Similarity demonstrated between isolated charge variants of MB02, a biosimilar of bevacizumab, and Avastin® following extended physicochemical and functional characterization.

The majority of recombinant mAb products contain heterogeneous charge variants, commonly the result of post-translational modifications occurring during cell culture and accumulated during production, formulation and storage. MB02 is a biosimilar mAb to bevacizumab. Similarity data of charge variants for biosimilars against its reference products must be generated to demonstrate consistency in product quality and to ensure efficacy and safety. The goal of this work was to isolate seven charge variants of MB02 and Avastin® by semi-preparative cation exchange chromatography followed by purity test and extended analytical characterization to prove similarity. Although poor purity obtained for minor variants complicated data interpretation, an in-depth insight into the charge variants pattern of MB02 compared to Avastin® was obtained, contributing to a better understanding of modifications associated to microheterogeneity. To our knowledge, this is the first comparative analytical study of individual charge variants of a bevacizumab biosimilar following a head-to head approach and the most comprehensive N-glycosylation assessment of IgG1 charge variants. Although modifications related to N- and C-terminal, N-glycans, size heterogeneity or deamidation were specifically enriched among low abundant charge variants, they did not affect binding affinity to VEGF or FcRn and in vitro potency compared with the main species or unfractionated material.

Design, parallel synthesis of Biginelli 1,4-dihydropyrimidines using PTSA as a catalyst, evaluation of anticancer activity and structure activity relationships via 3D QSAR studies.

Biginelli 1,4-dihydropyrimidines are extensively screened for their potential anticancer activity in the last decade. In this context, a series of Biginelli 1,4-dihydropyrimidines were designed and synthesised using PTSA as an efficient catalyst. The synthesised 1,4-dihydropyrimidines were screened for their anticancer activity against MCF-7 breast cancer cells by measuring cytotoxicity. The compounds exhibited activity ranging from weak to significant in terms of percentage cytotoxicity which is proportional to the anticancer activity. Amongst the screened compounds, compounds 4, 6 and 8 exhibited potential anticancer activity. Furthermore, CoMSIA studies were performed to derive the structure activity relationships in a 3D grid space by plotting experimental vs predicted cytotoxic activities. We have an opinion that, this developed model helps us in future to develop more potential 1,4-dihydropyrimidines for their cytotoxicity or anticancer activity.

Similarity demonstrated between isolated charge variants of MB02, a biosimilar of bevacizumab, and Avastin® following extended physicochemical and functional characterization.

The majority of recombinant mAb products contain heterogeneous charge variants, commonly the result of post-translational modifications occurring during cell culture and accumulated during production, formulation and storage. MB02 is a biosimilar mAb to bevacizumab. Similarity data of charge variants for biosimilars against its reference products must be generated to demonstrate consistency in product quality and to ensure efficacy and safety. The goal of this work was to isolate seven charge variants of MB02 and Avastin® by semi-preparative cation exchange chromatography followed by purity test and extended analytical characterization to prove similarity. Although poor purity obtained for minor variants complicated data interpretation, an in-depth insight into the charge variants pattern of MB02 compared to Avastin® was obtained, contributing to a better understanding of modifications associated to microheterogeneity. To our knowledge, this is the first comparative analytical study of individual charge variants of a bevacizumab biosimilar following a head-to head approach and the most comprehensive N-glycosylation assessment of IgG1 charge variants. Although modifications related to N- and C-terminal, N-glycans, size heterogeneity or deamidation were specifically enriched among low abundant charge variants, they did not affect binding affinity to VEGF or FcRn and in vitro potency compared with the main species or unfractionated material.

A novel one-pot de-blocking and conjugation reaction step leads to process intensification in the manufacture of PEGylated insulin IN-105.

Bio-catalytic in vitro multistep reactions can be combined in a single step in one pot by optimizing multistep reactions under identical reaction condition. Using this analogy, the process of making PEGylated insulin, IN-105, was simplified. Instead of taking the purified active insulin bulk powder as the starting material for the conjugation step, an insulin process intermediate, partially purified insulin ester, was taken as starting material. Process intensification (PI) was established by performing a novel de-blocking (de-esterification) of the partially purified insulin ester and conjugation at B-29 Lys residue of B chain with a short-chain methoxy polyethylene glycol (mPEG) in a single-pot reactor. The chromatographic profile at the end of the reaction was found similar irrespective of whether both the reactions were performed sequentially or simultaneously. The conjugated product of interest, IN-105 (conjugation at LysB(29)), was purified from the heterogeneous mixture of conjugated products. The new manufacturing process was deduced to be more simplified and economical in making the insulin conjugates as several downstream purification steps could be circumvented. The physicochemical characteristics of IN-105 manufactured through this economic process was found to be indifferent from the product formed through the traditional process where the conjugation starting material was purified from bulk insulin.

A review of bridging clinical studies between different presentations of biological products approved by the United States Food and Drug Administration (US FDA).

INTRODUCTION: There is growing interest in the development of multiple presentations for biological products for subcutaneous (SC) injection for life cycle management and product differentiation. Bridging clinical studies are required to extrapolate the existing data package to new presentations. AREAS COVERED: This review compiles information of bridging clinical studies conducted for biological products administered by the SC route and approved in more than one presentation by the United States Food and Drug Administration's Center for Drug Evaluation and Research up until 31 December 2021. Information regarding indication(s), presentation(s), approval pathways, approval timelines, and various aspects of bridging clinical studies was collected from published documents. EXPERT OPINION: The type of bridging clinical study can depend on the extent of differences between presentations, existing data packages, and the stage of the product development. Design of a bridging clinical study should be based on the characteristics of a biological product and should be aimed at detecting the relevant differences between presentations. Single-dose comparative pharmacokinetics in normal healthy volunteers is the most common bridging clinical study design. Covariates like body weight and injection site should be considered during the design of these studies. The impact of the different user interfaces of presentations should also be considered.

Physicochemical and functional characterization of trastuzumab-dkst, a trastuzumab biosimilar.

Aims: Preclinical comparative similarity studies of trastuzumab-dkst, a Herceptin® biosimilar, are reported. Materials & methods: Primary sequence and higher order structure and pharmacological mechanisms of action were compared using multiple techniques. Pharmacokinetics and repeat-dose toxicity were assessed in cynomolgus monkeys. Results: Primary structures were identical; secondary and tertiary structures were highly similar. Non-significant differences were observed for charge heterogeneity. Twelve of 13 glycan species were highly similar, with slightly higher total mannose levels in trastuzumab-dkst. FcγR and FcRn binding activity was highly similar. Each drug equally inhibited HER2+ cell proliferation, demonstrating equivalent relative potency in mediating HER2+ cell cytolysis by antibody-dependent cellular cytotoxicity. Pharmacokinetic and toxicological profiles in cynomolgus monkeys were similar. Conclusion: Trastuzumab-dkst, US-licensed trastuzumab and EU-approved trastuzumab demonstrate high structural and functional similarity.

Probing deamidation in therapeutic immunoglobulin gamma (IgG1) by 'bottom-up' mass spectrometry with electron transfer dissociation.

Aspartic acid formed by nonenzymatic deamidation of asparagine often isomerizes to isoaspartic acid through a succinimide intermediate. Accumulation of isoaspartic acid initiates aggregation and degradation in proteins. Deamidation at the antigen-binding region reduces the efficacy and also upregulates immunogenicity of monoclonal antibodies. We report an improved 'bottom-up' tandem mass spectrometric method to detect and decipher the position of isoaspartate formation in therapeutic immunoglobulin gamma in a single chromatographic run. Differentiation between aspartate and isoaspartate residues through collision-induced tandem mass spectrometry is formidable due to their identical mass. Signature backbone cleavage ions, c(n) + 57 and z(l-n) - 57, produced upon radical-mediated fragmentation, were used to delineate the site of isomerization. It is more conclusive than monitoring the relative peak intensity and the decrease in hydrophobicity of the isoaspartate-containing peptide in a chromatographic elution. Collectively, this methodology provides a useful tool to monitor deamidation and isomerization in biopharmaceuticals during their production, downstream processing and storage.

Identification of the dipeptidyl aminopeptidase responsible for N-terminal clipping of recombinant Exendin-4 precursor expressed in Pichia pastoris.

Exendin-4 is a naturally occurring 39 amino acid peptide that is useful for the control of Type 2 diabetes. Recombinant Exendin-4, with an extra glycine at the carboxy-terminus (Exdgly), was expressed in the methylotropic yeast Pichia pastoris. A high proportion of the Exdgly molecules secreted into medium were found to be clipped, lacking the first two amino acids (His-Gly) from the N-terminus. Disruption of the P. pastoris homolog of the Saccharomyces cerevisiae dipeptidyl aminopeptidase (STE13) gene in Pichia genome resulted in a clone that expressed N-terminally intact Exdgly. Elimination of N-terminal clipping enhanced the yield and simplified the purification of Exdgly from P. pastoris culture supernatant.

Novel glitazones as PPARγ agonists: molecular design, synthesis, glucose uptake activity and 3D QSAR studies.

BACKGROUND: An alarming requirement for finding newer antidiabetic glitazones as agonists to PPARγ are on its utmost need from past few years as the side effects associated with the available drug therapy is dreadful. In this context, herein, we have made an attempt to develop some novel glitazones as PPARγ agonists, by rational and computer aided drug design approach by implementing the principles of bioisosterism. The designed glitazones are scored for similarity with the developed 3D pharmacophore model and subjected for docking studies against PPARγ proteins. Synthesized by adopting appropriate synthetic methodology and evaluated for in vitro cytotoxicity and glucose uptake assay. Illustrations about the molecular design of glitazones, synthesis, analysis, glucose uptake activity and SAR via 3D QSAR studies are reported. RESULTS: The computationally designed and synthesized ligands such as 2-(4-((substituted phenylimino)methyl)phenoxy)acetic acid derivatives were analysed by IR, 1H-NMR, 13C-NMR and MS-spectral techniques. The synthesized compounds were evaluated for their in vitro cytotoxicity and glucose uptake assay on 3T3-L1 and L6 cells. Further the activity data was used to develop 3D QSAR model to establish structure activity relationships for glucose uptake activity via CoMSIA studies. CONCLUSION: The results of pharmacophore, molecular docking study and in vitro evaluation of synthesized compounds were found to be in good correlation. Specifically, CPD03, 07, 08, 18, 19, 21 and 24 are the candidate glitazones exhibited significant glucose uptake activity. 3D-QSAR model revealed the scope for possible further modifications as part of optimisation to find potent anti-diabetic agents.

Identification and quantification of product-related quality attributes in bio-therapeutic monoclonal antibody via a simple, and robust cation-exchange HPLC method compatible with direct online detection of UV and native ESI-QTOF-MS analysis.

Modern analytical ion-exchange chromatography is one of the conventional tools used for assessment of product-related quality attributes in bio-therapeutic monoclonal antibodies (mAbs). Here, we present an approach to resolve, identify, and quantify product-related substances of therapeutic mAb at its intact molecular level by cation exchange (CIEX) HPLC coupled directly to electrospray ionization - quadrupole time of flight mass spectrometry (ESI-QTOF-MS). This method utilizes pH gradient elution mode comprised of ammonium formate buffer components, and a weak cation exchange column as stationary phase. Furthermore, ion-mobility mass spectrometry (IM-MS) provided additional insights on its higher order structure. Also, orthogonal assays such as conventional CIEX-HPLC, high resolution capillary isoelectric focusing, peptide mapping, spectroscopic, and fluorescence methods were used considerably to support the findings. Additionally, an in vitro assay was included to assess the associated impact on Fc mediated function. Overall, the developed method with simultaneous detection of UV peak area percentage at 280 nm and native ESI-MS is found to be a rapid and robust analytical tool for direct assessment of structural and purity attributes, process optimization, product development, and to decipher the relevant role of micro-variants on quality, stability, and clinical outcomes.

Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics.

We present here extensive mass spectrometric studies on the formation of a Tris conjugate with a therapeutic monoclonal antibody. The results not only demonstrate the reactive nature of the Tris molecule but also the sequence and reaction conditions that trigger this reactivity. The results corroborate the fact that proteins are, in general, prone to conjugation and/or adduct formation reactions and any modification due to this essentially leads to formation of impurities in a protein sample. Further, the results demonstrate that the conjugation reaction happens via a succinimide intermediate and has sequence specificity. Additionally, the data presented in this study also shows that the Tris formation is produced in-solution and is not an in-source phenomenon. We believe that the facts given here will open further avenues on exploration of Tris as a conjugating agent as well as ensure that the use of Tris or any ionic buffer in the process of producing a biopharmaceutical drug is monitored closely for the presence of such conjugate formation. Graphical Abstract ᅟ.

Mass spectrometric distinction of in-source and in-solution pyroglutamate and succinimide in proteins: a case study on rhG-CSF.

Formation of cyclic intermediates involving water or ammonia loss is a common occurrence in any reaction involving terminal amines or hydroxyl group containing species. Proteins that have both these functional groups in abundance are no exception, and presence of amino acids such as asparagine, glutamines, aspartic acids, and glutamic acids aid in formation of such intermediates. In the biopharma scenario, such intermediates lead to product- or process-related impurities that might be immunogenic. Mass spectroscopy is a powerful technique that is used to decipher the presence and physicochemical characteristics of such impurities. However, such intermediates can also form in situ during mass spectrometric analysis. We present here the detection of in-source and in-solution formation of succinimide and pyroglutamate in the protein granulocyte colony stimulating factor. We also propose an approach for quick differentiation of such in-situ species from the tangible impurities. We believe that this will not only reduce the time spent in unambiguous identification of succinimide- and/or pyroglutamate-related impurity in bio-pharmaceutics but also provide a platform for similar studies on other impurities that may form due to stabilized intermediates.

Synthesis, glucose uptake activity and structure-activity relationships of some novel glitazones incorporated with glycine, aromatic and alicyclic amine moieties via two carbon acyl linker.

Three series of novel glitazones were designed and prepared by using appropriate synthetic schemes to incorporate glycine, aromatic and alicyclic amines via two carbon linker. Compounds were synthesized both under conventional and microwave methods. Nineteen out of twenty four synthesized compounds were evaluated for their in vitro glucose uptake activity using isolated rat hemi-diaphragm. Compounds, 6, 9a, 13a, 13b, 13c, 13f and 13h exhibited significant glucose uptake activity. Illustration about their synthesis and in vitro glucose uptake activity is described along with the structure-activity relationships.

Development of a process to manufacture PEGylated orally bioavailable insulin.

To make insulin orally bioavailable, insulin was modified by covalent attachment (conjugation) of a short-chain methoxy polyethylene glycol (mPEG) derivative to the ε-amino group of a specific amino acid residue (LysB(29)). During the conjugation process, activated PEG can react with any of the free amino groups, the N-terminal of the B chain (PheB(1)), the N-terminal of the A chain (GlyA(1)), and the ε-amino group of amino acid (LysB(29)), resulting in a heterogeneous mixture of conjugated products. The abundance of the desired product (Methoxy-PEG(3)-propionyl--insulin at LysB(29):IN-105) in the conjugation reaction can be controlled by changing the conjugation reaction conditions. Reaction conditions were optimized for maximal yield by varying the proportions of protein to mPEG molecule at various values of pH and different salt and solvent concentrations. The desired conjugated molecule (IN-105) was purified to homogeneity using RP-HPLC. The purified product, IN-105, was crystallized and lyophilized into powder form. The purified product was characterized using multiple analytical methods including ESI-TOF and peptide mapping to verify its chemical structure. In this work, we report the process development of new modified insulin prepared by covalent conjugation of short chain mPEG to the insulin molecule. The attachment of PEG to insulin resulted in a conjugated insulin derivative that was biologically active, orally bioavailable and that showed a dose-dependent glucose lowering effect in Type 2 diabetes patients.