Stability assessment of FDA-approved ramucirumab monoclonal antibody; validated SE-HPLC method for degradation pattern evaluation.

Ramucirumab (RAMU) is a recently US Food and Drug Administration-approved monoclonal antibody that is included in various anticancer protocols. It has a structural complexity and high degradation risk that have a significant effect on its safety and effectiveness. The major aim of this work was to assess the degradation pattern of RAMU based on physicochemical characterization. Mechanical agitation, repeated freeze-thaw cycles, pH and temperature were the selected stress conditions to which RAMU samples were subjected. The SE-HPLC method was applied and validated to monitor the RAMU monomer along with its aggregates and/or fragments. The purity of the separated peaks together with system suitability parameters were determined through the calculation of percentage purity and percentage drop in RAMU concentration. The results were interpreted by correlating them with those of dynamic light scattering and reducing and non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Samples incubated at pH 2.0-10.0 and 37°C for up to 4 weeks were analysed, recording detection of reversed phase (RP) aggregates and low molecular weight peptide fragments. Similarly, samples under short-term storage conditions of 4 weeks at different temperatures (-20, 2-8, 25, 37 and 50°C) showed low molecular weight peptide fragments but to a lesser extent. These results highlight the alarming effect on RAMU multidose vial efficacy and safety.

Simple Addition of Glycine in Trifluoroacetic Acid-Containing Mobile Phases Enhances the Sensitivity of Electrospray Ionization Mass Spectrometry for Biopharmaceutical Characterization.

Trifluoroacetic acid (TFA) is a commonly used mobile phase additive in liquid chromatography-mass spectrometry (LC-MS)-based biopharmaceutical characterization to enhance reversed-phase chromatographic performance of peptide separation; however, it leads to significant mass spectrometry signal suppression during electrospray ionization. Previous studies have shown that introducing large amounts of carboxylic acids or ammonium hydroxide to LC eluents postcolumn can improve MS sensitivity. In this study, we discovered glycine as a simple additive for TFA mobile phases, which mitigates ion suppression through a similar mechanism but in a more effective way than weak acid or weak base and boosts mass spectrometry responses (signal-to-noise ratio) of peptides by more than 1 order of magnitude on average after directly adding a small amount (e.g., 2 mM) into TFA mobile phases without compromising the chromatographic performance of peptide separation. Other small molecule additives in TFA mobile phases, such as amino acids containing extended side chains with different chemical properties and glycine-based variants, were also evaluated. The results demonstrated that glycine offered the best response boosting on peptides. The discovery of this glycine additive in TFA mobile phases provides a simple and conventional approach to achieve greater mass spectrometry detection sensitivity than TFA mobile phases for LC-MS-based characterization of biopharmaceuticals.

Middle Level IM-MS and CIU Experiments for Improved Therapeutic Immunoglobulin Subclass Fingerprinting.

Most of the current FDA and EMA approved therapeutic monoclonal antibodies (mAbs) are based on humanized or human IgG1, 2, or 4 subclasses and engineered variants. On the structural side, these subclasses are characterized by specific interchain disulfide bridge connections. Different analytical techniques have been reported to assess intact IgGs subclasses, with recently special interest in native ion mobility (IM) and collision induced unfolding (CIU) mass spectrometry (MS). However, these two techniques exhibit significant limitations to differentiate mAb subclasses at the intact level. In the present work, we aimed at developing a unique IM-MS-based approach for the characterization of mAb subclasses at the middle level. Upon IdeS-digestion, the unfolding patterns of the F(ab')2 and Fc domains were simultaneously analyzed in a single run to provide deeper structural insights of the mAb scaffold. The unfolding patterns associated with the F(ab')2 domains are completely different in terms of unfolding energies and number of transitions. Thereby, F(ab')2 regions are the diagnostic domain to provide specific unfolding signatures to differentiate IgG subclasses and provide more confident subclass categorization than CIU on intact mAbs. In addition, the potential of middle-level CIU was evaluated through the characterization of eculizumab, a hybrid therapeutic IgG2/4 mAb. The unfolding signatures of both domains were allowed to corroborate, within a single run, the hybrid nature of eculizumab as well as specific subclass domain assignments to the F(ab')2 and Fc regions. Altogether, our results confirm the suitability of middle-level CIU of F(ab')2 domains for subclass categorization of canonical and more complex new generation engineered antibodies and related products.

Laboratory coagulation tests and emicizumab treatment A United Kingdom Haemophilia Centre Doctors' Organisation guideline.

INTRODUCTION: The factor VIII mimetic emicizumab (Hemlibra, Hoffman-la Roche, Basel, Switzerland) has a novel mode of action that affects the laboratory monitoring of patients receiving this treatment. AIM: This guideline from the United Kingdom Haemophilia Centre Doctors Organisation (UKHCDO) aims to provide advice for clinical and laboratory staff on appropriate use of laboratory assays in patients with Haemophilia A treated with emicizumab. METHODOLOGY: The guideline was prepared by a review of the available literature and discussion and revision by the authors. RESULTS: The guideline describes the effect of emicizumab on commonly used coagulations tests and provides recommendations on the use of assays for measurement of factor VIII and factor VIII inhibitor in the presence of emicizumab. The guideline also provides recommendations on measurement of emicizumab. CONCLUSION: Knowledge of the effect of emicizumab on coagulation tests and factor assays is required to ensure appropriate testing and monitoring of therapy in patients receiving this drug.

An in-line enzymatic microreactor for the middle-up analysis of monoclonal antibodies by capillary electrophoresis.

Monoclonal antibodies (mAbs) are undergoing rapid growth in the pharmaceutical industry due to their clinical efficiency. Concomitantly, robust, cost-effective, and high throughput analytical methods are needed for their quality control. Among all analytical techniques, capillary electrophoresis (CE) presents alternative and attractive features because the capillary can be used both as a microreactor and as a support for separation. Transverse diffusion of laminar flow profiles was applied for the middle-up analysis of mAbs for the first time. Infliximab was selected as the model mAb. All middle-up analysis steps (enzymatic digestion, electrophoretic separation and UV detection) were integrated into the same capillary. The conditions for the separation of infliximab subunits (pH, ionic strength, and type of background electrolyte) and in-line digestion parameters (reactant injection conditions, time, temperature and enzyme/mAb ratio) were optimized. The in-line methodology was compared to the off-line methodology and evaluated in terms of proteolysis efficiency, repeatability, and applicability to different mAbs. Finally, the methodology was transferred to capillary electrophoresis coupled to mass spectrometry (sheathless interface) to identify infliximab subunits. The in-line methodology was successfully implemented with a simplified injection scheme, temperature control, fast enzymatic reaction and high resolution of separation of infliximab subunits under pseudo-native MS compatible conditions. In comparison with the off-line methodology, reactant consumption was reduced by a factor of 1000, and the numbers of theoretical plates were increased by a factor of 2.

PNL2: A Useful Adjunct Biomarker to HMB45 in the Diagnosis of Uterine Perivascular Epithelioid Cell Tumor (PEComa).

Perivascular epithelioid cell tumors (PEComa) are rare neoplasms characterized by co-expression of melanocytic and muscle markers. HMB45 and Melan-A are used to confirm a PEComa diagnosis; however, both are often focally expressed and sensitivity for Melan-A is low. PNL2 is a reliable biomarker for epithelioid melanoma and renal angiomyolipoma/PEComa. The objective of this study was to determine PNL2 utility in diagnosing uterine PEComas as well as distinguishing PEComas from uterine smooth muscle tumors (SMTs). Twenty-one uterine PEComas and 45 SMTs were analyzed for PNL2; a subset was also stained for HMB45, Melan-A, Cathepsin-K, Desmin, and h-Caldesmon. Cases were scored as negative (0), focal (<10% of tumor cells), or patchy to diffusely positive (>10% of tumor cells). PEComas were positive for PNL2, HMB45, and Melan-A in 86%, 100%, and 57% of cases, respectively. In PEComas, PNL2 was patchy to diffusely positive more frequently (10/18, 56%) than Melan-A (4/12, 33%). In contrast, 2 of 45 (4%) SMTs were focally PNL2 positive; HMB45 was focally positive in 4 SMTs (11%) and all were negative for Melan-A. Desmin and h-Caldesmon were positive in 90% and 57% of PEComas, and 91% and 82% of SMTs. Cathepsin-K was positive in 100% of PEComas and 93% of SMTs. PNL2 is a useful biomarker for the diagnosis of uterine PEComa, with comparable sensitivity and specificity to HMB45. In contrast, PNL2 stains more PEComas when compared with Melan-A. Cathepsin-K, Desmin, and h-Caldesmon are of little utility for distinguishing PEComas and SMTs; however, lack of Cathepsin-K argues against PEComa. These results suggest that PNL2 should be used in conjunction with HMB45 in the diagnosis of PEComa of the uterine corpus.

Vedolizumab quantitation using high-resolution accurate mass-mass spectrometry middle-up protein subunit: method validation.

Background While quantitation methods for small-molecule and tryptic peptide bottom-up mass spectrometry (MS) have been well defined, quantitation methods for top-down or middle-up MS approaches have not been as well defined. Therapeutic monoclonal antibodies (t-mAbs) are a group of proteins that can be used to both demonstrate the advantages of top-down or middle-up detection methods over classic tryptic peptide bottom-up along with the growing need for robust quantitation strategies/software for these top-down or middle-up methods. Bottom-up proteolytic digest methods for the t-mAbs tend to suffer from challenges such as limited peptide selection due to potential interference from the polyclonal immunoglobulin background, complicated workflows, and inadequate sensitivity and specificity without laborious purification steps, and therefore have prompted the search for new detection and quantitation methods. Time-of-flight along with Orbitrap MS have recently evolved from the research and/or pharmaceutical setting into the clinical laboratory. With their superior mass measurement accuracy, resolution and scanning speeds, these are ideal platforms for top-down or middle-up characterization and quantitation. Methods We demonstrate a validated, robust, middle-up protein subunit detection and quantitation method for the IgG1 t-mAb, vedolizumab (VEDO), which takes advantage of the high resolution of the Orbitrap MS detection and quantitation software to increase specificity. Results Validated performance characteristics met pre-defined acceptance criteria with simple workflows and rapid turnaround times: characteristics necessary for implementation into a high-volume clinical MS laboratory. Conclusions While the extraction method can easily be used with other IgG1 t-mAbs, the detection and quantitation method may become an option for measurement of other proteins.

Parsimonious Charge Deconvolution for Native Mass Spectrometry.

Charge deconvolution infers the mass from mass over charge (m/z) measurements in electrospray ionization mass spectra. When applied over a wide input m/z or broad target mass range, charge-deconvolution algorithms can produce artifacts, such as false masses at one-half or one-third of the correct mass. Indeed, a maximum entropy term in the objective function of MaxEnt, the most commonly used charge deconvolution algorithm, favors a deconvolved spectrum with many peaks over one with fewer peaks. Here we describe a new "parsimonious" charge deconvolution algorithm that produces fewer artifacts. The algorithm is especially well-suited to high-resolution native mass spectrometry of intact glycoproteins and protein complexes. Deconvolution of native mass spectra poses special challenges due to salt and small molecule adducts, multimers, wide mass ranges, and fewer and lower charge states. We demonstrate the performance of the new deconvolution algorithm on a range of samples. On the heavily glycosylated plasma properdin glycoprotein, the new algorithm could deconvolve monomer and dimer simultaneously and, when focused on the m/z range of the monomer, gave accurate and interpretable masses for glycoforms that had previously been analyzed manually using m/z peaks rather than deconvolved masses. On therapeutic antibodies, the new algorithm facilitated the analysis of extensions, truncations, and Fab glycosylation. The algorithm facilitates the use of native mass spectrometry for the qualitative and quantitative analysis of protein and protein assemblies.

A Novel Online Four-Dimensional SEC×SEC-IM×MS Methodology for Characterization of Monoclonal Antibody Size Variants.

The determination of size variants is a major critical quality attribute of a therapeutic monoclonal antibody (mAb that may affect the drug product safety, potency, and efficacy. Size variant characterization often relies on size-exclusion chromatography (SEC), which could be hampered by difficult identification of peaks. On the other hand, mass spectrometry (MS)-based techniques performed in nondenaturing conditions have proven to be valuable for mAb-related compound characterization. On the basis of the observation that limited SEC performance was observed in nondenaturing MS compatible ammonium acetate buffer compared with classical phosphate salts, a multidimensional analytical approach was proposed. It combines comprehensive online two-dimensional chromatography (SEC×SEC), with ion mobility and mass spectrometry (IM-MS) in nondenaturing conditions for the characterization of a variety of mAbs. We first exemplify the versatility of our approach for simultaneous detection, identification, and quantitation of adalimumab size variants. Benefits of the SEC×SEC-native IM×MS were further highlighted on forced degraded pembrolizumab and bevacizumab samples, for which the 4D setup was mandatory to obtain an extensive and unambiguous identification, and accurate quantitation of unexpected high/low molecular weight species (HMWS and LMWS). In this specific context, monomeric conformers were detected by IM-MS as HMWS or LMWS. Altogether, our results emphasize the power of comprehensive 2D LC×LC setups hyphenated to IM×MS in nondenaturing conditions with unprecedented performance including: (i) maintaining optimal SEC performance (under classical nonvolatile salt conditions), (ii) performing online native MS identification, and (iii) providing IM-MS conformational characterization of all separated size variants.

Use of Cyclodextrin as a Novel Agent in the SEC-HPLC Mobile Phase to Mitigate the Interactions of Proteins or Peptide or their Impurities with the Residual Silanols of Commercial SEC-HPLC Columns with Improved Separation and Resolution.

PURPOSE: Accurate quantification of the intact proteins, antibodies or peptides and their impurities without interaction to silanols of HPLC column. METHODS: Hydroxypropyl ß Cyclodextrin (HPCD) is added in the mobile phase at different concentrations. Different commercial SEC-HPLC columns and biologics with a molecular weight ranging from 5.8 kDa to 150kDa were assessed with and without cyclodextrin. RESULTS: Addition of non-ionic sugars such as Hydroxypropyl ß Cyclodextrin in the mobile phase, resulted improved peak performance such as theoretical plates, peak resolution, peak width, peak height, and improved quantification of aggregates in biologics such as antibodies Humira and Actemra, and peptides such as insulin. There is an increase in peak height, reduced retention time, increased plate and reduced peak width with increasing concentration of cyclodextrin studied. DISCUSSION: High ionic strength, basic amino acids such as arginine, organic solvents (with a concentration low enough not to precipitate protein), sodium perchlorate and ion pairing agents in the mobile phase used for separation of peptides, proteins and antibodies to prevent silanol interaction. These commonly used solutions are not always successful, as they not only interact with the biologic, but are sometimes, not compatible. The non-ionic cyclodextrin itself does not cause protein aggregation but prevents the nonspecific binding or interaction of protein itself and thereby allowing for improved resolution, and accurate quantification of aggregates in antibodies, and peptides. The data on the separation in presence of cyclodextrin in the mobile phase showed higher peak resolution, improved peak shape, accurate apparent molecular weight, improved efficiency, and less peak tailing for biological products. CONCLUSION: Hydroxypropyl ß Cyclodextrin in the mobile phase, resulted improved SEC-HPLC resolution, and quantitation of aggregates in biologics by preventing the interaction of biologics to silanol of the commercial SEC-HPLC columns.

Development of an Enzyme-Linked Immune Sorbent Assay to Measure Nivolumab and Pembrolizumab Serum Concentrations.

BACKGROUND: Treatment with monoclonal antibodies (mAbs) against programmed cell death protein 1 receptor is subject to high variation in treatment outcome among cancer patients. For these agents, no exposure-response (ER) relationships have been investigated in routine health care settings. However, ER relationships have been identified for several other mAbs used in oncology. Methods to conveniently measure serum concentrations of anti-programmed cell death protein 1 mAbs in routine health care may clarify possible ER relationships. Therefore, the authors aimed to develop an enzyme-linked immune sorbent assay (ELISA) for the measurement of both nivolumab and pembrolizumab serum concentrations of treated cancer patients. METHODS: Optimal capture antigen and detection antibody concentrations were selected based on titrations. Nivolumab calibration standards ranging from 0.2 to 300 ng/mL were tested in duplicate. Accuracy was assessed in 2 recovery experiments. Intra- and interassay variations were assessed on 3 different days by 2 independent technicians. The developed ELISA was also set up for pembrolizumab calibration curves. Cross-reactivity of nivolumab measurements with ipilimumab was assessed. Of one nivolumab treated patient, serum concentrations in follow up samples were measured and presented. RESULTS: Nivolumab calibration standards of 0.20-25 ng/mL were used. Nivolumab trough concentrations after 1 cycle in 8 patients ranged from 17.3 to 31.1 mcg/mL. The range of accuracy was 84%-105%, whereas intra- and interassay variations showed a coefficient of variation of 5.5% and 10.1%, respectively. No cross-reactivity with ipilimumab was detected. Pembrolizumab trough concentrations (n = 8) ranged from 9.1 to 19.7 mcg/mL after 1 infusion. CONCLUSIONS: The in-house-developed ELISA provides the opportunity to measure both nivolumab and pembrolizumab serum concentrations. This may help identify possible ER relationships in treated cancer patients and may potentially lead to dose adjustments in the future.

The NISTmAb Reference Material 8671 lifecycle management and quality plan.

Comprehensive analysis of monoclonal antibody therapeutics involves an ever expanding cadre of technologies. Lifecycle-appropriate application of current and emerging techniques requires rigorous testing followed by discussion between industry and regulators in a pre-competitive space, an effort that may be facilitated by a widely available test metric. Biopharmaceutical quality materials, however, are often difficult to access and/or are protected by intellectual property rights. The NISTmAb, humanized IgG1κ Reference Material 8671 (RM 8671), has been established with the intent of filling that void. The NISTmAb embodies the quality and characteristics of a typical biopharmaceutical product, is widely available to the biopharmaceutical community, and is an open innovation tool for development and dissemination of results. The NISTmAb lifecyle management plan described herein provides a hierarchical strategy for maintenance of quality over time through rigorous method qualification detailed in additional submissions in the current publication series. The NISTmAb RM 8671 is a representative monoclonal antibody material and provides a means to continually evaluate current best practices, promote innovative approaches, and inform regulatory paradigms as technology advances. Graphical abstract The NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing.

Development of orthogonal NISTmAb size heterogeneity control methods.

The NISTmAb is a monoclonal antibody Reference Material from the National Institute of Standards and Technology; it is a class-representative IgG1κ intended to serve as a pre-competitive platform for harmonization and technology development in the biopharmaceutical industry. The publication series of which this paper is a part describes NIST's overall control strategy to ensure NISTmAb quality and availability over its lifecycle. In this paper, the development of a control strategy for monitoring NISTmAb size heterogeneity is described. Optimization and qualification of size heterogeneity measurement spanning a broad size range are described, including capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), size exclusion chromatography (SEC), dynamic light scattering (DLS), and flow imaging analysis. This paper is intended to provide relevant details of NIST's size heterogeneity control strategy to facilitate implementation of the NISTmAb as a test molecule in the end user's laboratory. Graphical abstract Representative size exclusion chromatogram of the NIST monoclonal antibody (NISTmAb). The NISTmAb is a publicly available research tool intended to facilitate advancement of biopharmaceutical analytics. HMW = high molecular weight (trimer and dimer), LMW = low molecular weight (2 fragment peaks). Peak labeled buffer is void volume of the column from L-histidine background buffer.

Biosensing Probe for Quality Control Monitoring of the Structural Integrity of Therapeutic Antibodies.

Ideal quality control of therapeutic antibodies involves analytical techniques with high-sensitivity, high-resolution, and high-throughput performance. Few technologies that assess the physicochemical heterogeneity of antibodies, however, meet all the required demands. We developed a biosensing method for the quality control of therapeutic antibodies based on an artificial protein, AF.2A1, which discriminates between the native and the non-native three-dimensional structures of immunoglobulin G (IgG). AF.2A1 specifically recognized non-native IgG spiked into a solution of native IgG, thereby making it possible to detect contamination by a small amount of non-native IgG, which is difficult using conventional size-based separation or spectroscopic techniques. Using AF.2A1 as an analytical probe, we determined the concentration of non-native IgG formed under various pH conditions. The probe was also applicable to accelerated tests of the long-term stability of a therapeutic antibody, allowing monitoring of the formation of non-native IgG at elevated temperatures and extended periods of storage. AF.2A1, a proteinous probe, can be combined with established methods or devices to achieve high-throughput assays and provides the potential for probe-based biosensing for the quality control of therapeutic antibodies.

Full antibody primary structure and microvariant characterization in a single injection using transient isotachophoresis and sheathless capillary electrophoresis-tandem mass spectrometry.

Here we report the complete characterization of the primary structure of a multimeric glycoprotein in a single analysis by capillary electrophoresis (CE) coupled to mass spectrometry (MS). CE was coupled to electrospray ionization tandem MS by means of a sheathless interface. Transient isotachophoresis (t-ITP) was introduced in this work as an electrokinetically based preconcentration technique, allowing injection of up to 25% of the total capillary volume. Characterization was based on an adapted bottom-up proteomic strategy. Using trypsin as the sole proteolytic enzyme and data from a single injection per considered protein, 100% of the amino acid sequences of four different monoclonal antibodies could be achieved. Furthermore, illustrating the effectiveness and overall capabilities of the technique, the results were possible through identification of peptides without tryptic miscleavages or posttranslational modifications, demonstrating the potency of the technique. In addition to full sequence coverages, posttranslational modifications (PTMs) were simultaneously identified, further demonstrating the capacity of this strategy to structurally characterize glycosylations as well as faint modifications such as asparagine deamidation or aspartic acid isomerization. Together with the exquisite detection sensitivity observed, the contributions of both the CE separation mechanism and selectivity were essential to the result of the characterization with regard to that achieved with conventional MS strategies. The quality of the results indicates that recent improvements in interfacing CE-MS coupling, leading to a considerably improved sensitivity, allows characterization of the primary structure of proteins in a robust and faster manner. Taken together, these results open new research avenues for characterization of proteins through MS.

Charge profiling and stability testing of biosimilar by capillary isoelectric focusing.

CIEF was developed for the rapid analysis of charge heterogeneity of trastuzumab biosimilar using commercially available fluorocarbon-coated capillary. The CIEF master mix was composed of 0.30% w/v methyl cellulose, 2.3 M urea, 56.8 mM l-arginine, 1.52 mM iminodiacetic acid, 4.5% v/v carrier ampholytes (broad-range pI 3-10 and narrow-range pI 8-10.5 with ratio of 3:1), and 0.45% v/v 10.0, 9.5, 7.0, 5.5, 4.1 pI markers. To get a robust method to analyze charge heterogeneity, some separation parameters, including focusing time and separation temperature, were investigated and optimized. The optimized method gave good precision in estimated pI values of charge variants with RSDs of not more than 0.16% intraday analysis (n = 6) and < 0.18% interday analysis (n = 9). In addition, the applications of this method including purity, stability, lot consistency, peptide N-glycosidase F digest, and C-terminal lysine variants characterization were also investigated.

Analysis of monoclonal antibody by a novel CE-UV/MALDI-MS interface.

mAbs are highly complex proteins that present a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product- and time-consuming. CE-MS couplings, especially to MALDI, appear really attractive methods for the characterization of biological samples. In this work, we report the last instrumental development and performance of the first totally automated off-line CE-UV/MALDI-MS/MS. This interface is based on the removal of the original UV cell of the CE apparatus, modification of the spotting device geometry, and creation of an integrated delivery matrix system. The performance of the method was evaluated with separation of five intact proteins and a tryptic digest mixture of nine proteins. Intact protein application shows the acquisition of electropherograms with high resolution and high repeatability. In the peptide mapping approach, a total number of 154 unique identified peptides were characterized using MS/MS spectra corresponding to average sequence coverage of 64.1%. Comparison with NanoLC/MALDI-MS/MS showed complementarity at the peptide level with an increase of 42% when using CE/MALDI-MS coupling. Finally, this work represents the first analysis of intact mAb charge variants by CZE using an MS detection. Moreover, using a peptide mapping approach CE-UV/MALDI-MS/MS fragmentation allowed 100% sequence coverage of the light chain and 92% of the heavy chain, and the separation of four major glycosylated peptides and their structural characterization.

Quality control and stability studies with the monoclonal antibody, trastuzumab: application of 1D- vs. 2D-gel electrophoresis.

Recombinant monoclonal antibodies (rmAbs) are medicinal products obtained by rDNA technology. Consequently, like other biopharmaceuticals, they require the extensive and rigorous characterization of the quality attributes, such as identity, structural integrity, purity and stability. The aim of this work was to study the suitability of gel electrophoresis for the assessment of charge heterogeneity, post-translational modifications and the stability of the therapeutic, recombinant monoclonal antibody, trastuzumab. One-dimensional, SDS-PAGE, under reducing and non-reducing conditions, and two-dimensional gel electrophoresis were used for the determination of molecular mass (Mr), the isoelectric point (pI), charge-related isoform patterns and the stability of trastuzumab, subjected to stressed degradation and long-term conditions. For the assessment of the influence of glycosylation in the charge heterogeneity pattern of trastuzumab, an enzymatic deglycosylation study has been performed using N-glycosidase F and sialidase, whereas carboxypeptidase B was used for the lysine truncation study. Experimental data documented that 1D and 2D gel electrophoresis represent fast and easy methods to evaluate the quality of biological medicinal products. Important stability parameters, such as the protein aggregation, can be assessed, as well.

Assessment of chemical stability of monoclonal antibody and antibody drug conjugate administered by pressurized intraperitoneal aerosol chemotherapy.

Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a new therapeutic approach for patients with peritoneal cancer. So far, most published studies investigated the administration of established cytostatic agents through PIPAC. This study aimed to evaluate the effect of PIPAC on two breakthrough anti-cancer agents, specifically anti-PD1 pembrolizumab, and anti-HER2 antibody-drug conjugate (ADC) - trastuzumab-deruxtecan. We conducted systematic analyses on samples of pembrolizumab and trastuzumab-deruxtecan at clinically relevant concentrations before and after PIPAC administration using an experimental setup of a hermetic container system, mimicking the abdominal cavity and using identical features as in clinical use. We utilized a range of chromatographic and spectroscopic techniques to explore potential alterations in the primary, secondary, and tertiary structures of the drugs, focusing on post-translational modifications resulting from the aerosolization. Our findings indicate that PIPAC did not compromise the integrity of tested biopharmaceuticals. The size variants of both drugs, assessed by size exclusion chromatography (SEC), remained unchanged. Reversed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC) revealed no significant differences in hydrophobicity variants, the average drug-to-antibody ratio (DAR), or DAR distribution before and after PIPAC treatment. Circular dichroism (CD) spectroscopy confirmed that the secondary and tertiary structures were preserved. While pembrolizumab showed no change in charge variants post-PIPAC, trastuzumab-deruxtecan exhibited a non-negligible change in the quantity of charge variants on the monoclonal antibody itself, while the payload remained unchanged. This shift could possibly be related to the metallic composition of the CapnoPen® device (made of nickel and chromium) used in PIPAC and for these experiments. Together, our results suggest that PIPAC does not alter the structure of pembrolizumab and trastuzumab-deruxtecan, paving the way for future clinical trials.