos (Bio-Manguinhos), da Fundação Oswaldo Cruz.

Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However, challenges such as immunogenicity and high production costs complicate the commercial viability of AAV-based therapies. To overcome these barriers, improvements in production yield, driven through the availability of robust and sensitive characterization techniques that allow for the monitoring of critical quality attributes to deepen product and process understanding are crucial. Among the main attributes affecting viral production and performance, the ratio between empty and full capsids along with capsid protein stoichiometry are emerging as potential parameters affecting product quality and safety. This study focused on the production of AAV vectors using the baculovirus expression vector system (BEVS) in Sf9 cells and the complete characterization of AAV5 variants using novel liquid chromatography and mass spectrometry techniques (LC-MS) that, up to this point, had only been applied to reference commercially produced virions. When comparing virions produced using ATG, CTG or ACG start codons of the cap gene, we determined that although ACG was the most productive in terms of virus yield, it was also the least effective in transducing mammalian cells. This correlated with a low VP1/VP2 ratio and a higher percentage of empty capsids. Overall, this study provides insights into the impact of translational start codon modifications during rAAV5 production using the BEVS, the associated relationship with capsid packaging, capsid protein stoichiometry and potency. The developed characterization workflow using LC-MS offers a comprehensive and transferable analysis of AAV-based gene therapies, with the potential to aid in process optimization and facilitate the large-scale commercial manufacturing of these promising treatments.

The spread of multidrug-resistant strains of Neisseria gonorrhoeae, the etiologic agent of gonorrhea, represents a global health emergency. Therefore, the development of a safe and effective vaccine against gonorrhea is urgently needed. In previous studies, murine monoclonal antibody (mAb) 2C7 was raised against gonococcal lipooligosaccharide (LOS). mAb 2C7 elicits complement-dependent bactericidal activity against gonococci, and its glycan epitope is expressed by almost every clinical isolate. Furthermore, we identified a peptide, cyclic peptide 2 (CP2) that mimicked the 2C7 LOS epitope, elicited bactericidal antibodies in mice, and actively protected in a mouse vaginal colonization model. In this study, we performed structural analyses of mAb 2C7 and its complex with the CP2 peptide by X-ray crystallography, NMR spectroscopy, and molecular dynamics (MD) simulations. The crystal structure of Fab 2C7 bound to CP2 showed that the peptide adopted a beta-hairpin conformation and bound the Fab primarily through hydrophobic interactions. We employed NMR spectroscopy and MD simulations to map the 2C7 epitope and identify the bioactive conformation of CP2. We also used small-angle X-ray scattering (SAXS) and native mass spectrometry to obtain further information about the shape and assembly state of the complex. Collectively, our new structural information suggests strategies for humanizing mAb 2C7 as a therapeutic against gonococcal infection and for optimizing peptide CP2 as a vaccine antigen.

Chinese hamster ovary (CHO) cells are used to produce almost 90% of therapeutic monoclonal antibodies (mAbs) and antibody fusion proteins (Fc-fusion). The annotation of non-canonical translation events in these cellular factories remains incomplete, limiting our ability to study CHO cell biology and detect host cell protein (HCP) impurities in the final antibody drug product. We utilised ribosome footprint profiling (Ribo-seq) to identify novel open reading frames (ORFs) including N-terminal extensions and thousands of short ORFs (sORFs) predicted to encode microproteins. Mass spectrometry-based HCP analysis of eight commercial antibody drug products (7 mAbs and 1 Fc-fusion protein) using the extended protein sequence database revealed the presence of microprotein impurities. We present evidence that microprotein abundance varies with growth phase and can be affected by the cell culture environment. In addition, our work provides a vital resource to facilitate future studies of non-canonical translation and the regulation of protein synthesis in CHO cell lines.

Poloxamer 188, also known as Pluronic F-68, is an excipient added to the biotherapeutic protein-manufacturing process. Poloxamer 188 (P188) is a nonionic triblock copolymer surfactant that can be used as a shear protective excipient in bioreactors. In the current study, a method for the process clearance monitoring of poloxamer 188 during downstream processing of biotherapeutics using liquid chromatography-triple-quadrupole mass spectrometry was developed and validated. Chromatographic separation of P188 was achieved using a Phenomenex, Luna 3 μm phenyl-hexyl, 150 × 2 mm column, and quantitation was achieved using a triple-quadrupole mass spectrometer operated in selected reaction monitoring mode. Linearity was assessed over a working range of 250-10,000 ng/mL. Precision and accuracy were within 15% of the theoretical spike levels assessed across the three different concentration levels. For this study, two different IgG1 antibodies were used for the method validation activities. Analyte specificity and selectivity were deemed acceptable based on no extraneous peaks present. System suitability was evaluated throughout this study in anticipation of the introduction of this method into the quality control environment. This method was successfully validated and used to monitor the clearance of poloxamer 188 in a tangential flow filtration purification step during biotherapeutic downstream processing. In addition, the capability of the method to successfully support poloxamer 188 mixing studies is presented in this work.