Background Lysine variants of monoclonal antibodies (mAbs) result from incomplete clipping of the C-terminal lysine residues of the heavy chain. Although the structure of the lysine variants has been determined for several mAb products, a detailed study that investigates the impact of lysine charge variants on PK/PD and preclinical safety is yet to be published. Objective An in-depth investigation of the impact of C- terminal lysine clipping of mAbs on safety and efficacy for bevacizumab charge variants. Method Charge variant isolation using semi-preparative chromatography is followed by a comparative analysis of FcRn binding, pharmacokinetics, and pharmacodynamics in relevant animal models. Results K1 variant exhibited improved FcRn binding affinity (4-fold), half-life (1.3-fold), and anti-tumor activity (1.3-fold) as compared to the K0 (main) product. However, the K2 variant, even though exhibited higher FcRn affinity (2-fold), displayed lower half-life (1.6-fold) and anti-tumor activity at medium and low doses. Differential proteomic analysis revealed that seven pathways (such as glycolysis, gluconeogenesis, carbon metabolism, synthesis of amino acids) were significantly enriched. Higher efficacy of the K1 variant is likely due to higher bioavailability of the drug, leading to complete downregulation of the pathways that facilitate catering of the energy requirements of the proliferating tumor cells. On the contrary, the K2 variant exhibits a shorter half-life, resulting only in partial reduction in the metabolic/energy requirements of the growing tumor cells. Conclusion Overall, we conclude that the mAb half-life, dosage, and efficacy of a biotherapeutic product are significantly impacted by the charge variant profile of a biotherapeutic product.

The present study investigates the impact of charge variants on bevacizumab's structure, stability, and biological activity. Five basic and one acidic charge variants were separated using semi-preparative cation exchange chromatography using linear pH gradient elution with purity > 85%. Based on the commercial biosimilar product's composition, two basic variants, one acidic and the main bevacizumab product, were chosen for further investigation. Intact mass analysis and tryptic peptide mapping established the basic variants' identity as those originating from an incomplete clipping of either one or both C-terminal lysine residues in the heavy chain of bevacizumab. Based on peptide mapping data, the acidic variant formation was attributed to deamidation of asparagine residue (N84), oxidation of M258, and preservation of C-terminal lysine residue, located on the heavy chain of bevacizumab. None of the observed charge heterogeneities in bevacizumab were due to differences in glycosylation among the variants. The basic (lysine) variants exhibited similar structural, functional, and stability profiles as the bevacizumab main product. But it was also noted that both the variants did not improve bevacizumab's therapeutic utility when pooled in different proportions with the main product. The acidic variant was found to have an equivalent secondary structure with subtle differences in the tertiary structure. The conformational difference also translated into a ~ 62% decrease in biological activity. Based on these data, it can be concluded that different charge variants behave differently with respect to their structure and bioactivity. Hence, biopharmaceutical manufacturers need to incorporate this understanding into their process and product development guidelines to maintain consistency in product quality.

BACKGROUND Inactive aggregated protein deposits formed during fermentation of recombinant bacteria - inclusion bodies (IBs) - continue to be a significant source of active proteins. Understanding how the microenvironment, in particular growth medium, affects the quality and quantity of the IBs would be desirable for optimal production of proteins. In this study, we explore the effect of various chemically defined media components on IBs using a variety of analytical techniques. Expression of a biotherapeutic, ranibizumab, has been chosen as a case study. RESULTS A repertoire of nutrients, including but not limited to carbon and nitrogen source, vitamins, cofactors, buffering agents and minerals, were examined. Sucrose, biotin, pantothenate, glutathione, K2HPO4, K2SO4, cyanocobalamin and CaSO4 were shortlisted based on experimentation. A design of experiments (DOE) study was performed to identify optimal concentrations of and interactions between these nutrients. Higher concentrations of sucrose, biotin and pantothenate, and lower concentrations of cyanocobalamin, CaSO4 and glutathione, resulted in larger IBs and greater product expression. Further, K2HPO4 and K2SO4 concentrations did not seem to have a meaningful impact on product formation. CONCLUSIONS Medium composition affects size and quality of IBs. The size of IBs exhibits a positive correlation with the quantity of product expressed, as well as the purity of the product, but does not correlate with the total amount of host cell proteins produced. IB size could be used as a key process attribute during bioreactor optimization, and the otherwise difficult task of measuring product concentration in the fermentation broth could be avoided. (c) 2020 Society of Chemical Industry

Purpose To understand the impact of methionine oxidation in GCSF on efficacy (neutrophil production/activation) and safety (biochemical and histopathological changes). Methods Nine GCSF biosimilars were analyzed for the levels of residual iron and copper content. Oxidation in GCSF was induced by H 2 O 2 treatment and four samples were prepared: wtGCSF (no oxidation), MetO (1138), MetO (1,138,127) and MetO (1138,127,122). These samples were used to evaluate binding affinity with the GCSF receptor (GCSFR) using biolayer interferometry, thermal stability using circular dichroism and in vitro potency using a relevant cell-based assay. In vivo pharmacodynamics examined changes in neutrophil production upon GCSF methionine oxidation, with the outcome correlated with the differential expression of genes implicated in the GCSF mediated neutrophil activation/ maturation. Pre-clinical safety studies including biochemical and histopathological changes were also performed. Results Met 122 and Met 127 have the most deleterious effect on the potency. Lower binding affinity with GCSFR was identified as the underlying cause for lower efficacy and potency. Role of Asp 110 in GCSF as the critical residue having adverse impact on efficacy in context of methionine oxidation has been elucidated. Impairment of in vitro binding affinity with GCSF manifests as in vivo pharmacodynamic differences via differential expression of downstream genes required for neutrophil maturation. Conclusion The data from the present study suggests that methionine oxidation in GCSF is a critical quality attribute that needs careful monitoring and control during commercial manufacturing and subsequent supply chain stages.

Ranibizumab is a biotherapeutic Fab fragment used for the treatment of age-related macular degeneration and macular oedema. It is currently expressed in the gram-negative bacterium, Escherichia coli. However, low expression levels result in a high manufacturing cost. The protein expression can be increased by manipulating nutritional requirements (carbon source, nitrogen source, buffering agent), process parameters (pH, inducer concentration, agitation, temperature), and the genetic make-up of the producing strain. Further, understanding the impact of these factors on product quality is a requirement as per the principles of Quality by Design (QbD). In this paper, we examine the effect of various media components and process parameters on the expression level and quality of the biotherapeutic. First, risk analysis was performed to shortlist different media components based on the literature. Next, experiments were performed to screen these components. Eight components were identified for further investigation and were examined for their effect and interactions using a Fractional Factorial experimental design. Sucrose, biotin, and pantothenate were found to have the maximum effect during Fab production. Furthermore, cyanocobalamin glutathione and biotin-glutathione were the most significant interactions observed. Product identification was performed with Liquid Chromatography–Mass Spectrometry (LC-MS), the expression level was quantified using Bio-layer Interferometry, Reverse Phase-HPLC, and SDS-PAGE, and product quality were measured by RP-HPLC. Overall, a five-fold enhancement of the target protein titer was obtained (from 5 mg/L to 25 mg/L) using the screened medium components vis-a-vis the basal medium, thereby demonstrating the efficacy of the systematic approach purported by QbD.

2-Mercaptobenzothiazole (MBT) complexes of Ag(I) and Au(III) were synthesized by wet chemical method. The structural, optical, 1 HNMR, ICP – MS and electrochemical studies of the complexes were carried out. The TUNEL assay studies of Ag(I)MBT and Au(III)MBT complexes on A549 cell line indicated induced apoptosis in the cells. TUNEL assay showed 60% cell viability for Ag(I)MBT whereas 80% for Au(III)MBT. Thus Ag(I)MBT can induce cell apoptosis in cells at a higher rate than Au(III)MBT. Therefore these complexes studied here can be a viable option as anti – proliferating agent.

The ability of an enzyme linked immunosorbent assay (ELISA) to measure the concentration of charge variants of a monoclonal antibody (mAb) biotherapeutic has been investigated. Percentage recovery was found to be in the range of 80%-120% with inter and intra assay variation between 5% and 15%. Linear regression analysis of ELISA output at different dilutions yielded a good fit (R-2 > 0.98). Assay output was not hindered by the presence of serum proteins and other analytes such as GCSF, demonstrating acceptable precision and specificity. It was concluded that the charge variants of the mAb can be accurately quantified by ELISA.

Accurate fingerprinting of critical raw materials that have significant impact on process performance and product quality is a necessary precursor for implementation of QbD in process and product development. This article presents a review of major developments in this space in the last 10 years, with a special emphasis on those in last 5 years. A step by step approach for managing raw materials in the QbD paradigm has been proposed. We think that it is necessary for the biotech industry to better manage variability originating from raw materials if holistic implementation of QbD is to be achieved.

Estimating impact of the various product-related variants and impurities on a biotherapeutic’s safety and efficacy is an essential requirement in the quality by design paradigm. In view of the limited role that clinical studies offer in this regard, we demonstrate a preclinical approach to achieve this for granulocyte colony-stimulating factor (GCSF). While our repeated-dose toxicity data suggest that these variants do not elicit any adverse effects or histopathological changes, aggregated GCSF impurity caused sluggishness in animal behavior manifested by a possible muscular injury. Cell assay data revealed that the cys-64-cys74 disulfide bond in reduced GCSF imparts stabilization in absence of the cys-36-cys42 bond. PK data demonstrate variability in half lives of different species when compared to the native GCSF. PD data along with differential expression of JAK-2 and STAT5a genes show that all the tested variants triggered the required signal transduction pathways for neutrophil proliferation and activation.

Charge variants, namely acidic and basic variants, are typically found in mAb therapeutics. Charge heterogeneity is typically not regarded to affect safety and efficacy of the product. As a result, the commonly followed approach involves assignment of a specification for the variants based on statistical analysis of variability in levels that is seen during commercial manufacturing. This is followed by monitoring of product quality to demonstrate consistency. This paper aims to demonstrate that this perception of charge variants warrants a more in‐depth investigation to evaluate the role charge variants play in safety and efficacy of a mAb therapeutic. In addition, a novel procedure has been suggested for making this assessment and alleviate the problems that are traditionally faced when isolating these variants for characterization. The suggested procedure utilizes the principles of bioseparations, cell biology, and statistics and it is demonstrated that this is significantly more efficient than the approach practiced today.

BackgroundCurrent chemotherapeutic agents based on apoptosis induction are lacking in desired efficacy. Therefore, there is continuous effort to bring about new dimension in control and gradual eradication of cancer by means of ever evolving therapeutic strategies. Various forms of PCD are being increasingly implicated in anti-cancer therapy and the complex interplay among them is vital for the ultimate fate of proliferating cells. We elaborated and illustrated the underlying mechanism of the most potent Andrographolide analogue (AG-4) mediated action that involved the induction of dual modes of cell death-apoptosis and autophagy in human leukemic U937 cells.Principal FindingsAG-4 induced cytotoxicity was associated with redox imbalance and apoptosis which involved mitochondrial depolarisation, altered apoptotic protein expressions, activation of the caspase cascade leading to cell cycle arrest. Incubation with caspase inhibitor Z-VAD-fmk or Bax siRNA decreased cytotoxic efficacy of AG-4 emphasising critical roles of caspase and Bax. In addition, AG-4 induced autophagy as evident from LC3-II accumulation, increased Atg protein expressions and autophagosome formation. Pre-treatment with 3-MA or Atg 5 siRNA suppressed the cytotoxic effect of AG-4 implying the pro-death role of autophagy. Furthermore, incubation with Z-VAD-fmk or Bax siRNA subdued AG-4 induced autophagy and pre-treatment with 3-MA or Atg 5 siRNA curbed AG-4 induced apoptosis-implying that apoptosis and autophagy acted as partners in the context of AG-4 mediated action. AG-4 also inhibited PI3K/Akt/mTOR pathway. Inhibition of mTOR or Akt augmented AG-4 induced apoptosis and autophagy signifying its crucial role in its mechanism of action.ConclusionsThus, these findings prove the dual ability of AG-4 to induce apoptosis and autophagy which provide a new perspective to it as a potential molecule targeting PCD for future cancer therapeutics.

-----------------------------------------------------ABSTRACT-----------------------------------------------------As the competition in market is growing at a very fast pace, one can survive in today’s industrial world by adopting the philosophy of Lean Manufacturing. In order to stay competitive, producing cheaper products at a faster rate Lean Manufacturing would help the industry. This paper highlights the Lean Manufacturing with its principles, tools & techniques so as to give an advantage by using these tools and techniques. With the help of these tools & techniques a better idea can be generated about the product flow in the organisation, value addition to the product through various processes and the time taken to produce the same. Lean Manufacturing helps to identify the different types of wastes and defects during manufacturing and take the remedial steps to eliminate them.