Biophysical Characterization of the Contribution of the Fab Region to the IgG-FcγRIIIa Interaction.

The cell-surface receptor FcγRIIIa is crucial to the efficacy of therapeutic antibodies as well as the immune response. The interaction of the Fc region of IgG molecules with FcγRIIIa has been characterized, but until recently, it was thought that the Fab regions were not involved in the interaction. To evaluate the influence of the Fab regions in a biophysical context, we carried out surface plasmon resonance analyses using recombinant FcγRIIIa ligands. A van't Hoff analysis revealed that compared to the interaction of the papain-digested Fc fragment with FcγRIIIa, the interaction of commercially available, full-length rituximab with FcγRIIIa had a more favorable binding enthalpy, a less favorable binding entropy, and a slower off rate. Similar results were obtained from analyses of IgG1 molecules and an IgG1-Fc fragment produced by Expi293 cells. For further validation, we also prepared a maltose-binding protein-linked IgG1-Fc fragment (MBP-Fc). The binding enthalpy of MBP-Fc was nearly equal to that of the IgG1-Fc fragment for the interaction with FcγRIIIa, indicating that such alternatives to the Fab domains as MBP do not positively contribute to the IgG-FcγRIIIa interactions. Our investigation strongly suggests that the Fab region directly interacts with FcγRIIIa, resulting in an increase in the binding enthalpy and a decrease in the dissociation rate, at the expense of favorable binding entropy.

Effect of Fatty Acid Composition in Polysorbate 80 on the Stability of Therapeutic Protein Formulations.

PURPOSE: Polysorbate excipients are commonly used as surfactants to stabilize therapeutic proteins in formulations. Degradation of polysorbates could lead to particle formation and instability of the drug formulation. We investigated how the fatty acid composition of polysorbate 80 impacts the degradation profile, particle formation, and product stability under stress conditions. METHODS: Two polysorbate 80-containing therapeutic protein formulations were reformulated with either Polysorbate 80 NF synthesized from a fatty acid mixture that contains mainly oleic acid (≥58%) or a version of polysorbate 80 synthesized with high oleic acid (>98%). Stress conditions, including high temperature and esterase spiking, were applied and changes to both the polysorbate and the therapeutic protein product were investigated for stability, purity, innate immune response and biological activity. RESULTS: The addition of esterase and storage at 37°C led to significant hydrolysis of the polysorbate and increases in sub-visible particle formation for both polysorbates tested. The fatty acid composition of polysorbate 80 did not directly alter the stability profile of either therapeutic protein as measured by size exclusion chromatography, or significantly impact innate immune response or biological activity. However, formulations with Polysorbate 80 NF showed greater propensity for sub-visible particle formation under stress conditions. CONCLUSIONS: These results suggest that composition of fatty acids in polysorbate 80 may be a promoter for sub-visible particulate formation under the stress conditions tested but may not impact protein aggregation or biological activity.

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).

Co-delivery of rituximab targeted curcumin and imatinib nanostructured lipid carriers in non-Hodgkin lymphoma cells.

The aim of the present study was production of nanostructured lipid carriers (NLCs) of curcumin and imatinib for co-administration in non-Hodgkin lymphoma cells. NLCs were prepared and conjugated to rituximab to target CD20 receptors of lymphoma cell lines. Oleic acid or Labrafac and glyceryl monostearate or lecithin were used for production of NLCs. The antibody coupling efficiency to NLCs and their physical characteristics were studied. The cytotoxicity of NLCs on Jurkat T cells (CD20 receptor negative) and Ramos B cells (CD20 receptor positive) was studied by MTT assay. The cellular uptake was determined by fluorescent microscopy. The results indicated both curcumin and imatinib targeted NLCs had a significant cytotoxic effect much higher than the free drugs and non-targeted NLCs on Ramos cells. In both cell lines, the cytotoxicity of the co-administrated drugs was significantly higher than each drug alone. In Ramos cells the co-administration of curcumin (15 µg/ml)/imatinib (5 µg/ml) decreased the free curcumin IC50 from 8.3 ± 0.9 to 1.9 ± 0.2 µg/ml, and curcumin targeted NLCs from 6.7 ± 0.1 to 1.3 ± 0.2 µg/ml. In this case the IC50 of imatinib was reduced from 11.1 ± 0.7 to 2.3 ± 0.1 µg/ml and imatinib targeted NLCs from 4.3 ± 0.1 to 1.4 ± 0.0 µg/ml. The co-administration of ritoximab conjugated NLCs of curcumin and imatinib may enhance cytotoxicity of imatinib in treatment of non-Hodgkin lymphoma.

Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics.

Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera® and Reditux™ displayed differences at the molecular level. MabThera® showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux™ but not in MabThera®. This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes.

Preparation and In Vitro Evaluation of RITUXfab-Decorated Lipoplexes to Improve Delivery of siRNA Targeting C1858T PTPN22 Variant in B Lymphocytes.

Autoimmune endocrine disorders, such as type 1 diabetes (T1D) and thyroiditis, at present are treated with only hormone replacement therapy. This emphasizes the need to identify personalized effective immunotherapeutic strategies targeting T and B lymphocytes. Among the genetic variants associated with several autoimmune disorders, the C1858T polymorphism of the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene, encoding for Lyp variant R620W, affects the innate and adaptive immunity. We previously exploited a novel personalized immunotherapeutic approach based on siRNA delivered by liposomes (lipoplexes) that selectively inhibit variant allele expression. In this manuscript, we improved lipoplexes carrying siRNA for variant C1858T by functionalizing them with Fab of Rituximab antibody (RituxFab-Lipoplex) to specifically target B lymphocytes in autoimmune conditions, such as T1D. RituxFab-Lipoplexes specifically bind to B lymphocytes of the human Raji cell line and of human PBMC of healthy donors. RituxFab-Lipoplexes have impact on the function of B lymphocytes of T1D patients upon CpG stimulation showing a higher inhibitory effect on total cell proliferation and IgM+ plasma cell differentiation than the not functionalized ones. These results might open new pathways of applicability of RituxFab-Lipoplexes, such as personalized immunotherapy, to other autoimmune disorders, where B lymphocytes are the prevalent pathogenic immunocytes.

NMR Spectroscopy for Protein Higher Order Structure Similarity Assessment in Formulated Drug Products.

Peptide and protein drug molecules fold into higher order structures (HOS) in formulation and these folded structures are often critical for drug efficacy and safety. Generic or biosimilar drug products (DPs) need to show similar HOS to the reference product. The solution NMR spectroscopy is a non-invasive, chemically and structurally specific analytical method that is ideal for characterizing protein therapeutics in formulation. However, only limited NMR studies have been performed directly on marketed DPs and questions remain on how to quantitively define similarity. Here, NMR spectra were collected on marketed peptide and protein DPs, including calcitonin-salmon, liraglutide, teriparatide, exenatide, insulin glargine and rituximab. The 1D 1H spectral pattern readily revealed protein HOS heterogeneity, exchange and oligomerization in the different formulations. Principal component analysis (PCA) applied to two rituximab DPs showed consistent results with the previously demonstrated similarity metrics of Mahalanobis distance (DM) of 3.3. The 2D 1H-13C HSQC spectral comparison of insulin glargine DPs provided similarity metrics for chemical shift difference (Δδ) and methyl peak profile, i.e., 4 ppb for 1H, 15 ppb for 13C and 98% peaks with equivalent peak height. Finally, 2D 1H-15N sofast HMQC was demonstrated as a sensitive method for comparison of small protein HOS. The application of NMR procedures and chemometric analysis on therapeutic proteins offer quantitative similarity assessments of DPs with practically achievable similarity metrics.

Identification and Quantification of Oxidation Products in Full-Length Biotherapeutic Antibodies by NMR Spectroscopy.

Therapeutic proteins are an indispensable class of drugs and often therapeutics of last resort. They are sensitive to oxidation, which is of critical concern, because it can affect drug safety and efficacy. Protein oxidation, with methionine and tryptophan as the most susceptible moieties, is mainly monitored by HPLC-MS techniques. However, since several oxidation products display the same mass difference, their identification by MS is often ambiguous. Therefore, an alternative analytical method able to unambiguously identify and, ideally, also quantify oxidation species in proteins is highly desired. Here, we present an NMR-based approach to monitor oxidation in full-length proteins under denaturing conditions, as demonstrated on two biotherapeutic monoclonal antibodies (mAbs). We show that methionine sulfoxide, methionine sulfone, N-formylkynurenine, kynurenine, oxindolylalanine, hydroxypyrroloindole, and 5-hydroxytryptophan result in characteristic chemical shift correlations suited for their identification and quantification. We identified the five most abundant oxidation products in forced degradation studies of two full-length therapeutic mAbs and can also unambiguously distinguish oxindolylalanine from 5-hydroxytryptophan, which are undistinguishable by MS due to the same mass shift. Quantification of the abundant methionine sulfoxide by NMR and MS gave highly comparable values. These results underline the suitability of NMR spectroscopy for the identification and quantification of critical quality attributes of biotherapeutics.

Well-Orientation Strategy for Direct Immobilization of Antibodies: Development of the Immunosensor Using the Boronic Acid-Modified Magnetic Graphene Nanoribbons for Ultrasensitive Detection of Lymphoma Cancer Cells.

This work presents an effective strategy for the well-oriented immobilization of antibodies in which boronic acid is directly attached to the surface and with no need of the long and flexible spacer. A magnetic graphene nanoribbon-boronic-acid-based immunosensor was developed and tested for the impedimetric detection of lymphoma cancer cells, a blood cancer biomarker. Magnetic graphene nanoribbons (MGNRs) were modified with boronic acid (BA) to create a supporting matrix that is utilized by immobilizing anti-CD20 antibodies with good orientation. The prepared biosensing layer (MGNR/BA/Ab) with well-oriented antibodies was premixed into whole blood samples to interact with lymphoma cancer cell receptors. In the presence of target cell receptors, an immunocomplex was formed between anti-CD20 antibodies and lymphoma cancer cell receptors. Then, the biosensing layer was magnetically collected on a screen-printed carbon electrode (SPCE) and placed in a homemade electrochemical cell configuration to measure impedimetric signals. The fabrication steps of the immunosensor were characterized by various techniques, such as resonance light scattering, fluorescence, electrochemical impedance spectroscopy, and cyclic voltammetry. The assay is highly sensitive: the calculated limit of detection of lymphoma cancer cells was as low as 38 cells/mL, and the detection was linear from 100 to 1 000 000 cells/mL. The specificity of the immunosensor is also very high, and there is no interference effect with several potential interferents, such as the breast cancer (MCF-7), human embryonic kidney (HEK293), and leukemia (HL-60 and KCL-22) cell lines. The performance of the immunosensor for lymphoma cancer cells in clinical blood samples is consistent with that of commercial flow cytometric assays.

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.

Highland games: A benchmarking exercise in predicting biophysical and drug properties of monoclonal antibodies from amino acid sequences.

Biopharmaceutical product and process development do not yet take advantage of predictive computational modeling to nearly the degree seen in industries based on smaller molecules. To assess and advance progress in this area, spirited coopetition (mutually beneficial collaboration between competitors) was successfully used to motivate industrial scientists to develop, share, and compare data and methods which would normally have remained confidential. The first "Highland Games" competition was held in conjunction with the October 2018 Recovery of Biological Products Conference in Ashville, NC, with the goal of benchmarking and assessment of the ability to predict development-related properties of six antibodies from their amino acid sequences alone. Predictions included purification-influencing properties such as isoelectric point and protein A elution pH, and biophysical properties such as stability and viscosity at very high concentrations. Essential contributions were made by a large variety of individuals, including companies which consented to provide antibody amino acid sequences and test materials, volunteers who undertook the preparation and experimental characterization of these materials, and prediction teams who attempted to predict antibody properties from sequence alone. Best practices were identified and shared, and areas in which the community excels at making predictions were identified, as well as areas presenting opportunities for considerable improvement. Predictions of isoelectric point and protein A elution pH were especially good with all-prediction average errors of 0.2 and 1.6 pH unit, respectively, while predictions of some other properties were notably less good. This manuscript presents the events, methods, and results of the competition, and can serve as a tutorial and as a reference for in-house benchmarking by others. Organizations vary in their policies concerning disclosure of methods, but most managements were very cooperative with the Highland Games exercise, and considerable insight into common and best practices is available from the contributed methods. The accumulated data set will serve as a benchmarking tool for further development of in silico prediction tools.

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.

Assessment of biosimilarity under native and heat-stressed conditions: rituximab, bevacizumab, and trastuzumab originators and biosimilars.

Biosimilars are highly similar to, but not identical with, their originator products. As a result, structural differences between originators and biosimilars can be difficult to detect and characterize without the appropriate analytical tools. Therefore, we first focus on identifying initial structural differences between rituximab, bevacizumab, and trastuzumab originator and biosimilar pairs and later address how these differences change after applying thermal stress at 40 °C with orbital shaking for 4 weeks. Prior to incubation, we detected comparable secondary and tertiary structures for each pair and identified different levels of soluble aggregates, charge variants, and molecular weight variants due to differences in glycoforms and the number of C-terminal lysine groups. Over the course of incubation, we compared differences in charge variants and unfolding patterns. Taken together, our study provides a comparability exercise, providing information on the minor differences present between originator and biosimilar products and how those differences are impacted by stress.

Exploring sample preparation and data evaluation strategies for enhanced identification of host cell proteins in drug products of therapeutic antibodies and Fc-fusion proteins.

Manufacturing of biopharmaceuticals involves recombinant protein expression in host cells followed by extensive purification of the target protein. Yet, host cell proteins (HCPs) may persist in the final drug product, potentially reducing its quality with respect to safety and efficacy. Consequently, residual HCPs are closely monitored during downstream processing by techniques such as enzyme-linked immunosorbent assay (ELISA) or high-performance liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS). The latter is especially attractive as it provides information with respect to protein identities. Although the applied HPLC-MS/MS methodologies are frequently optimized with respect to HCP identification, acquired data is typically analyzed using standard settings. Here, we describe an improved strategy for evaluating HPLC-MS/MS data of HCP-derived peptides, involving probabilistic protein inference and peptide detection in the absence of fragment ion spectra. This data analysis workflow was applied to data obtained for drug products of various biotherapeutics upon protein A affinity depletion. The presented data evaluation strategy enabled in-depth comparative analysis of the HCP repertoires identified in drug products of the monoclonal antibodies rituximab and bevacizumab, as well as the fusion protein etanercept. In contrast to commonly applied ELISA strategies, the here presented workflow is process-independent and may be implemented into existing HPLC-MS/MS setups for drug product characterization and process development. Graphical abstract.

Analytical and functional similarity of biosimilar ABP 798 with rituximab reference product.

ABP 798 is a biosimilar candidate to rituximab reference product (RP). This comprehensive analytical similarity assessment was designed to assess the structural and functional similarity of ABP 798, rituximab (US), and rituximab (EU) using sensitive state-of-the-art analytical techniques capable of detecting small differences in product attributes. The similarity assessment was performed to evaluate product quality attributes associated with Fab, Fab/Fc, and Fc domains, including those known to affect the mechanisms of action. ABP 798 has the same amino acid sequence and exhibits similar secondary and tertiary structures, similar glycan and post-translational modification profiles, and biological activities as rituximab RP. There are minor differences in biochemical attributes, which are not considered clinically meaningful. The results of the analytical and functional similarity assessment demonstrate that ABP 798 is highly analytically similar to rituximab RP. These results support the totality of evidence and the scientific justification for extrapolation of ABP 798 to all therapeutic indications approved for rituximab.

Immunofluorescent labeling of CD20 tumor marker with quantum dots for rapid and quantitative detection of diffuse large B-cell non-Hodgkin's lymphoma.

Fluorescent semiconductor quantum dots (QDs) are newfound nanocrystal probes which have been used in bioimaging filed in recent years. The purpose of this study is to evaluate the diagnostic value of specific QDs coupled to rituximab monoclonal antibody against CD20 tumor markers for patients with diffuse large B-cell lymphoma (DLBCL). In current study rituximab-conjugated quantum dots (QDs-rituximab) were prepared against CD20 tumor markers for detection of CD20-positive cells (human Raji cell line) using flowcytometry. A total of 27 tumor tissue samples were collected from patients with DLBCL and 27 subjects with negative pathological tests as healthy ones, which stained by QD-rituximab. The detection signals were obtained from QDs using fluorescence microscopy. The flowcytometry results demonstrated a remarkable difference in fluorescent intensity and FL2-H + (CD20-positive cells percentage) between two groups. Both factors were significantly higher in Raji in comparison with K562 cell line (P < 0.05). Lot of green fluorescence signals was observed due to the selectively binding of QD-rituximab to CD20 tumor markers which overexpressed in tumor tissues and a few signals observed on the defined healthy ones. Based on these observations the cut-off point was 46.8 dots and the sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 89.5%, 91.3%, and 100%, respectively (LR+, 9.52; LR-, 0). The QD -rituximab could be beneficial as a bioimaging tool with high sensitivity to provide an accurate molecular imaging technique for identifying CD20 tumor markers for early diagnosis of the patients with DLBCL.

Unambiguous Identification of Pyroglutamate in Full-Length Biopharmaceutical Monoclonal Antibodies by NMR Spectroscopy.

Biotherapeutic proteins are an indispensable class of pharmaceuticals that present a high degree of structural complexity and are prone to chemical modifications during production, processing, and storage, which have to be tightly controlled. Pyroglutamate (pGlu), a cyclization product of N-terminal Gln or Glu residues, is a widespread post-translational modification in proteins, including monoclonal antibodies (mAbs). The unambiguous identification and quantification of this modification in proteins is challenging, since the mass difference of -17 Da or -18 Da, when formed from Gln or Glu, respectively, is not unique. Moreover, deamidation and dehydration occur not only during cyclization to pGlu, but also during other reactions leading to different types of modifications, like succinimide or isopeptide bond moieties due to cross-linking between Asn or Gln and Lys side chains. Here we report the unambiguous identification and quantification of pGlu in intact mAbs with natural isotope distribution by NMR spectroscopy. The assignment of all 1H, 13C and 15N random coil chemical shifts of pGlu in short reference peptides led to the identification of unique chemical shift pairs that are distinct from the random coil chemical shifts of the natural amino-acid residues. These characteristic correlations are suited for the detection of pGlu in denatured proteins. We achieved complete denaturation of mAbs using a straightforward protocol, and could detect and quantify pGlu, in agreement with available mass spectrometric data. The application to the mAbs rituximab and adalimumab illustrates the potential of our approach for the characterization of biotherapeutics containing isotopes at natural abundance.

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.

Comparative research on 99mTc-Rituximab and 99mTc-sulfur colloid in sentinel lymph node imaging of breast cancer.

BACKGROUND: 99mTc-Rituximab is a new specific radiopharmaceutical that binds to the CD20 receptor which is highly expressed on the surface of B cells. We conducted a study in which 99mTc-Rituximab was compared with filtered 99mTc-sulfur colloid (fTcSC) for sentinel lymph node (SLN) detection in patients with breast cancer. METHOD: The study is divided into three parts. 1. Initially, 25 patients were selected for an internal controlled trial to received both 99mTc-Rituximab and fTcSC, the interval time is separated by ≥2 days. 2. Then, 91 patients were selected for a randomized controlled trial (41 and 50 patients in the 99mTc-Rituximab and fTcSC groups, respectively). All patients were administered either agent at the 6- and 12-o' clock positions by subareolar injection technique. SLN mapping was then performed 2 h after injection. 3. Serial dynamic images were further acquired for 2 h in 31 patients (22 and 9 patients from 99mTc-Rituximab and fTcSC cohorts, respectively). RESULTS: The identification rate of lymphoscintigraphy and SLNB in all and axilla regions for 99mTc-Rituximab and 99mTc-SC were 98.5% vs 98.7, 100% vs 98.4%, respectively. The mean number of SLNs identified by 99mTc-Rituximab and fTcSC was respectively 2.72 and 3.28, with a significant difference of P = 0.013 (paired sample t-test). The difference exists in the internal mammary and clavicular area, not in the axillary. The mean number of axillary sentinel lymph node biopsy (SLNB) for 99mTc-Rituximab and fTcSC was 2.95 vs 3.14, respectively, and no significant difference existed. 99mTc-Rituximab also exhibited a significantly faster injection site clearance rate when compared with fTcSC (0.193 ± 0.057 h- 1 vs 0.021 ± 0.007 h- 1, respectively). CONCLUSION: No significant difference was observed in identification rate and number of axillary SLN imaging and SLNB, between the two tracers. Compared to fTcSC, 99mTc-Rituximab based imaging demonstrated a fewer number of secondary lymph nodes and had faster injection site clearance rate. TRIAL REGISTRATION: www.chictr.org.cn, ChiCTR1900024990 (retrospectively registered August 6, 2019).

Differentiating the Effects of Oxidative Stress Tests on Biopharmaceuticals.

PURPOSE: A rapid and broadly applicable method to assess relevant oxidative damage in biopharmaceuticals is important for lifecycle management of product quality. Multiple methods are currently employed as stress tests to induce oxidative damage for assessment of stability, safety, and efficacy. We compared two common methods for inducing oxidative damage to assess differences in impact on bioactivity and structure of the biopharmaceuticals. METHODS: Biopharmaceuticals were treated with either metal-catalyzed oxidation (MCO) conditions or the reactive-oxygen species (ROS) inducer 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), then analyzed for changes in structure and bioactivity. RESULTS: We demonstrate that commonly used chemical methods for assessing oxidation yield distinct oxidation profiles for each of the biotechnology products analyzed, including monoclonal antibodies. We further report oxidant- and product-specific changes in bioactivity under oxidizing conditions, along with differential oxidation on the molecular subunits of monoclonal antibodies. CONCLUSION: Our results highlight the need for product-specific optimization and selection of orthogonal, relevant oxidizers when characterizing stress responses in biopharmaceuticals.