Residual protein analysis by SDS-PAGE in clinically manufactured BM-MSC products.

Residual substances that are considered hazardous to the recipient must be removed from final cellular therapeutic products manufactured for clinical purposes. In doing so, quality rules determined by competent authorities (CAs) for the clinical use of tissue- and cell-based products can be met. In our study, we carried out residual substance analyses, and purity determination studies of trypsin and trypsin inhibitor in clinically manufactured bone marrow-derived mesenchymal stromal/stem cell products, using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. Despite being a semiquantitative method, SDS-PAGE has several benefits over other methods for protein analysis, such as simplicity, convenience of use, and affordability. Due to its convenience and adaptability, SDS-PAGE is still a commonly used method in many laboratories, despite its limits in dynamic range and quantitative precision. Our goal in this work was to show that SDS-PAGE may be used effectively for protein measurement, especially where practicality and affordability are the major factors. The results of our study suggest a validated method to guide tissue and cell manufacturing sites for making use of an agreeable, accessible, and cost-effective method for residual substance analyses in clinically manufactured cellular therapies.

SARS-CoV-2 Mutations and their Viral Variants.

Mutations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occur spontaneously during replication. Thousands of mutations have accumulated and continue to since the emergence of the virus. As novel mutations continue appearing at the scene, naturally, new variants are increasingly observed. Since the first occurrence of the SARS-CoV-2 infection, a wide variety of drug compounds affecting the binding sites of the virus have begun to be studied. As the drug and vaccine trials are continuing, it is of utmost importance to take into consideration the SARS-CoV-2 mutations and their respective frequencies since these data could lead the way to multi-drug combinations. The lack of effective therapeutic and preventive strategies against human coronaviruses (hCoVs) necessitates research that is of interest to the clinical applications. The reason why the mutations in glycoprotein S lead to vaccine escape is related to the location of the mutation and the affinity of the protein. At the same time, it can be said that variations should occur in areas such as the receptor-binding domain (RBD), and vaccines and antiviral drugs should be formulated by targeting more than one viral protein. In this review, a literature survey in the scope of the increasing SARS-CoV-2 mutations and the viral variations is conducted. In the light of current knowledge, the various disguises of the mutant SARS-CoV-2 forms and their apparent differences from the original strain are examined as they could possibly aid in finding the most appropriate therapeutic approaches.

Host variations in SARS-CoV-2 infection.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the zoonotic pathogen that causes the "Coronavirus Disease of 2019 (COVID-19)", and COVID-19 itself is yet to be thoroughly understood. Both the disease as well as the mechanisms by which the host interacts with the SARS-CoV-2 have not been fully enlightened. The epidemiological factors -e.g. age, sex, race-, the polymorphisms of the host proteins, the blood types and individual differences have all been in discussions about affecting the progression and the course of COVID-19 both individually and collectively, as their effects are mostly interwoven. We focused mainly on the effect of polymorphic variants of the host proteins that have been shown to take part in and/or affect the pathogenesis of COVID-19. Additionally, how the procedures of diagnosing and treating COVID-19 are affected by these variants and what possible changes can be implemented are the other questions, which are sought to be answered.

Quality Management Systems (QMSs) of Human-Based Tissue and Cell Product Manufacturing Facilities.

In the broadest sense, a quality management system (QMS) runs by continuous interaction of elements based on processes, procedures, policies, guidelines, and resources that are compiled to guide an organization under the scope of its operational mission, vision, and objectives. QMSs in the biopharmaceutical sector defines a written and applied set of rules which aids in improvement of the quality of biopharmaceutical process engineering, while primarily assuring human tissue- and cell-based starting material and end product safety and minimizing the risk of human medicinal product recall, in the most cost-effective ways possible. This chapter aims to outline the crucial position of a QMS under the scope of good practices (GxPs) in the biopharmaceutical industry, as regards human tissue- and cell-based products.

Understanding factors affecting human chondrocyte culturing: an experimental study.

Over the years, surgical strategies have been developed in hope of full regeneration of the injured cartilage. In our study, we aimed to develop an optimized chondrocyte culture isolation technique as an active ingredient of a standardized autologous chondrocte implantation product, which is able to maintain the phenotype along with the molecular features of the cartilage. We compared different enzymes, which suggested optimal performance with collagenase type II at 5 mg/ml concentration. Thereafter, we observed that COL2 and GAG expression is substantially reduced with passaging. There was a need to omit passaging to reach the optimal isolation method. We then tested various growth factors and media in order to maintain the natural character of chondrocytes. Our study also suggested the highest COL2 and GAG expressions with the highest recovery in the presence of Advanced DMEM. Autologous chondrocyte implantation manufacturing approval was recently received from the national competent authority, making it possible to utilize the process engineering protocol developed with this study at our Tissue and Cell Manufacturing Center as a part of the autologous chondrocyte implantation manufacturing standard operation procedure (SOP).

Where is human-based cellular pharmaceutical R&D taking us in cartilage regeneration?

Lately, cellular-based cartilage joint therapies have gradually gained more attention, which leads to next generation bioengineering approaches in the development of cell-based medicinal products for human use in cartilage repair. The greatest hurdles of chondrocyte-based cartilage bioengineering are: (i) preferring the cell source; (ii) differentiation and expansion processes; (iii) the time necessary for chondrocyte expansion pre-implantation; and (iv) fixing the chondrocyte count in accordance with the lesion surface area of the patient in question. The chondrocyte presents itself to be the focal starting material for research and development of bioengineered cartilage-based medicinal products which promise the regeneration and restoration of non-orthopedic cartilage joint defects. Even though chondrocytes seem to be the first choice, inevitable complications related to proliferation, dedifferentation and redifferentiation are probable. Detailed studies are a necessity to fully investigate detailed culturing conditions, the chondrogenic strains of well-defined phenotypes and evaluation of the methods to be used in biomaterial production. Despite a majority of the current methods which aid amelioration of joint functionality, they are insufficient in fully restoring the natural structure and composition of the joint cartilage. Hence current studies have trended towards gene therapy, mesenchymal stem cells and tissue engineering practices. There are many studies addressing the outcomes of chondrocytes in the clinical scene, and many vital biomaterials have been developed for structuring the bioengineered cartilage. This study aims to convey to the audience the practical significance of chondrocyte-based clinical applications.

A CD34+ Cell Enrichment Protocol of Hematopoietic Stem Cells in a Well-Established Quality Management System.

Allogeneic stem cell transplantation applications have improved tremendously over the past quarter of a century. The use of new immunosuppressive protocols and elimination of T cells by CD34+ cell enrichment or T cell depletion on apheresis products increases the chance of using partially matched or haploidentical grafts. This is without increasing the risk of graft-versus-host disease, which is observed as a major complication of hematopoietic stem cell transplantation. The aim of this protocol is to evaluate the results obtained from 6 different process cycles performed on 6 different days. We used the CliniMACS Plus system located in our Cell and Tissue Manufacturing Center Quality Control Unit which is already calibrated as a class D room and includes a class A microbiological safety cabinet inside. The average purity of the end products was 95.66%, excluding only one end product which was 70%; this was higher than the values in current studies in the field. Superior to the reported studies, the CD3 quantity in each end product was below the dedicated thresholds. BactecTM FX40 blood culture system test results were detected as negative for each end product. Endotoxin testing suggested the absence of endotoxin within the products. The consistent outcomes obtained from these 6 different process cycles confirmed that the CliniMACS® Plus process cycles performed in accordance with our well-defined quality management system procedure is sufficient for the routine application of high-quality and safe CD34+ enrichment processes within our clean room area.

Cellular therapies: Day by day, all the way.

Tremendous effort and knowledge have elucidated a new era of 'cellular therapy,' also called "live" or "living" drugs. There are currently thousands of active clinical trials that are ongoing, seeking hope for incurable conditions thanks to the increased accessibility and reliability of gene editing and cellular reprogramming. Accomplishments in various adoptive T cell immunotherapies and chimeric antigen receptor (CART) T cell therapies oriented researchers to the field. Cellular therapies are believed to be the next generation of curative therapeutics in many ways, the classification and nomenclature for these applications have not yet reached a consensus. Trends in recent years are moving towards making tissues and cell processes only in centers with production permits. It is quite promising that competent authorities have increased licensing activities of tissue and cell establishments in their countries, under good practice (GxP) rules, and preclinical and clinical trials involving cell-based therapies have led to significant investments. Despite the initiatives undertaken and the large budgets that have been allocated, only limited success has been achieved in cellular therapy compared to conventional drug development. Cost, and cost effectiveness, are important issues for these novel therapies to meet unmet clinical needs, and there are still many scientific, translational, commercializational, and ethical questions that do not have answers. The main objectives of this review is to underline the current position of cellular therapies in research, highlight the timely topic of immunotherapy and chimeric antigen receptor (CAR) T-cell treatment, and compile information related to regulations and marketing of cellular therapeutic approaches worldwide.

An Overview of the Orphan Medicines Market in Turkey.

BACKGROUND: The health policies of many countries and regions have already defined orphan drugs for rare diseases. Although there is no official definition of orphan drugs in Turkey, all orphan drugs are covered by reimbursement, regardless of their market authorization status. Thus, a pharmacoeconomic analysis does not have to be presented to the Social Security Institution (Sosyal Güvenlik Kurumu) for reimbursement decisions on orphan drugs. OBJECTIVE: The aim of this study was to shed light on the use of orphan drugs to aid classifications of rare diseases and assessments of orphan drugs in Turkey. METHODS: Data for sales of authorized orphan drugs and all other drugs were extracted from the IMS Turkey for 2008, 2009, and 2010. Nonauthorized orphan drug sales data were extracted from records of the Turkish Pharmacists' Association for the same years. Government prices were obtained from the Sosyal Güvenlik Kurumu. RESULTS: The European Medicines Agency has classified more than 60 orphan drugs for rare diseases. Of these, 50 entered the Turkish market in recent years, half of which were authorized. The remaining drugs were imported through the early access procedure. Antineoplastic agents accounted for the largest percentage of orphan drugs, with 58% of the total market share. In 2010, there were 18 such agents in use, at a cost of €120 million. CONCLUSIONS: Although legislation is not yet in place for orphan drugs in Turkey, recognized pricing and reimbursement policies are in operation. This situation facilitates an analysis of orphan drug prices and reimbursement policies in Turkey.