Therapeutic Monoclonal Antibodies for Metabolic Disorders: Major Advancements and Future Perspectives.

PURPOSE OF REVIEW: Globally, the prevalence of metabolic disorders is rising. Elevated low-density lipoprotein (LDL) cholesterol is a hallmark of familial hypercholesterolemia, one of the most prevalent hereditary metabolic disorders and another one is Diabetes mellitus (DM) that is more common globally, characterised by hyperglycemia with low insulin-directed glucose by target cells. It is still known that low-density lipoprotein cholesterol (LDL-C) increases the risk of cardiovascular disease (CVD). LDL-C levels are thought to be the main therapeutic objectives. RECENT FINDINGS: The primary therapy for individuals with elevated cholesterol levels is the use of statins and other lipid lowering drugs like ezetimibe for hypercholesterolemia. Even after taking statin medication to the maximum extent possible, some individuals still have a sizable residual cardiovascular risk. To overcome this proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors-monoclonal antibodies (mAbs) are a novel class of systemic macromolecules that have enhanced LDL-C-lowering efficacy. Along with this other inhibitor are used like Angiopoeitin like 3 inhibitors. Research on both humans and animals has shown that anti-CD3 antibodies can correct autoimmune disorders like diabetes mellitus. Individuals diagnosed with familial hypercholesterolemia (FH) may need additional treatment options beyond statins, especially when facing challenges such as statin tolerance or the inability of even the highest statin doses to reach the desired target cholesterol level. Here is the summary of PCSK9, ANGPTL-3 and CD3 inhibitors and their detailed information. In this review we discuss the details of PCSK9, ANGPTL-3 and CD3 inhibitors and the current therapeutic interventions of using the monoclonal antibodies in case of the metabolic disorder. We further present the present studies and the future prospective of the same.

LC-MS Characterization and Stability Assessment Elucidate Correlation Between Charge Variant Composition and Degradation of Monoclonal Antibody Therapeutics.

Aggregation stability of monoclonal antibody (mAb) therapeutics is influenced by many critical quality attributes (CQA) such as charge and hydrophobic variants in addition to environmental factors. In this study, correlation between charge heterogeneity and stability of mAbs for bevacizumab and trastuzumab has been investigated under a variety of stresses including thermal stress at 40 °C, thermal stress at 55 °C, shaking (mechanical), and low pH. Size- and charge-based heterogeneities were monitored using analytical size exclusion chromatography (SEC) and cation exchange chromatography (CEX), respectively, while dynamic light scattering was used to assess changes in hydrodynamic size. CEX analysis revealed an increase in cumulative acidic content for all variants of both mAbs post-stress treatment attributed to increased deamidation. Higher charge heterogeneity was observed in variants eluting close to the main peak than the ones eluting further away (25-fold and 42-fold increase in acidic content for main and B1 of bevacizumab and 19-fold for main of trastuzumab, respectively, under thermal stress; 50-fold increase in acidic for main and B1 of bevacizumab and 10% rise in basic content of main of trastuzumab under pH stress). Conversely, variants eluting far away from main exhibit greater aggregation as compared to close-eluting ones. Aggregation kinetics of variants followed different order for the different stresses for both mAbs (2nd order for thermal and pH stresses and 0th order for shaking stress). Half-life of terminal charge variants of both mAbs was 2- to 8-fold less than main indicating increased degradation propensity.

Taking the individual bias out of examining comparability of biosimilars: A case study on monoclonal antibody therapeutics.

Biosimilar manufacturers need to perform analytical and functional similarity assessments against the reference product. Successful demonstration allows for an abbreviated clinical path, thereby translating to affordable biosimilars. Current practices for regulatory concurrence on analytical similarity data are based on chart visualization and open to individual (human) bias. Here, we present a novel, chemometric approach for assessing biosimilarity that aims to simplify assessment and eliminate individual bias from decision making through application of weighted principal component analysis. Through the proposed approach, chemical information across the analytical characterization platform and drug products can be collated into a single plot for quantitative biosimilarity assessment. The proposed one-plot analysis offers a holistic visualization of 1) inter-product variability (w.r.t reference product) in cases where multiple batches per product have been investigated and 2) intra-product variability for each critical quality attribute (CQA) wherein information from orthogonal tools can be incorporated within the same plot. This allows for numerical grading of similarity for biosimilars of any given reference product. Although the proposed statistical approach is novel, it builds on standardized measures of CQA, criticality, and analytical procedures, thus making this approach easy to incorporate within the existing regulatory framework.

Assessing the Structural and Functional Similarity of Insulin Glargine Biosimilars.

BACKGROUND: A biosimilar product is expected to exhibit similar safety, efficacy, and quality as that of the approved reference product. Only a few reports of thorough evaluation of the quality of insulin glargine biosimilars are available in literature. Here, we examine the structural and functional similarity of biosimilars of insulin glargine, the first basal long-acting insulin analogue with respect to its innovator product (Lantus® from Sanofi Aventis). METHODS: Structural similarity was established using mass spectrometry, chromatographic, and spectroscopic techniques. Stability was compared by performing accelerated thermal stress studies. Functional similarity was established via in vitro assay. RESULTS: Biosimilar 4 exhibited greater content of high molecular weight species (HMWs) (0.80%) and related substances (RS) (0.45±0.06%) vs others (HMWs of 0.04% and RS of 0.17%). Biosimilars 1 and 3 exhibited higher rate of impurity generation (0.78% and 0.73% per week, respectively), as compared with other drug products (0.02% to 0.43% per week). Furthermore, %aggregation at 14 days was found to statistically correlate (R2= 0.99, root mean square error (RMSE) = 0.095) with %aggregation at 0 day (linearly) and the number of months from expiry (nonlinearly), highlighting the overpowering impact of the latter. CONCLUSIONS: While an overall structural and functional similarity was observed across insulin glargine biosimilars with respect to the innovator product, low amounts of product-related variants were seen in some biosimilars and these impact product stability. The %aggregation at 14 days exhibits statistical correlation with %aggregation at 0 day and the number of months from expiry. The order of biosimilarity was denoted as Lantus®>Biosimilar 2>Biosimilar 4>Biosimilar 1>Biosimilar 3.

Elucidating chemical and disulfide heterogeneities in rituximab using reduced and non-reduced peptide mapping.

Peptide mapping by liquid chromatography-mass spectrometry is the gold standard to characterize post-translational modifications (PTMs) and disulfide bonds. The structural integrity, heterogeneity, and quality of biotherapeutic proteins are evaluated via reduced and non-reduced peptide mapping methods. However, non-enzymatic artifacts are often induced during sample preparation when alkaline pH conditions are used. To minimize these artifacts, methods using various acidic pH conditions have been developed by multiple researchers. However, these may lead to missed and non-specific cleavages during the analysis. In this study, an improved reduced and non-reduced peptide mapping method has been proposed to characterize endogenous chemical modifications and native disulfide bonds of monoclonal antibody-based products. Solubilization has been carried out at acidic pH conditions under high temperature, followed by the addition of tris (2-carboxyethyl) phosphine as a reducing agent and a low alkylating agent. It was observed that the non-enzymatic PTMs and non-native disulfide scrambled peptides significantly reduced under trypsin plus endoproteinase Lys-C digestion conditions at acidic pH as compared to the traditional methods. The results demonstrate that the proposed reduced and non-reduced peptide mapping method using trypsin plus Lys-C could identify and quantify various chemical and disulfide heterogeneities with minimal artifacts.

Analytical Similarity Assessment of Biosimilars: Global Regulatory Landscape, Recent Studies and Major Advancements in Orthogonal Platforms.

Biopharmaceuticals are one of the fastest-growing sectors in the biotechnology industry. Within the umbrella of biopharmaceuticals, the biosimilar segment is expanding with currently over 200 approved biosimilars, globally. The key step towards achieving a successful biosimilar approval is to establish analytical and clinical biosimilarity with the innovator. The objective of an analytical biosimilarity study is to demonstrate a highly similar profile with respect to variations in critical quality attributes (CQAs) of the biosimilar product, and these variations must lie within the range set by the innovator. This comprises a detailed comparative structural and functional characterization using appropriate, validated analytical methods to fingerprint the molecule and helps reduce the economic burden towards regulatory requirement of extensive preclinical/clinical similarity data, thus making biotechnological drugs more affordable. In the last decade, biosimilar manufacturing and associated regulations have become more established, leading to numerous approvals. Biosimilarity assessment exercises conducted towards approval are also published more frequently in the public domain. Consequently, some technical advancements in analytical sciences have also percolated to applications in analytical biosimilarity assessment. Keeping this in mind, this review aims at providing a holistic view of progresses in biosimilar analysis and approval. In this review, we have summarized the major developments in the global regulatory landscape with respect to biosimilar approvals and also catalogued biosimilarity assessment studies for recombinant DNA products available in the public domain. We have also covered recent advancements in analytical methods, orthogonal techniques, and platforms for biosimilar characterization, since 2015. The review specifically aims to serve as a comprehensive catalog for published biosimilarity assessment studies with details on analytical platform used and critical quality attributes (CQAs) covered for multiple biotherapeutic products. Through this compilation, the emergent evolution of techniques with respect to each CQA has also been charted and discussed. Lastly, the information resource of published biosimilarity assessment studies, created during literature search is anticipated to serve as a helpful reference for biopharmaceutical scientists and biosimilar developers.

Usability of NISTmAb reference material for biosimilar analytical development.

With the number of approved biosimilars rapidly increasing in the various regulatory jurisdictions, public perception of safety and efficacy of these products have been gradually improving. Establishment of analytical similarity with the innovator product is a key aspect of biosimilar development. It serves as the basis for reduction in the size and/or scope of clinical trials. However, the clinical performance must be consistent with the innovator product. A considerable amount of effort precedes this exercise and requires a suitable reference material to support analytical method development. In view of the high cost as well as limited availability of innovator product in the market, there is a need for suitable reference material which could serve this purpose. The National Institute of Standards and Technology (NIST) have recently created NIST monoclonal antibody (NISTmAb) to support analytical method development and commercialization of monoclonal antibody-based biotherapeutics. In this paper, it is intended to evaluate lifecycle appropriate implementation of NISTmAb, a class-specific reference material in biosimilar analytical development. The results show that NISTmAb can be useful in developing initial platform analytical methods and as an external control for class-specific products. However, as expected, it cannot replace an innovator product or in-house product-specific reference material for analytical similarity assessment. As per regulatory guideline, a product-specific optimization will continue to be a prerequisite for specific analytical properties.

Assessment of structural and functional similarity of biosimilar products: Rituximab as a case study.

Biosimilars are products that are similar in terms of quality, safety, and efficacy to an already licensed reference/ innovator product and are expected to offer improved affordability. The most significant source of reduction in the cost of development of a biosimilar is the reduced clinical examination that it is expected to undergo as compared to the innovator product. However, this clinical relief is predicated on the assumption that there is analytical similarity between the biosimilar and the innovator product. As a result, establishing analytical similarity is arguably the most important step towards successful development of a biosimilar. Here, we present results from an analytical similarity exercise that was performed with five biosimilars of rituximab (Ristova®, Roche), a chimeric mouse/ human monoclonal antibody biotherapeutic, that are available on the Indian market. The results show that, while the biosimilars exhibited similarity with respect to protein structure and function, there were significant differences with respect to size heterogeneity, charge heterogeneity and glycosylation pattern.

Preparation and Biochemical Property of Penicillin G Amidase-Loaded Alginate and Alginate/Chitosan Hydrogel Beads.

BACKGROUND: Penicillin G amidase (PGA) (EC 3.5.1.11) are enzymes that are mainly involved in the synthesis of semi-synthetic }-lactam antibiotics. Soluble PGA is costly and lacks long term operational stability. We revised most of the patents related to Penicillin G amidase (PGA) immobilization in the section "Recent Patents on Immobilized Penicillin G Amidase". OBJECTIVE: The aim of this work was to study comparative biochemical property of PGA enzyme immobilized in two hydro-gel beads - Ca-alginate and alginate+chitosan hybrid and morphologically characterised by SEM. METHODS: PGA immobilized in alginate+chitosan hybrid bead shows high pH and thermal stability. Km, Vmax and Effectiveness factor (1) value of free PGA were 56.19 mg/ml, 1.786 U/ml and 1, respectively. These parameters for PGA immobilized alginate beads were 64.84 mg/ml, 0.781U/ml and 0.437, respectively and for PGA immobilized alginate+chitosan hybrid beads were 87.08 mg/ml, 0.622 U/ml and 0.348, respectively. RESULTS: Immobilized PGA on alginate+chitosan hybrid beads gave the highest thermal stability, reusability and storage stability than alginate immobilized PGA. CONCLUSION: The entrapment of PGA on alginate+chitosan hybrid beads revealed several advantages and could be used in 6APA (6- aminopenicillanic acid) production.

Assessing analytical comparability of biosimilars: GCSF as a case study.

The biosimilar industry is witnessing an unprecedented growth with the newer therapeutics increasing in complexity over time. A key step towards development of a biosimilar is to establish analytical comparability with the innovator product, which would otherwise affect the safety/efficacy profile of the product. Choosing appropriate analytical tools that can fulfil this objective by qualitatively and/or quantitatively assessing the critical quality attributes (CQAs) of the product is highly critical for establishing equivalence. These CQAs cover the primary and higher order structures of the product, product related variants and impurities, as well as process related impurities, and host cell related impurities. In the present work, we use such an analytical platform for assessing comparability of five approved Granulocyte Colony Stimulating Factor (GCSF) biosimilars (Emgrast, Lupifil, Colstim, Neukine and Grafeel) to the innovator product, Neupogen(®). The comparability studies involve assessing structural homogeneity, identity, secondary structure, and product related modifications. Physicochemical analytical tools include peptide mapping with mass determination, circular dichroism (CD) spectroscopy, reverse phase chromatography (RPC) and size exclusion chromatography (SEC) have been used in this exercise. Bioactivity assessment include comparison of relative potency through in vitro cell proliferation assays. The results from extensive analytical examination offer robust evidence of structural and biological similarity of the products under consideration with the pertinent innovator product. For the most part, the biosimilar drugs were found to be comparable to the innovator drug anomaly that was identified was that three of the biosimilars had a typical variant which was reported as an oxidized species in the literature. But, upon further investigation using RPC-FLD and ESI-MS we found that this is likely a conformational variant of the biotherapeutic been studied.