Impact of stirring material on formation of submicron and subvisible aggregates in mAbs by quantitative laser diffraction, dynamic light scattering and background membrane imaging.

Aggregation of monoclonal antibodies (mAbs) is the driving force for their undesirable immunogenic effects. There are multiple factors responsible for aggregation in therapeutic proteins. One significant cause is the process-related shear and interfacial stress generated due to impellers and stirrers. This investigation focuses on understanding the possible aggregation arising upon stirring mAb formulations using stirrers made of different materials. We used quantitative laser diffraction (qLD) to monitor and quantify the stirring induced formation of submicron and subvisible aggregates in the size range from 100 nm to 10 µm. We analysed various aspects of aggregate generation, such as onset of aggregation, particle size distribution, and concentration of aggregates generated using stirrers of different materials. We observed that mixing with stainless steel stirrers resulted in a quicker onset of aggregation and led to significantly higher concentrations of aggregates. Analysis of the stirred samples using dynamic light scattering (DLS) and background imaging technique (BMI) were conducted to complement the qLD analysis. All the three techniques resulted in a similar trend, showing presence of larger and higher quantities of aggregates in steel stirred samples, as compared to those stirred using PEEK and glass. Additionally, we performed SEC-HPLC to quantify the soluble fraction of monomer and recorded that the least amount was present in the steel stirred samples. This work highlights the need for optimizing the materials used for fabricating the stirrers/impellers.

Analytical techniques for screening of cannabis and derivatives from human hair specimens.

Cannabis and associated substances are some of the most frequently abused drugs across the globe, mainly due to their anxiolytic and euphorigenic properties. Nowadays, the analysis of hair samples has been given high importance in forensic and analytical sciences and in clinical studies because they are associated with a low risk of infection, do not require complicated storage conditions, and offer a broad window of non-invasive detection. Analysis of hair samples is very easy compared to the analysis of blood, urine, and saliva samples. This review places particular emphasis on methodologies of analyzing hair samples containing cannabis, with a special focus on the preparation of samples for analysis, which involves screening and extraction techniques, followed by confirmatory assays. Through this manuscript, we have presented an overview of the available literature on the screening of cannabis using mass spectroscopy techniques. We have presented a detailed overview of the advantages and disadvantages of this technique, to establish it as a suitable method for the analysis of cannabis from hair samples.

Current concepts in the management of cataract with keratoconus.

This review analyzed all pertinent articles on keratoconus (KCN) and cataract surgery. It covers preoperative planning, intraoperative considerations, and postoperative management, with the aim of providing a simplified overview of treating such patients. Preoperatively, the use of corneal cross-linking, intrastromal corneal ring segments, and topo-guided corneal treatments can help stabilize the cornea and improve the accuracy of biometric measurements. It is important to consider the advantages and disadvantages of traditional techniques such as penetrating keratoplasty and deep anterior lamellar keratoplasty, as well as newer stromal augmentation techniques, to choose the most appropriate surgical approach. Obtaining reliable measurements can be difficult, especially in the advanced stages of the disease. The choice between toric and monofocal intraocular lenses (IOLs) should be carefully evaluated. Monofocal IOLs are a better choice in patients with advanced disease, and toric lenses can be used in mild and stable KCN. Intraoperatively, the use of a rigid gas permeable (RGP) lens can overcome the challenge of image distortion and loss of visual perspective. Postoperatively, patients may need updated RGP or scleral lenses to correct the corneal irregular astigmatism. A thorough preoperative planning is crucial for good surgical outcomes, and patients need to be informed regarding potential postoperative surprises. In conclusion, managing cataracts in KCN patients presents a range of challenges, and a comprehensive approach is essential to achieve favorable surgical outcomes.

From Efficiency to Yield: Exploring Recent Advances in CHO Cell Line Development for Monoclonal Antibodies.

The increasing demand for biosimilar monoclonal antibodies (mAbs) has prompted the development of stable high-producing cell lines while simultaneously decreasing the time required for screening. Existing platforms have proven inefficient, resulting in inconsistencies in yields, growth characteristics, and quality features in the final mAb products. Selecting a suitable expression host, designing an effective gene expression system, developing a streamlined cell line generation approach, optimizing culture conditions, and defining scaling-up and purification strategies are all critical steps in the production of recombinant proteins, particularly monoclonal antibodies, in mammalian cells. As a result, an active area of study is dedicated to expression and optimizing recombinant protein production. This review explores recent breakthroughs and approaches targeted at accelerating cell line development to attain efficiency and consistency in the synthesis of therapeutic proteins, specifically monoclonal antibodies. The primary goal is to bridge the gap between rising demand and consistent, high-quality mAb production, thereby benefiting the healthcare and pharmaceutical industries.

Drug-loaded vegan gummies for personalized dosing of simethicone: A feasibility study of semi-solid extrusion-based 3D printing of pectin-based low-calorie drug gummies.

Chewable gummies are an attractive dosage form for all age groups because of their appearance and texture. Although, this dosage form has been highly preferred administering nutraceuticals, its application in the pharmaceutical sector is worth exploring. In this study, simethicone (SMT), an OTC drug prescribed for anti-flatulence was incorporated in pectin- based, low-calorie, 3D printed gummies. Semi-solid extrusion (SSE)-based 3D printing was used to dispense personalized dose of SMT i.e 40 mg for children and 125 mg for adults. Formulation optimization was carried out based on the texture profile of the gummies, using a texture analyzer. The inks were thoroughly characterized for their rheological behavior since it is a critical attribute for SSE-based 3D printing. Printing parameters like the printing speed, layer height and the type of the nozzle were optimized based on the printing accuracy achieved. The printed gummies were further evaluated for their disintegration time, drug content, weight variation, water activity and total microbial count. SSE-based 3D printing was found to be an effective tool to print pectin-based shear thinning gels for accurate drug dispensing. The texture profile of the printed gummies was comparable to the gummies prepared by conventional method as well as the marketed samples.

Microfluidic technology for cell biology-related applications: a review.

Fluid flow at the microscale level exhibits a unique phenomenon that can be explored to fabricate microfluidic devices integrated with components that can perform various biological functions. In this manuscript, the importance of physics for microscale fluid dynamics using microfluidic devices has been reviewed. Microfluidic devices provide new opportunities with regard to spatial and temporal control over cell growth. Furthermore, the manuscript presents an overview of cellular stimuli observed by combining surfaces that mimic the complex biochemistries and different geometries of the extracellular matrix, with microfluidic channels regulating the transport of fluids, soluble factors, etc. We have also explained the concept of mechanotransduction, which defines the relation between mechanical force and biological response. Furthermore, the manipulation of cellular microenvironments by the use of microfluidic systems has been highlighted as a useful device for basic cell biology research activities. Finally, the article focuses on highly integrated microfluidic platforms that exhibit immense potential for biomedical and pharmaceutical research as robust and portable point-of-care diagnostic devices for the assessment of clinical samples.

Taking the leap toward human-specific nonanimal methodologies: The need for harmonizing global policies for microphysiological systems.

With a recent amendment, India joined other countries that have removed the legislative barrier toward the use of human-relevant methods in drug development. Here, global stakeholders weigh in on the urgent need to globally harmonize the guidelines toward the standardization of microphysiological systems. We discuss a possible framework for establishing scientific confidence and regulatory approval of these methods.

Large-Scale Purification and Characterization of Recombinant Receptor-Binding Domain (RBD) of SARS-CoV-2 Spike Protein Expressed in Yeast.

SARS-CoV-2 spike protein is an essential component of numerous protein-based vaccines for COVID-19. The receptor-binding domain of this spike protein is a promising antigen with ease of expression in microbial hosts and scalability at comparatively low production costs. This study describes the production, purification, and characterization of RBD of SARS-CoV-2 protein, which is currently in clinical trials, from a commercialization perspective. The protein was expressed in Pichia pastoris in a large-scale bioreactor of 1200 L capacity. Protein capture and purification are conducted through mixed-mode chromatography followed by hydrophobic interaction chromatography. This two-step purification process produced RBD with an overall productivity of ~21 mg/L at >99% purity. The protein's primary, secondary, and tertiary structures were also verified using LCMS-based peptide mapping, circular dichroism, and fluorescence spectroscopy, respectively. The glycoprotein was further characterized for quality attributes such as glycosylation, molecular weight, purity, di-sulfide bonding, etc. Through structural analysis, it was confirmed that the product maintained a consistent quality across different batches during the large-scale production process. The binding capacity of RBD of spike protein was also assessed using human angiotensin-converting enzyme 2 receptor. A low binding constant range of KD values, ranging between 3.63 × 10-8 to 6.67 × 10-8, demonstrated a high affinity for the ACE2 receptor, revealing this protein as a promising candidate to prevent the entry of COVID-19 virus.

Cell-based Therapies for Corneal and Retinal Disorders.

Maintenance of the visual function is the desired outcome of ophthalmologic therapies. The shortcomings of the current treatment options, like partial recovery, post-operation failure, rigorous post-operative care, complications, etc., which are usually encountered with the conventional treatment options has warranted newer treatment options that may eliminate the root cause of diseases and minimize the side effects. Cell therapies, a class of regenerative medicines, have emerged as cutting-edge treatment option. The corneal and retinal dystrophies during the ocular disorders are the major cause of blindness, worldwide. Corneal disorders are mainly categorized mainly into corneal epithelial, stromal, and endothelial disorders. On the other hand, glaucoma, retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, Stargardt Disease, choroideremia, Leber congenital amaurosis are then major retinal degenerative disorders. In this manuscript, we have presented a detailed overview of the development of cell-based therapies, using embryonic stem cells, bone marrow stem cells, mesenchymal stem cells, dental pulp stem cells, induced pluripotent stem cells, limbal stem cells, corneal epithelial, stromal and endothelial, embryonic stem cell-derived differentiated cells (like retinal pigment epithelium or RPE), neural progenitor cells, photoreceptor precursors, and bone marrow-derived hematopoietic stem/progenitor cells etc. The manuscript highlights their efficiency, drawbacks and the strategies that have been explored to regain visual function in the preclinical and clinical state associated with them which can be considered for their potential application in the development of treatment.

Statistically-aided development of protein A affinity chromatography for enhancing recovery and controlling quality of a monoclonal antibody.

Protein A chromatography is widely used for isolation of monoclonal antibodies (mAbs) from cell culture components. In this study, the effect of different process parameters of the Protein A purification namely, binding pH, elution pH, flow rate, neutralization pH and tween concentration, on the concentration and quality of the purified mAb were evaluated. Using design of experiments approach, the critical process parameters of protein A chromatography were identified and experimentally optimized. Their impact on quality attributes, such as size variants and charge variants, of the mAb was studied. Multivariate data analysis was subsequently performed using multiple linear regression and partial least squares regression methods. It was observed that the elution pH primarily governed the concentration of the purified mAb and the content of monomers and aggregates, while the tween concentration primarily influenced the main peak of the charge variants. This is the first study that evaluates the impact of tween concentration in buffers on the protein A chromatography purification step. These studies helped in identifying the design space and defining the target robust and optimal setpoints of the responses, which were subsequently verified experimentally. These setpoints not only passed the target criteria but also resulted in the highest recoveries during the investigation. Through this statistically-aided approach, an optimized and robust protein A chromatography process was rationally developed for purification of mAbs, while achieving the desired product quality. This study highlights the influence of multiple parameters of the protein A purification process on critical quality attributes of mAbs, such as the size and charge variants, which has been a very scarcely explored area.

Controlling monoclonal antibody aggregation during cell culture using medium additives facilitated by the monitoring of aggregation in cell culture matrix using size exclusion chromatography.

Controlling monoclonal antibody aggregation at the upstream stage itself can significantly reduce the burden on downstream processing and can improve the process yield. Hence, we have investigated the use of sugar osmolytes (glucose, mannose, sucrose and maltose) and formulation excipients (mannitol, polysorbate 20 and polysorbate 80) as medium additives to reduce protein aggregation during cell culture. Aggregate content in cell culture samples was estimated using a high-resolution size-exclusion chromatography technique, which efficiently resolved the antibody monomer and aggregates in the cell culture matrix i.e., without purification. Glucose, mannose, maltose and the polysorbates effectively reduced the mean aggregate content over the course of the culture. Sugar-based additives exhibited a higher degree of variation during aggregate quantitation as compared to polysorbate additives, rendering the latter a preferred additive. Therefore, this study demonstrated the potential of sugar osmolytes and formulation excipients as media additives during cell culture to reduce aggregate formation, without negatively impacting cell growth and antibody production, facilitated by the monitoring of aggregate content in cell culture samples without purification.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Third International Conference on 3Rs Research and Progress, Vishakhapatnam, 2022.

Animal experimentation has been integral to drug discovery and development and safety assessment for many years, since it provides insights into the mechanisms of drug efficacy and toxicity (e.g. pharmacology, pharmacokinetics and pharmacodynamics). However, due to species differences in physiology, metabolism and sensitivity to drugs, the animal models can often fail to replicate the effects of drugs and chemicals in human patients, workers and consumers. Researchers across the globe are increasingly applying the Three Rs principles by employing innovative methods in research and testing. The Three Rs concept focuses on: the replacement of animal models (e.g. with in vitro and in silico models or human studies), on the reduction of the number of animals required to achieve research objectives, and on the refinement of existing experimental practices (e.g. eliminating distress and enhancing animal wellbeing). For the last two years, Oncoseek Bio-Acasta Health, a 3-D cell culture-based cutting-edge translational biotechnology company, has organised an annual International Conference on 3Rs Research and Progress. This series of global conferences aims to bring together researchers with diverse expertise and interests, and provides a platform where they can share and discuss their research to promote practices according to the Three Rs principles. In November 2022, the 3rd international conference, Advances in Animal Models and Cutting-Edge Research in Alternatives, took place at the GITAM University in Vishakhapatnam (AP, India) in a hybrid format (i.e. online and in-person). These conference proceedings provide details of the presentations, which were categorised under five different topic sessions. It also describes a special interactive session on in silico strategies for preclinical research in oncology, which was held at the end of the first day.

Surfactants reduce aggregation of monoclonal antibodies in cell culture medium with improvement in performance of mammalian cell culture.

Therapeutic monoclonal antibodies (mAbs) are biologics produced using mammalian cells and represent an important class of biotherapeutics. Aggregation in mAbs is a major challenge that can be mitigated by rigorous and reproducible upstream and downstream approaches. The impact of frequently used surfactants, like polysorbate 20, polysorbate 80, poloxamer 188, and 2-hydroxypropyl-beta-cyclodextrin, on aggregation of mAbs during cell culture was investigated in this study. Their impact on cell proliferation, viability, and mAb titer was also investigated. Polysorbate 20 and polysorbate 80 at the concentration of 0.01 g/L and poloxamer 188 at the concentration of 5 g/L were found to be effective in reducing aggregate formation in cell culture medium, without affecting the cell growth or viability. Furthermore, their presence in culture media resulted in increased cell proliferation as compared to the control group. Addition of these surfactants at the specified concentrations increased monomer production while decreasing high molecular weight species in the medium. After mAbs were separated, using protein "A" chromatography, flasks with surfactant exhibited improved antibody stability, when analyzed by DLS. Thus, while producing aggregation-prone mAbs via mammalian cell culture, these excipients may be employed as cell culture medium supplements to enhance the quality and yield of functional mAbs.

Comparison between carbohydrate and salt-based macromolecular crowders for cell preservation at higher temperatures.

In this investigation, the macromolecular crowding effect of a carbohydrate-based polymer, pullulan, and a salt-based polymer, poly-(4-styrenesulfonic-acid) sodium salt (PSS) was compared for the storage of A549 lung carcinoma cells, at temperatures greater than that of liquid nitrogen storage tanks. A DoE-CCD response surface model was used to optimise medium compositions comprising DMSO and a macromolecular crowder (MMC; pullulan, PSS and their combinations). The effect of adding MMCs was evaluated in terms of post-preservation viability, apoptotic population and growth curve analysis. The optimised medium consisting of 10% DMSO and 3% pullulan in the basal medium (BM) could facilitate long-term cell preservation for 90 days at - 80 °C, resulting in cell viability of ∼83%. The results also showed a significant decrease in the apoptotic population at all time points for the optimised composition of the freezing medium. These results indicated that adding 3% pullulan to the freezing medium improved the post-thaw viability and reduced the apoptotic cell population. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03571-6.

Rapid visual detection of Mycobacterium tuberculosis DNA using gold nanoparticles.

Tuberculosis (TB) is one of the world's deadliest infections caused by Mycobacterium tuberculosis (MTB). Though curable, the disease goes undetected in early stages owing to the lack of rapid, simple, cost-effective, and sensitive detection methods. In this investigation, we describe a procedure which is superior, more sensitive, and easier to handle, as compared to the previously reported, nanoparticle-based visual colorimetric assays for rapid detection of TB DNA, after its PCR amplification. This assay employs plasmonic gold nanoparticles (GNP) as a colorimetric agent and ethanol to promote aggregation of GNPs, thereby specifically detecting the amplified MTB DNA. An unambiguous response was achieved within 3 min after adding the DNA amplicon to the reaction tube. This conclusion was supported by spectroscopic data. The assay is sensitive up to ∼340 femtomole levels of MTB DNA, which was amplified using 0.125 ng µL-1 of the MTB DNA template. Thus, the technique developed here may be employed as a sensitive screening tool for early diagnosis of TB infection and is valuable for low-resource settings in remote areas, because of its simplicity. This ethanol-based visual TB DNA detection method is more sensitive, and fool-proof as compared to the commonly used salt-based colorimetric TB DNA assays, to the best of our knowledge.

Disease-related biomarkers as experimental endpoints in 3D skin culture models.

The success of in vitro 3D models in either recapitulating the normal tissue physiology or altered physiology or disease condition depends upon the identification and/or quantification of relevant biomarkers that confirm the functionality of these models. Various skin disorders, such as psoriasis, photoaging, vitiligo, etc., and cancers like squamous cell carcinoma and melanoma, etc. have been replicated via organotypic models. The disease biomarkers expressed by such cell cultures are quantified and compared with the biomarkers expressed in cultures depicting the normal tissue physiology, to identify the most prominent variations in their expression. This may also indicate the stage or reversal of these conditions upon treatment with relevant therapeutics. This review article presents an overview of the important biomarkers that have been identified in in-vitro 3D models of skin diseases as endpoints for validating the functionality of these models. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00574-2.

In vitro triple culture model of retinoblastoma for pre-clinical investigations.

BACKGROUND: Retinoblastoma (Rb) is a rare cancer of the retina that occurs during early childhood. The disease is relatively rare but aggressive, accounting for ∼3% of childhood cancers. Treatment modalities encompass the administration of large doses of chemotherapeutic drugs, which result in multiple side-effects. Therefore, it is essential to have safe and effective newer therapies and suitable physiologically relevant, alternative-to-animal, in vitro cell culture-based models to enable rapid and efficient evaluation of potential therapies. METHODOLOGY: This investigation was focused on the development of a triple co-culture model comprising Rb, retinal epithelium, and choroid endothelial cells, using a protein coating cocktail, to recapitulate this ocular cancer under in vitro conditions. This resulting model was used for screening drug toxicity, based on the growth profile of Rb cells, using carboplatin as the model drug. Further, a combination of bevacizumab and carboplatin was evaluated using the developed model, to lower the concentration of carboplatin and thereby reduce its physiological side-effects. MAJOR RESULTS: The effect of drug treatment on the triple co-culture was assessed by increase in the apoptotic profile of Rb cells. Further, the barrier properties were found to be lower with a decrease in the angiogenetic signals that included expression of vimentin. Measurement of cytokine levels signified reduced inflammatory signals due to the combinatorial drug treatment. CONCLUSIONS: These findings validated that the triple co-culture Rb model was suitable for evaluating anti-Rb therapeutics and could thereby decrease the immense load on animal trials, which are the primary screens employed for evaluating retinal therapies.

Developmental cataract in congenital ichthyosis.

A teenage boy who was previously diagnosed to have congenital ichthyosis presented to the eye clinic with complaints of gradually decreasing vision in both eyes since childhood. The best-corrected distance visual acuity was 20/125 in the right eye and 20/40 in the left eye. Clinical examination revealed developmental cataracts in both eyes. He underwent cataract surgery in the right eye and visual acuity improved to 20/25. Hence, we conclude that congenital ichthyosis can be associated with developmental cataracts. Cataract surgery helps in restoring vision in those with visually significant cataracts.

A case study: Correlation of the nutrient composition in Chinese Hamster Ovary cultures with cell growth, antibody titre and quality attributes using multivariate analyses for guiding medium and feed optimization in early upstream process development.

In this case-study, we demonstrate an approach for identifying correlations between nutrients/metabolites in the spent medium of CHO cell cultures and cell growth, mAb titre and critical quality attributes, using multivariate analyses, which can aid in selection of targets for medium and feed optimization. An extensive LC-MS-based method was used to analyse the spent medium composition. Partial least squares (PLS) model was used to identify correlations between nutrient composition and cell growth and mAb titre and orthogonal projections to latent structures (OPLS) model was used to determine the effect of the changing nutrient composition during the culture on critical quality attributes. The PLS model revealed that the initial concentrations of several amino acids as well as pyruvic acid and pyridoxine, governed the early cell growth, while the concentrations of TCA cycle intermediates and several vitamins highly influenced the stationary phase, in which mAb production was maximum. For the first time, with the help of the OPLS model, we were able to draw correlations between nutrients/metabolites during the culture and critical quality attributes, for example, optimizing the supply of certain amino acids and vitamins could reduce impurities while simultaneously increasing desirable glycoforms. The unique correlations obtained from such an exploratory analysis, utilizing conditions that are commonly adopted in early process development, present opportunities for optimizing the compositions of the growth media and the feed media for enhancing cell growth, mAb production and quality, thereby proving to be a useful preliminary step in bioprocess optimization. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00561-z.

Assessment of Urinary Biomarkers for Infectious Diseases Using Lateral Flow Assays: A Comprehensive Overview.

Screening of biomarkers is a powerful approach for providing a holistic view of the disease spectrum and facilitating the diagnosis and prognosis of the state of infectious diseases. Unaffected by the homeostasis mechanism in the human body, urine accommodates systemic changes and reflects the pathophysiological condition of an individual. Easy availability in large volumes and non-invasive sample collection have rendered urine an ideal source of biomarkers for various diseases. Infectious diseases may be communicable, and therefore early diagnosis and treatment are of immense importance. Current diagnostic approaches preclude the timely identification of clinical conditions and also lack portability. Point-of-care (POC) testing solutions have gained attention as alternative diagnostic measures due to their ability to provide rapid and on-site results. Lateral flow assays (LFAs) are the mainstay in POC device development and have attracted interest owing to their potential to provide instantaneous results in resource-limited settings. The discovery and optimization of a definitive biomarker can render POC testing an excellent platform, thus impacting unwarranted antibiotic administration and preventing the spread of infectious diseases. This Review summarizes the importance of urine as an emerging biological fluid in infectious disease research and diagnosis in clinical settings. We review the academic research related to LFAs. Further, we also describe commercial POC devices based on the identification of urinary biomarkers as diagnostic targets for infectious diseases. We also discuss the future use of LFAs in developing more effective POC tests for urinary biomarkers of various infections.