Development of a multidimensional online method for the characterization and quantification of monoclonal antibodies using immobilized flow-through enzyme reactors.

Complete characterization and quantification of monoclonal antibodies often rely on enzymatic digestion with trypsin. In order to accelerate and automate this frequently performed sample preparation step, immobilized enzyme reactors (IMER) compatible with standard HPLC systems were used. This allows an automated online approach in all analytical laboratories. We were able to demonstrate that the required digestion time for the model monoclonal antibody rituximab could be reduced to 20 min. Nevertheless, a previous denaturation of the protein is required, which also needs 20 min. Recoveries were determined at various concentrations and were 100% ± 1% at 100 ng on column, 96% ± 7% at 250 ng on column and 98% ± 2% at 450 ng on column. Despite these good recoveries, complete digestion was not achieved, resulting in a poorer limit of quantification. This is 50 ng on column under optimized IMER conditions, whereas an offline digest on the same system achieved 0.3 ng on column. Furthermore, our work revealed that TRIS buffers, when used with an IMER system, led to alteration of the peptides and induced modifications in the peptides. Therefore, the addition of TRIS should be avoided when working at elevated temperatures of about 60 °C. Nevertheless, our results have shown that the recovery is not significantly influenced whether TRIS is used or not (recovery: 96 ± 7% with TRIS vs. 100 ± 9% without TRIS).

Characterization of Glycosylated Proteins at Subunit Level by HILIC/MS.

Hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry (MS) is considered as the reference analytical technique for glycans profiling, especially for the characterization of glycosylated protein therapeutics such as monoclonal antibodies (mAbs) and mAbs-related products. Although HILIC/MS is mainly known to profile enzymatically released and fluorescently labeled N-glycans, the recent commercialization of new widepore HILIC amide bonded stationary phases packed with sub-2 µm particles has allowed for remarkable separations also at the subunit level. Here, we describe a simple protocol to perform the mAb glycans profiling at subunit level by HILIC/MS.

Anti-fouling poly adenine coating combined with highly specific CD20 epitope mimetic peptide for rituximab detection in clinical patients' plasma.

In this study, a high-performance anti-fouling coating based on poly adenine (polyAn) as well as a highly specific cluster of differentiation 20 (CD20) epitope mimetic peptide (CN14) were employed to synergistically construct a facile biosensor for the rapid and sensitive determination of rituximab in lymphoma patients' plasma. The well-designed and optimized polyAn coating displayed excellent stability, hydrophilicity, thanks to its intrinsic affinity with gold surface and thoroughly exposed hydrophilic phosphate groups. Moreover, the proposed strategy avoids the necessity to modify binding groups (e.g. thiol), making it more facile, repeatable and efficient. When dealing with complex clinical plasma samples, the polyAn coating demonstrated better anti-fouling performance and lower background signal in comparison with mercaptan and bovine serum albumin coatings. The dissociation constant (~60 nM) between CN14 and rituximab was measured by microscale thermophoresis and their binding mechanism was further explained using computer simulation. The constructed GE/CN14/polyA20 biosensor displayed satisfactory performance with detection limit of 35.26 ng/mL. Finally, the proposed biosensor was successfully applied for rapidly determining rituximab in lymphoma patients' plasma, and exhibited comparable accuracy to the commercial ELISA, but has advantages including a shorter detection time, wider detection range and lower cost. It's worth noting that the anti-fouling polyAn coating can be tailored according to the surface property of sensing interface and can be easily expanded to other gold electrode related biosensors.

Sedimentation Velocity Analytical Ultracentrifugation Analysis of Marketed Rituximab Drug Product Size Distribution.

PURPOSE: Analytical methods suitable for intact drug products are often necessary to evaluate the equivalence in physicochemical properties between two drug products (DP) containing the same drug substance (DS), e.g., an innovator biologic drug and its proposed biosimilar. Analytical Ultracentrifugation (AUC) is a biophysics technique applied to the analysis of size and shape of biomolecules. However, the application of AUC to formulated monoclonal antibody (mAb) DP at high concentration has not been reported. METHODS: A sedimentation velocity (SV) AUC procedure with a short-pathlength centerpiece was applied to two marketed rituximab DPs, Rituxan® (US) and Reditux® (India), without any buffer exchange or dilution. Detailed precision analysis was performed. RESULTS: Highly reproducible sedimentation coefficient values (S) and peak areas were obtained for the dominant (> 84%) monomeric rituximab peak. The minor mAb fragment peaks had large variation in both S values and peak areas (3-12%). The identification of oligomer peaks was only reproducible once the abundance was higher than 2%. CONCLUSIONS: SV-AUC provides an orthogonal characterization tool for protein size distribution, composition and assay, which could be informative for biosimilar drug developers who mostly only have access to formulated mAb. However, AUC needs thorough validation on its accuracy, precision and sensitivity.

Analytical comparability study of anti-CD20 monoclonal antibodies rituximab and obinutuzumab using a stability-indicating orthogonal testing protocol: Effect of structural optimization and glycoengineering.

Glycoengineering and biosimilarity are the key factors for growing, promising and progressive approaches in monoclonal antibodies development. In this study, the physicochemical stability of anti-CD20 rituximab (RTX); originator and biosimilar was compared to its glycoengineered humanized version; obinutuzumab (OBZ). An orthogonal stability-indicating protocol using a set of validated bioanalytical techniques; size exclusion high performance liquid chromatography (SE-HPLC), reversed phase liquid chromatography (RP-HPLC), quantitative gel electrophoresis by TapeStation, receptor binding assay and dynamic light scattering (DLS) was used to investigate the effect of different stress factors on the pattern and kinetics of degradation. SE-HPLC results supported with spectral purity showed similar degradation extent with a different pattern of degradation between RTX and OBZ. A lower tendency to form degraded fragments and a relatively higher favorability for degradation through aggregate formation has been revealed in case of OBZ. Results were in agreement with those of DLS and receptor binding assay which showed specificity to the intact antibodies in the presence of their degradation products. Furthermore, results were additionally confirmed through denaturing quantitative gel electrophoresis which suggested reducible covalent bonds as the mechanism for aggregates formation. RP-HPLC results showed two oxidized forms via excessive oxidation of RTX and OBZ with nearly the same degradation percent. Comparability data of RTX and OBZ using the applied methodologies showed that although glycoengineering; carried out to enhance the therapeutic and biological activity of OBZ altered the pattern of degradation but did not significantly affect the overall stability. Results showed also consistent stability profile between the biosimilar and its originator RTX products.

An application of Nano Differential Scanning Fluorimetry for Higher Order Structure assessment between mAb originator and biosimilars: Trastuzumab and Rituximab as case studies.

Differential scanning fluorimetry (DSF) or thermal shift has emerged in recent years as a high-throughput screening method in biotherapeutic formulation studies. The present article reports on a fast-track assessment platform for rapid investigation of therapeutic proteins such as monoclonal antibodies (mAb) with minimal sample concentration, volume, and preparation. The proposed nanoDSF platform has been demonstrated for rapid assessment of two commercial IgG 1 drug products (DP), trastuzumab and rituximab, and their biosimilars with respect to their conformational and colloidal stability. Domain specific differences for each of the IgGs have been elucidated with respect to onset of domain unfolding (Tonset) and melting temperatures. These thermal unfolding and transition midpoint (Tm) measurements are based on the intrinsic aromatic amino acid residue fluorescence of proteins. Moreover, to understand the possibility of nanoDSF as a predictive tool, data from nanoDSF has been correlated with accelerated stability studies. Melting temperatures across brands were found to be highly comparable to the rate of heating, thereby exhibiting a significant domain specific effect on melting temperatures for both trastuzumab and rituximab. Conservation of higher order structure (HOS) through reversible unfolding was also examined and both the mAbs were found to regain tertiary structure up till the first transition midpoint. No clear correlation was found between formation of higher molecular weight species (HMWS) and unfolding parameters (Tonset and Tagg) for accelerated stability studies. Finally, a discussion on the need for fast predictive assessment of conformation and colloidal stability as well as a comparison of advantages and limitations of the technique with routine/classical tools such as circular dichroism spectrophotometry and differential scanning calorimetry has been presented.

Intact and middle-down CIEF of commercial therapeutic monoclonal antibody products under non-denaturing conditions.

A two-step CIEF with chemical mobilization was developed for charge profiling of the therapeutic mAb rituximab under non-denaturing separation conditions. CIEF of the intact mAb was combined with a middle-down approach analyzing Fc/2 and F(ab´)2 fragments after digest with a commercial cysteine protease (IdeS). CIEF methods were optimized separately for the intact mAb and its fragments due to their divergent pIs. Best resolution was achieved by combining Pharmalyte (PL) 8-10.5 with PL 3-10 for variants of intact rituximab and of F(ab´)2 fragments, respectively, whereas PL 6.7-7.7 in combination with PL 3-10 was used for Fc/2 variants. Charge heterogeneity in Fc/2 dominates over F(ab´)2 . In addition, a copy product of rituximab, and adalimumab were analyzed. Both mAbs contain additional alkaline C-terminal lysine variants as confirmed by digest with carboxypeptidase B. The optimized CIEF methods for intact mAb and Fc/2 were tested for their potential as platform approaches for these mAbs. The CIEF method for Fc/2 was slightly adapted in this process. The pI values for major intact mAb variants were determined by adjacent pI markers resulting in 9.29 (rituximab) and 8.42 (adalimumab). In total, seven to eight charge variants could be distinguished for intact adalimumab and rituximab, respectively.

In-depth analysis of monoclonal antibodies using microfluidic capillary electrophoresis and native mass spectrometry.

Charge variant profiling of therapeutic proteins is required by the International Council for Harmonisation guidelines and is traditionally performed by capillary electrophoresis or ion exchange chromatography. Recently, improvements in the hyphenation of capillary electrophoresis with mass spectrometry and the introduction of mass spectrometry compatible background electrolytes has allowed the implementation of native mass spectrometric determination of the charge variant profile obtained from the electrophoretic separation. The low flow operation of the microfluidic electrophoretic platform significantly boosts mass spectrometric sensitivity and increases the dynamic range, even when using sample amounts as low as 1 ng in capillary. In the current study, rituximab, trastuzumab and bevacizumab drug products were analysed using the ZipChip microfluidic CE-ESI-MS platform that facilitated confident identification of proteoforms with an average mass accuracy of <15 ppm. Up to 52 proteoforms were identified for trastuzumab drug product, while rituximab sample revealed the presence of fragments and sialylated N-glycans. Overall, the CE-ESI-MS platform proved to be a fast and robust tool for therapeutic protein charge variant profiling and facilitated efficient coupling with native mass spectrometry for the generation of highly informative characterisation data.

Detection of doxorubicin, cisplatin and therapeutic antibodies in formalin-fixed paraffin-embedded human cancer cells.

A major limitation in the pharmacological treatment of clinically detectable primary cancers and their metastases is their limited accessibility to anti-cancer drugs (cytostatics, inhibitory antibodies, small-molecule inhibitors) critically impairing therapeutic efficacies. Investigations on the tissue distribution of such drugs are rare and have only been based on fresh frozen material or methanol-fixed cell culture cells so far. In this paper, we expand the detection of cisplatin-induced DNA adducts and anthracyclines as well as therapeutic antibodies to routinely prepared formalin-fixed, paraffin-embedded sections (FFPE). Using pre-treated cell lines prepared as FFPE samples comparable to tissues from routine analysis, we demonstrate that our method allows for the detection of chemotherapeutics (anthracyclines by autofluorescence, cisplatin by immune detection of DNA adducts) as well as therapeutic antibodies. This methodology thus allows for analyzing archival FFPE tissues, as demonstrated here for the detection of cisplatin, doxorubicin and trastuzumab in FFPE sections of tumor xenografts from drug-treated mice. Analyzing human tumor samples, this will lead to new insights into the tissue penetration of drugs.

Development and validation of a mass spectrometric method to determine the identity of rituximab based on its microheterogeneity profile.

Analytical methods have been considered the "eyes" for development, characterization and batch releasing of biotherapeutics over the past 40 years. One of the most powerful analytical platform for biotherapeutic analysis is mass spectrometry coupled to liquid chromatography (LC-MS). Due to its wide flexibility and instrumental configurations, LC-MS can determine different physicochemical attributes of proteins, e.g. molecular mass, primary sequence, and posttranslational modifications. Intact molecular mass analysis of therapeutic proteins is essential to confirm their identity. Analytical methods must be validated to support drug quality information during its approval process. Although there are international guidelines that provide general information on validation of analytical methods, practical examples about the design, selection of validation attributes and acceptance criteria of identity LC-MS methods are scarce. Here, according to the recommendations of Q2R1 ICH guideline, we showcase the validation of an LC-MS-TOF method to identity rituximab by determining its intact and deglycosylated molecular mass profiles. The proposed method specifically identified the m/z profile and deconvoluted mass profile of rituximab from deglycosylated rituximab and from excipient blank (specificity) with a maximum error of 76.63 ppm (accuracy) and a maximum Relative Standard Deviation (RSD) of 0.00315% (precision). Besides, the system suitability test, which was based on the expected mass value of the mass calibrator, confirmed the reliability of the analytical results. In summary, validation showed that the proposed method is suitable for identifying rituximab based on its glycosylated (intact) and deglycosylated mass profile.

Optimal Collision Energies and Bioinformatics Tools for Efficient Bottom-up Sequence Validation of Monoclonal Antibodies.

Rigorous validation of amino acid sequence is fundamental in the characterization of original and biosimilar protein biopharmaceuticals. Widely accepted workflows are based on bottom-up mass spectrometry, and they often require multiple techniques and significant manual work. Here, we demonstrate that optimization of a set of tandem mass spectroscopy (MS/MS) collision energies and automated combination of all available information in the measurements can increase the sequence validated by one technique close to the inherent limits. We created a software (called "Serac") that consumes results of the Mascot database search engine and identifies the amino acids validated by bottom-up MS/MS experiments using the most rigorous, industrially acceptable definition of sequence coverage (we term this "confirmed sequence coverage"). The software can combine spectra at the level of amino acids or fragment ions to exploit complementarity, provides full transparency to justify validation, and reduces manual effort. With its help, we investigated collision energy dependence of confirmed sequence coverage of individual peptides and full proteins on trypsin-digested monoclonal antibody samples (rituximab and trastuzumab). We found the energy dependence to be modest, but we demonstrated the benefit of using spectra taken at multiple energies. We describe a workflow based on 2-3 LC-MS/MS runs, carefully selected collision energies, and a fragment ion level combination, which yields ∼85% confirmed sequence coverage, 25%-30% above that from a basic proteomics protocol. Further increase can mainly be expected from alternative digestion enzymes or fragmentation techniques, which can be seamlessly integrated to the processing, thereby allowing effortless validation of full sequences.

Validation of Rapi-Fluor method for glycan profiling and application to commercial antibody drugs.

N-glycans influence the activity of antibody drugs such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Thus, glycan profiling is considered a critical quality attribute (CQA) and requires routine and comprehensive monitoring. In this report, we validate the new glycan profiling method called Rapi-Fluor method, which reduced the sample preparation time and increased the FLR and MS intensities compared with conventional 2-AB method. Optimized glycan release, labeling, hydrophilic interaction liquid chromatography (HILIC) enrichment, and HILIC separation resulted in low variation and short preparation time. The method evaluated for human IgG standard varied from 100 µg/mL to 4000 µg/mL in 25 µL of water. The determination of coefficient (r2 > 0.9992), recovery (88.992% ~ 111.198%), limit of detection (LOD < 193.274 µg/mL), limit of quantification (LOQ < 585.679 µg/mL), and precision (Intra-day < 2.317%RSD and Inter-day < 4.287%RSD) were evaluated with four major glycans from antibody drugs. In addition, the method was used for glycan profiling of five different commercial antibodies. The method yielded precise results for IgG glycan analysis and demonstrated effective glycan profiling of commercial antibody drugs.

Simple and ultra-fast recognition and quantitation of compounded monoclonal antibodies: Application to flow injection analysis combined to UV spectroscopy and matching method.

Compounding of monoclonal antibody (mAbs) constantly increases in hospital. Quality control (QC) of the compounded mAbs based on quantification and identification is required to prevent potential errors and fast method is needed to manage outpatient chemotherapy administration. A simple and ultra-fast (less than 30 s) method using flow injection analysis associated to least square matching method issued from the analyzer software was performed and evaluated for the routine hospital QC of three compounded mAbs: bevacizumab, infliximab and rituximab. The method was evaluated through qualitative and quantitative parameters. Preliminary analysis of the UV absorption and second derivative spectra of the mAbs allowed us to adapt analytical conditions according to the therapeutic range of the mAbs. In terms of quantitative QC, linearity, accuracy and precision were assessed as specified in ICH guidelines. Very satisfactory recovery was achieved and the RSD (%) of the intermediate precision were less than 1.1%. Qualitative analytical parameters were also evaluated in terms of specificity, sensitivity and global precision through a matrix of confusion. Results showed to be concentration and mAbs dependant and excellent (100%) specificity and sensitivity were reached within specific concentration range. Finally, routine application on "real life" samples (n = 209) from different batch of the three mAbs complied with the specifications of the quality control i.e. excellent identification (100%) and ±â€¯15% of targeting concentration belonging to the calibration range. The successful use of the combination of second derivative spectroscopy and partial least square matching method demonstrated the interest of FIA for the ultra-fast QC of mAbs after compounding using matching method.

Development of an analytical method to assess the occupational health risk of therapeutic monoclonal antibodies using LC-HRMS.

Monoclonal antibodies are a group of commonly used therapeutics, whose occupational health risk is still discussed controversially. The long-term low-dose exposure side effects are insufficiently evaluated; hence, discussions are often based on a theoretical level or extrapolating side effects from therapeutic dosages. While some research groups recommend applying the precautionary principle for monoclonal antibodies, others consider the exposure risk too low for measures taken towards occupational health and safety. However, both groups agree that airborne monoclonal antibodies have the biggest risk potential. Therefore, we developed a peptide-based analytical method for occupational exposure monitoring of airborne monoclonal antibodies. The method will allow collecting data about the occupational exposure to monoclonal antibodies. Thus, the mean daily intake for personnel in pharmacies and the pharmaceutical industry can be determined for the first time and will help to substantiate the risk assessment by relevant data. The introduced monitoring method includes air sampling, sample preparation and detection by liquid chromatography coupled with high-resolution mass spectrometry of individual monoclonal antibodies as well as sum parameter. For method development and validation, a chimeric (rituximab), humanised (trastuzumab) and a fully humanised (daratumumab) monoclonal antibody are used. A limit of detection between 1 µg per sample for daratumumab and 25 µg per sample for the collective peptide is achieved. Graphical abstract Demonstration of the analytical workflow, from the release of monoclonal antibodies to the detection as single substances as well as sum parameter.

Use of Cysteine Aminoethylation To Identify the Hypervariable Peptides of an Antibody.

Aminoethylation of cysteines can provide enzymatically cleavable sites. The ability to obtain peptides containing antibody complementarity determining regions (CDRs) with aminoethylated cysteines was investigated. Because cysteines are often located N-terminal to CDRs, digestion with Lys-N enables acquisition of peptides with CDRs. Lys-N peptides containing an aminoethylated cysteine at the N-terminus were also amidinated. Subsequent collisional activation yields a unique loss of 118 Da that originates from this modified residue, providing a signature ion for cysteine-containing peptides. The relative cleavage efficiencies for Lys-N and trypsin are also compared.

Preparation and characterization of micro-cell membrane chromatographic column with N-hydroxysuccinimide group-modified silica-based porous layer open tubular capillary.

Cell membrane chromatography (CMC) is an effective tool in screening active compounds from natural products and studying membrane protein interactions. Nevertheless, it always consumes a large amount of cells (e.g. 107-108) for column preparation. To overcome this, micro-CMC (mCMC), that employs a silica capillary as membrane carrier, was developed. However, both CMC and mCMC suffer from short column life span (e.g. 3days), mainly due to the falling-off of cellular membranes (CMs). This has greatly limited further application of CMC and mCMC, especially when the cells are hard to obtain. To solve this, N-hydroxysuccinimide (NHS)-modified silica-based porous layer open tubular capillary was first prepared for mCMC. The NHS groups can easily react with amino groups on CMs to form a stable covalent bond under a mild condition. So, CMs immobilized on the NHS-modified capillary are less likely to fall off. To verify this, SKBR3/mCMC (Her2 positive) and BALL1/mCMC (CD20 positive) columns were prepared. Two monoclonal antibody drugs, trastuzumab (anti-Her2) and rituximab (anti-CD20), were selected as analytes to characterize the columns. As a result, NHS-modified column for mCMC can afford higher chromatographic retention than non-modified column. Besides, the column life span was significantly improved to more than 16days for SKBR3/mCMC and 14days for BALL1/mCMC, while the compared column showed a sharp decline in retention factor in first 3days.

Extended stability of the rituximab biosimilar CT-P10 in its opened vials and after dilution and storage in polyolefin bags.

The stability of the rituximab biosimilar CT-P10, in 50mL vials at a concentration of 10mg/mL, and after dilution to final concentrations of 1 and 4mg/mL and storage in polyolefin bags at 4°C and 25°C was studied by several orthogonal and complementary methods. No significant change (as defined by a magnitude greater than the inter-batch variability) was observed, for each of the parameters characterizing physical and chemical stability studied, for the two concentrations and temperatures tested, or for any of the three batches tested. This implies that cold-chain rupture and exposure to room temperature up to 15 days both for vials and diluted bags have no deleterious consequence on the quality of the product. Moreover, this extended stability permits safe in-advance preparation, dose-banding or flat-dose, that to avoid unnecessary delays in the management of the patient, improvement of the pharmacy and nurse workload and money saving by avoiding non justified losses of this expensive drug.

High-throughput identification of monoclonal antibodies after compounding by UV spectroscopy coupled to chemometrics analysis.

Monoclonal antibodies (mAbs) compounded into the hospital pharmacy are widely used nowadays. Their fast identification after compounding and just before administration to the patient is of paramount importance for quality control at the hospital. This remains challenging due to the high similarity of the structure between mAbs. Analysis of the ultraviolet spectral data of four monoclonal antibodies (cetuximab, rituximab, bevacizumab, and trastuzumab) using unsupervised principal component analysis led us to focus exclusively on the second-derivative spectra. Partial least squares-discriminant analysis (PLS-DA) applied to these data allowed us to build models for predicting which monoclonal antibody was present in a given infusion bag. The calibration of the models was obtained from a k-fold validation. A prediction set from another batch was used to demonstrate the ability of the models to predict well. PLS-DA models performed on the spectra of the region of aromatic amino acid residues presented high ability to predict mAb identity. The region corresponding to the tyrosine residue reached the highest score of good classification with 89 %. To improve the score, standard normal variate (SNV) preprocessing was applied to the spectral data. The quality of the optimized PLS-DA models was enhanced and the region from the tyrosine/tryptophan residues allowed us excellent classification (100 %) of the four mAbs according to the matrix of confusion. The sensitivity and specificity performance parameters assessed this excellent classification. The usefulness of the combination of UV second-derivative spectroscopy to multivariate analysis with SNV preprocessing demonstrated the unambiguous identification of commercially available monoclonal antibodies. Graphical abstract PLS-DA models on the spectra of the region of aromatic amino acid residues allows mAb identification with high prediction.

SpeB proteolysis with imaged capillary isoelectric focusing for the characterization of domain-specific charge heterogeneities of reference and biosimilar Rituximab.

The charge variations of therapeutic monoclonal antibody reveal important information of the post-translational modifications that may potentially impact the potency and safety of pharmaceutical products, especially during the evaluation of biosimilarity of therapeutic proteins. In this work, a novel SpeB-based proteolysis strategy coupling with imaged capillary isoelectric focusing was developed for the determination of domain-specific charge heterogeneities of innovator and generic Rituximab drug products from United States, European and Indian markets. It was observed that innovator Rituximab from the United States and Europe share highly similar peak distributions and charge heterogeneities with 26.2-26.6% Fc/2, 28.9-29.3% LC and 44.4-44.5% Fd peak areas detected, respectively, while multiple basic variations of Fc/2 and less acidic LC and Fd species were found from generic Rituximab from India with 20.9% Fc/2, 32.3% LC and 46.9% Fd peak areas detected. It was also demonstrated that structural changes caused by Carboxypeptidase B treatment and deamidation study at pH extremes could be sensitively captured with the established method, with the results further indicating that the generic product's basic variations of Fc/2 were un-cleaved Lysine residues, while the lack of certain acidic peaks on LC and Fd probably was due to the lower level of deamidation. This new strategy could become a useful tool to reveal domain-specific charge heterogeneities profiles of a variety of therapeutic monoclonal antibodies in regulated environments.