Expediting online liquid chromatography for real-time monitoring of product attributes to advance process analytical technology in downstream processing of biopharmaceuticals.

The application of Process Analytical Technology (PAT) principles for manufacturing of biotherapeutics proffers the prospect of ensuring consistent product quality along with increased productivity as well as substantial cost and time savings. Although this paradigm shift from a traditional, rather rigid manufacturing model to a more scientific, risk-based approach has been advocated by health authorities for almost two decades, the practical implementation of PAT in the biopharmaceutical industry is still limited by the lack of fit-for-purpose analytical methods. In this regard, most of the proposed spectroscopic techniques are sufficiently fast but exhibit deficiencies in terms of selectivity and sensitivity, while well-established offline methods, such as (ultra-)high-performance liquid chromatography, are generally considered as too slow for this task. To address these reservations, we introduce here a novel online Liquid Chromatography (LC) setup that was specifically designed to enable real-time monitoring of critical product quality attributes during time-sensitive purification operations in downstream processing. Using this online LC solution in combination with fast, purpose-built analytical methods, sampling cycle times between 1.30 and 2.35 min were achieved, without compromising on the ability to resolve and quantify the product variants of interest. The capabilities of our approach are ultimately assessed in three case studies, involving various biotherapeutic modalities, downstream processes and analytical chromatographic separation modes. Altogether, our results highlight the expansive opportunities of online LC based applications to serve as a PAT tool for biopharmaceutical manufacturing.

Fast HPLC-based affinity method to determine capsid titer and full/empty ratio of adeno-associated viral vectors.

Recombinant adeno-associated viruses (rAAVs) are promising gene delivery vectors in the emerging field of in vivo gene therapies. To ensure their consistent quality during manufacturing and process development, multiple analytical techniques have been proposed for the characterization and quantification of rAAV capsids. Despite their indisputable capabilities for performing this task, current analytical methods are rather time-consuming, material intensive, complicated, and costly, restricting their suitability for process development in which time and sample throughput are severe constraints. To eliminate this bottleneck, we introduce here an affinity-based high-performance liquid chromatography method that allows the determination of the capsid titer and the full/empty ratio of rAAVs within less than 5 min. By packing the commercially available AAVX affinity resin into small analytical columns, the rAAV fraction of diverse serotypes can be isolated from process-related impurities and analyzed by UV and fluorescence detection. As demonstrated by both method qualification data and side-by-side comparison with AAV enzyme-linked immunosorbent assay results for rAAV8 samples as well as by experiments using additional rAAV2, rAAV8, and rAAV9 constructs, our approach showed good performance, indicating its potential as a fast, simple and efficient tool for supporting the development of rAAV gene therapies.

Effects of Different Container Types on (1→3)-ß-D-glucan Recovery.

It has long been known that containers for sample analysis or storage can play a role in endotoxin recovery and have to be taken into account when determining endotoxin concentrations. However, there is little data on the effects of containers regarding (1→3)-ß-D-glucan, which plays a role as a contaminant in endotoxin measurements. To determine the effect of the container on (1→3)-ß-D-glucan measurements, four different types of containers were investigated at different temperatures and stored for up to 28 days. For short-term storage for 3 h at room temperature, no effect of the container on the (1→3)-ß-D-glucan recovery could be observed, but for storage at -20 °C, the results indicate that the storage time and temperature influences (1→3)-ß-D-glucan detection. All containers showed a trend of lower recoveries over time, but the polyethylene container showed a significantly lower recovery compared to the other containers. We also showed that freeze/thaw cycles had a strong influence on the recovery of (1→3)-ß-D-glucan in polyethylene containers. Our study showed that the container can affect not only the detection of endotoxins but also the detection of (1→3)-ß-D-glucans.

A fast and sensitive high-throughput assay to assess polysorbate-degrading hydrolytic activity in biopharmaceuticals.

Hydrolysis of polysorbate in biopharmaceutical products has been ascribed to the enzymatic activity from trace levels of residual host cell proteins. In recent years, significant efforts to identify the causative enzymes typically used elaborate, material-intensive and time-consuming approaches. Therefore, the lack of fast and sensitive assays to monitor their activity remains a major bottleneck for supporting process optimization and troubleshooting activities where time and sample throughput are crucial constraints. To address this bottleneck, we developed a novel Electrochemiluminescence-based Polysorbase Activity (EPA) assay to measure hydrolytic activities in biotherapeutics throughout the drug substance manufacturing process. By combining the favorable features of an in-house designed surrogate substrate with a well-established detection platform, the method yields fast (∼36 h turnaround time) and highly sensitive readouts compatible with high-throughput testing. The assay capability for detecting substrate conversion in a precise and reliable manner was demonstrated by extensive qualification studies and by employing a number of recombinant hydrolases associated with polysorbate hydrolysis. In addition, high assay sensitivity and wide applicability were confirmed for in-process pool samples of three different antibody products by performing a head-to-head comparison between this method and an established liquid chromatography - mass spectrometry based assay for the quantification of free fatty acids. Overall, our results suggest that this new approach is well-suited to resolve differences in hydrolytic activity through all stages of purification.

Industry Perspective on the Use and Characterization of Polysorbates for Biopharmaceutical Products Part 2: Survey Report on Control Strategy Preparing for the Future.

Polysorbate (PS) 20 and 80 are the main surfactants used to stabilize biopharmaceutical products. Industry practices on various aspects of PS based on a confidential survey and following discussions by 16 globally acting major biotechnology companies is presented in two publications. Part 1 summarizes the current practice and use of PS during manufacture in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS.1 The current part 2 of the survey focusses on understanding, monitoring, prediction, and mitigation of PS degradation pathways in order to propose an effective control strategy. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature.

Detailed Analytical Characterization of a Bispecific IgG1 CrossMab Antibody of the Knob-into-Hole Format Applying Various Stress Conditions Revealed Pronounced Stability.

In recent years, a variety of new antibody formats have been developed. One of these formats allows the binding of one type of antibody to two different epitopes. This can for example be achieved by introduction of the "knob-into-hole" format and a combined CrossMab approach. Due to their complexity, these bispecific antibodies are expected to result in an enhanced variety of different degradation products. Reports on the stability of these molecules are still largely lacking. To address this, a panel of stress conditions, including elevated temperature, pH, oxidizing agents, and forced glycation via glucose incubation, to identify and functionally evaluate critical quality attributes in the complementary-determining and conserved regions of a bispecific antibody was applied in this study. The exertion of various stress conditions combined with an assessment by size exclusion chromatography, ion exchange chromatography, LC-MS/MS peptide mapping, and functional evaluation by cell-based assays was adequate to identify chemical modification sites and assess the stability and integrity, as well as the functionality of a bispecific antibody. Stress conditions induced size variants and post-translational modifications, such as isomerization, deamidation, and oxidation, albeit to a modest extent. Of note, all the observed stress conditions largely maintained functionality. In summary, this study revealed the pronounced stability of IgG1 "knob-into-hole" bispecific CrossMab antibodies compared to already marketed antibody products.

Industry Perspective on the use and Characterization of Polysorbates for Biopharmaceutical Products Part 1: Survey Report on Current State and Common Practices for Handling and Control of Polysorbates.

Polysorbates (PS) are widely used as a stabilizer in biopharmaceutical products. Industry practices on various aspects of PS are presented in this part 1 survey report based on a confidential survey and following discussions by 16 globally acting major biotechnology companies. The current practice and use of PS during manufacture across their global manufacturing sites are covered in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature. Part 2 of the survey report (upcoming) will focus on understanding, monitoring, prediction, and mitigation of PS degradation pathways to develop an effective control strategy.

Fast analysis of antibody-derived therapeutics by automated multidimensional liquid chromatography - Mass spectrometry.

Characterization of post-translational modifications (PTMs) of therapeutic antibodies is commonly performed by bottom-up approaches, involving sample preparation and peptide analysis by liquid chromatography-mass spectrometry (LC-MS). Conventional sample preparation requires extensive hands-on time and can increase the risk of inducing artificial modifications as many off-line steps - denaturation, disulfide-reduction, alkylation and tryptic digestion - are performed. In this study, we developed an on-line multidimensional (mD)-LC-MS bottom-up approach for fast sample preparation and analysis of (formulated) monoclonal antibodies and antibody-derived therapeutics. This approach allows on-column reduction, tryptic digestion and subsequent peptide analysis by RP-MS. Optimization of the 1D -and 2D flow and temperature improved the trapping of small polar peptides during on-line peptide mapping analysis. These adaptations increased the sequence coverage (95-98% versus 86-94% for off-line approaches) and allowed identification of various PTMs (i.e. deamidation of asparagine, methionine oxidation and lysine glycation) within a single analysis. This workflow enables a fast (<2 h) characterization of antibody heterogeneities within a single run and a low amount of protein (10 µg). Importantly, the new mD-LC-MS bottom-up method was able to detect the polar, fast-eluting peptides: Fc oxidation at Hc-Met-252 and the Fc N-glycosylation at Hc-Asn-297, which can be challenging using mD-LC-MS. Moreover, the method showed good comparability across the different measurements (RSD of retention time in the range of 0.2-1.8% for polar peptides). The LC system was controlled by only a standard commercial software package which makes implementation for fast characterization of quality attributes relatively easy.

Inter-laboratory study to evaluate the performance of automated online characterization of antibody charge variants by multi-dimensional LC-MS/MS.

An international study was conducted to evaluate the performance and reliability of an online multi-dimensional (mD)-LC-MS/MS approach for the characterization of antibody charge variants. The characterization of antibody charge variants is traditionally performed by time-consuming, offline isolation of charge variant fractions by ion exchange chromatography (IEC) that are subsequently subjected individually to LC-MS/MS peptide mapping. This newly developed mD-LC-MS/MS approach enables automated and rapid characterization of charge variants using much lower sample requirements. This online workflow includes sample reduction, digestion, peptide mapping, and subsequent mass spectrometric analysis within a single, fully-automated procedure. The benefits of using online mD-LC-MS/MS for variant characterization include fewer handling steps, a more than 10-fold reduction in required sample amount, reduced sample hold time as well as a shortening of the overall turnaround time from weeks to few days compared to standard offline procedures. In this site-to-site comparison study, we evaluated the online peptide mapping data collected from charge variants of trastuzumab (Herceptin®) across three international laboratories. The purpose of this study was to compare the overall performance of the online mD-LC-MS/MS approach for antibody charge variant characterization, with all participating sites employing different mD-LC-MS/MS setups (e.g., instrument vendors, modules, columns, CDS software). The high sequence coverage (95%-97%) obtained in each laboratory, enabled a reproducible generation of tryptic peptides and the comparison of values of the charge variants. Results obtained at all three participating sites were in good agreement, highlighting the reliability and performance of this approach, and correspond with data gained by the standard offline procedure. Overall, our results underscore of the benefit mD-LC-MS/MS technology for therapeutic antibody characterization, confirming its potential to become an important tool in the toolbox of protein characterization scientists.

Identification and Characterization of Polysorbate-Degrading Enzymes in a Monoclonal Antibody Formulation.

Degradation of polysorbate (PS) by hydrolytically active host cell proteins (HCPs) in drug products may impair the protein-stabilizing properties of PS and lead to the formation of particles due to the accumulation of poorly soluble free fatty acids upon long-term storage. The identification of the causative enzymes is challenging due to their low-abundance even when using state-of-the-art instrumentation and workflows. To overcome these challenges, we developed a rigorous enrichment strategy for HCPs, utilizing both Protein A and anti-HCP affinity chromatography, which facilitated the in-depth characterization of the HCP population in a monoclonal antibody formulation prone to PS hydrolysis. Based on the HCPs identified by liquid chromatography coupled to tandem mass spectrometry, a number of enzymes annotated as hydrolases were recombinantly expressed and characterized in terms of polysorbate degradation. Among the selected candidates, Lipoprotein Lipase, Lysosomal Acid Lipase (LIPA) and Palmitoyl-Protein Thioesterase 1 (PPT1) exhibited notable activity towards PS. To our knowledge, this is the first report to identify LIPA and PPT1 as residual HCPs that can contribute to PS degradation in a biological product.

Controlled polysorbate 20 hydrolysis - A new approach to assess the impact of polysorbate 20 degradation on biopharmaceutical product quality in shortened time.

Hydrolysis of polysorbate in biopharmaceutical liquid formulations upon long-term storage represents a risk factor, since reduction of the intact surfactant concentration may compromise protein stability. Moreover, accumulation of polysorbate degradation products is associated with the formation of particulates potentially affecting drug product stability and quality. These effects are conventionally assessed by real-time end-of-shelf life studies constituting an integral yet lengthy process of formulation development. To accelerate this procedure, we describe here a powerful tool to conduct shake stress studies based on the controlled hydrolysis of polysorbate 20 by beads-immobilized lipases. For this purpose, the production of stable, partially degraded material characterized by a representative presence of non-emulsifying degradants such as ethoxylated sorbitan and free fatty acids was monitored by state-of-the-art chromatographic methods ensuring realistic pharmaceutical conditions. Freeze-thaw, shaking and shipping stress studies of a mAb formulation did not only demonstrate that this approach is useful to determine the critical degradation level impairing drug product quality, but furthermore revealed significant differences in protective effects depending on the hydrolysis pattern. As these results emphasize, the outlined strategy may support formulation scientists to unveil the interrelationship between polysorbate hydrolysis products and stabilization of the active pharmaceutical ingredient in a holistic and time-saving manner.

Recent advances in LC-MS based characterization of protein-based bio-therapeutics - mastering analytical challenges posed by the increasing format complexity.

Alongside the success of protein-based bio-therapeutics over the last decades and facilitated by advances both in protein engineering and manufacturing, new product formats progressively enter into the biopharmaceutical industry's pipelines with major implications on the analytical methods used for their characterization. While conventional approaches have proved sufficient for standard (IgG-like) molecules, the increased complexity of novel formats requires proper adjustments of the employed methodologies, in particular with regard to separation-based techniques coupled to UV/FLD detection. After introducing the status quo for the characterization of biopharmaceuticals in quality control settings, this review provides a comprehensive portrayal of emerging LC-MS based technologies, which have already demonstrated their potential to complement the existing analytical toolbox. In this context, the benefits of native LC-MS and two-/multidimensional LC-MS applications to assess product attributes while preserving the higher-order structure are discussed based on challenges arising from the analysis of complex product formats.

From proof of concept to the routine use of an automated and robust multi-dimensional liquid chromatography mass spectrometry workflow applied for the charge variant characterization of therapeutic antibodies.

The identification and quantification of post-translational modifications (PTMs) is a crucial step required during the development of therapeutic proteins. In particular, the characterization of charge variants separated by cation exchange chromatography (CEX) is a tedious process commonly performed with an off-line manual fraction collection followed by peptide mapping. To improve the efficiency of this time-consuming approach, a generic on-line multi-dimensional LC/MS approach was developed for the characterization of various monoclonal antibody (mAb) isotypes and a bi-specific antibody (BsAb). Fractions collected from 1D CEX analysis were consecutively reduced on a 2D reversed phase liquid chromatography (RPLC) column (polyphenyl), digested within 1-2 min using a 3D immobilized trypsin cartridge, and finally the obtained peptides were separated on another 4D RPLC column (C18), and simultaneously identified with a Q Exactive™ mass spectrometer. 2D RPLC columns and 3D trypsin cartridges from different suppliers were compared, as well as the effects of reducing agents. The effect of 2D and 4D RPLC column temperature, and 2D RPLC column mass load were also systematically studied. Under optimal conditions, the multi-dimensional LC/MS system described in this paper is a robust tool for the on-line digestion of proteins and shows high repeatability. Similar levels of oxidation and deamidation were measured using the off-line and on-line approaches for the same stressed samples. The lower amounts of deamidation and isomerization measured at some asparagine and aspartic acid residues by the on-line approach compared to the manual off-line procedure suggest reduced artifacts using the on-line methodology. The multi-dimensional LC/MS described here enables fast, on-line, automated characterization of therapeutic antibodies without the need for off-line fraction collection and sample pre-treatment (manual approach). The entire workflow can be completed within less than one day, compared to weeks with the manual off-line procedure.

Rapid characterization of biotherapeutic proteins by size-exclusion chromatography coupled to native mass spectrometry.

High-molecular weight aggregates such as antibody dimers and other side products derived from incorrect light or heavy chain association typically represent critical product-related impurities for bispecific antibody formats. In this study, an approach employing ultra-pressure liquid chromatography size-exclusion separation combined with native electrospray ionization mass spectrometry for the simultaneous formation, identification and quantification of size variants in recombinant antibodies was developed. Samples exposed to storage and elevated temperature(s) enabled the identification of various bispecific antibody size variants. This test system hence allowed us to study the variants formed during formulation and bio-process development, and can thus be transferred to quality control units for routine in-process control and release analytics. In addition, native SEC-UV/MS not only facilitates the detailed analysis of low-abundant and non-covalent size variants during process characterization/validation studies, but is also essential for the SEC-UV method validation prior to admission to the market.

Formation and activation of fibroblast spheroids depend on fibronectin-integrin interaction.

Clustering of fibroblasts into spheroids induces a massive proinflammatory, proteolytic and growth-factor response, named nemosis, which promotes tumor cell invasiveness and differentiation of leukemia cells. We have now sought to investigate mechanisms leading to the formation of multicellular spheroids and subsequent activation of fibroblasts (nemosis). Cell lines either lacking fibronectin expression (FN-/-) or expressing FN with a mutated integrin-binding site (FNRGE/RGE) were unable to form compact spheroids. Furthermore, inhibition of FN synthesis by siRNA or functional inhibition of FN or its integrins impaired spheroid formation (alpha5, beta1) and quenched fibroblast activation (alphaV). The integrin ligand GRGDSP hexapeptide interfered with spheroid formation and induced activation of fibroblasts. Surprisingly, a 70 kDa FN fragment, which prevents deposition of FN matrix but does not interfere with FN-integrin interaction, prevented spheroid formation only marginally and did not block the activation. Our results present a new mechanism of fibroblast activation, which is initiated by interaction of FN with its integrin receptors.

The role of integrin binding sites in fibronectin matrix assembly in vivo.

The extracellular matrix (ECM) glycoprotein fibronectin (FN) requires the help of cells to assemble into a functional fibrillar matrix, which then orchestrates the assembly of other ECM proteins and promotes cell adhesion, migration and signalling. Fibrillogenesis is initiated and governed by cell surface integrins that bind to specific sites in the FN molecule. Recent studies identified novel integrin binding sites in FN that can also participate in FN fibril formation and in morphogenetic events during development.

The RGD motif in fibronectin is essential for development but dispensable for fibril assembly.

Fibronectin (FN) is secreted as a disulfide-bonded FN dimer. Each subunit contains three types of repeating modules: FN-I, FN-II, and FN-III. The interactions of alpha5beta1 or alphav integrins with the RGD motif of FN-III repeat 10 (FN-III10) are considered an essential step in the assembly of FN fibrils. To test this hypothesis in vivo, we replaced the RGD motif with the inactive RGE in mice. FN-RGE homozygous embryos die at embryonic day 10 with shortened posterior trunk, absent tail bud-derived somites, and severe vascular defects resembling the phenotype of alpha5 integrin-deficient mice. Surprisingly, the absence of a functional RGD motif in FN did not compromise assembly of an FN matrix in mutant embryos or on mutant cells. Matrix assembly assays and solid-phase binding assays reveal that alphavbeta3 integrin assembles FN-RGE by binding an isoDGR motif in FN-I5, which is generated by the nonenzymatic rearrangement of asparagines (N) into an iso-aspartate (iso-D). Our findings demonstrate that FN contains a novel motif for integrin binding and fibril formation whose activity is controlled by amino acid modification.