Monitoring EDTA process residuals in recombinant protein manufacturing using liquid chromatography

We have developed a chromatographic method for the high sensitivity quantitation of EDTA process residuals in recombinant protein manufacturing validation studies. The reversed-phase HPLC method is based upon the detection of Cu(2+)/EDTA complexes at 254 nm, and has been qualified for use on intermediates from a purification process for a recombinant protein expressed in E. coli. Quantitation of EDTA in recombinant protein process intermediates is linear in the range of 0.2 to 64 microM with LOD/LOQ values below 2.0 microM. The assay is suitable for use in process backgrounds containing Tris, HEPES, MES, NaCl, hexanediol, NH(4)SO(4), and PEG. EDTA spike recovery values in all process samples tested were greater than 90% at the 4.0 microM concentration. System suitability parameters for the chromatographic method were developed based upon peak area and retention time precision, column efficiency and USP tailing. Peak area precision and intermediate precision values across the linear range of the assay exhibited C.V. values less than 15% at any concentration tested in all sample backgrounds. The assay robustness was tested by transfer of the assay to a second laboratory and analyst with use of multiple process intermediate lots, reagent/column lots, and HPLC systems.     

麻疹疫苗生产工艺的改进研究

对麻疹疫苗生产工艺进行了改进研究,在病毒培养过程中,以病毒生长稳定剂替代白蛋白,减少维持液加量,延长病毒培养时间并缩短病毒释放时间,提高了病毒原液的滴度,由此可使分装量减少,而每一剂量中实际病毒含量并不减少,从而使麻疹疫苗的冻干条件得到改善,采用改进工艺后,麻疹疫苗的质量,中间产品和成品合格率均有较大的提高。     

Characterisation of an industrial affinity process used in the manufacturing of digoxin-specific polyclonal Fab fragments

This paper describes the effect of several variables on the affinity process for the production of the FDA approved biotherapeutic product Digoxin Immune Fab (Ovine) (DigiFab, Protherics Inc., TN, USA). The study considers the effects of column re-use on matrix capacity and on the subsequent recovery of the antibody product, and the impact of varying column loading on matrix performance. The methodology used could be equally applied to assess the feasibility of using an affinity matrix for commercial scale purification of alternative antibody derived biotherapeutics. The capacity and specific Fab recovery were calculated through 24h equilibrium and mass balance studies. Results were assessed against data obtained through confocal scanning laser microscopy. Scale-down experiments produced specific Fab recoveries and purities that were comparable with those at production scale. The matrix capacity was found to be 45+/-15 mg of Fab/ml of matrix. Through the use of fluorescent DigiFab and confocal scanning techniques, Fab uptake onto single affinity bead was evaluated. Average intensity values calculated for each sample provided direct real-time, measure of Fab binding and matrix capacity. The results suggest that the affinity matrix had a limited reuse life as a drop in recovery is observed following the completion of a small number of process cycles (30% after three runs). The findings support that which is seen at the current manufacturing scale, where the affinity column is used for a limited number of runs. Results from this study can be used as a basis for future optimisation of this purification process.     

双抗夹心ELISA法在无细胞百日咳疫苗生产质控中的应用

实验中对无细胞百日咳疫苗的脱毒工序优化后,采用双抗夹心ELISA法来检测百日咳有效组分含量,同时采用效价试验方法来验证结果。ELISA法定量测定有效成分的结果和效价试验结果均证明达到《中国药典》三部2005版的要求。由于双抗夹心ELISA法特异性强,灵敏度高,适用于无细胞百日咳疫苗生产各个环节的质量控制。     

Mixing in process vessels used in biopharmaceutical manufacturing

The use of nonbaffled vessels for mixing applications is becoming common in the biopharmaceutical industry but is not sufficiently well studied. Orientation of the impellers off-centered and/or at an angle is necessary to enhance mixing and eliminate swirling that would result without a baffle in a standard tank. This study focuses on characterizing mixing in vessels with the hydrofoil axial flow impellers mounted off-center at 10 degrees to the vertical. Geometrically similar vessels ranging from 100 to 5000 L working volume were used in this study. Mixing performance was successfully correlated to vessel geometric factors.     

Tumor-infiltrating lymphocytes: Streamlining a complex manufacturing process

Adoptive cell therapy of tumor-infiltrating lymphocytes has shown promise for treatment of refractory melanoma and other solid malignancies; however, challenges to manufacturing have limited its widespread use. Traditional manufacturing efforts were lengthy, cumbersome and used open culture systems. We describe changes in testing and manufacturing that decreased the process cycle time, enhanced the robustness of critical quality attribute testing and facilitated a functionally closed system. These changes have enabled export of the manufacturing process to support multi-center clinical trials.     

Fully integrated downstream process to enable next-generation manufacturing

Next-generation manufacturing (NGM) has evolved over the past decade to a point where large biopharmaceutical organizations are making large investments in the technology and considering implementation in clinical and commercial processes. There are many well-considered reasons to implement NGM. For the most part, organizations will not fund NGM unless the implementation benefits the funding organization by providing reduced costs, reduced time, or additional needed capabilities. Productivity improvements gained from continuous purification are shown in this work, which used a new system that fully integrates and automates several downstream unit operations of a biopharmaceutical process to provide flexibility and easy implementation of NGM. The equipment and automation needed to support NGM can be complicated and expensive. Biopharmaceutical Process Development considered two options as follows: (1) design its own NGM system or (2) buy a prebuilt system. PAK BioSolutions offers a turn-key automated and integrated system that can operate up to four continuous purification stages simultaneously, while maintaining a small footprint in the manufacturing plant. The system provides significant cost benefits (~10× lower) compared with the alternative—integration of many different pieces of equipment through a Distributed Control System that would require significant engineering time for design, automation, and integration. Integrated and Continuous Biomanufacturing can lead to significant reductions in facility size, reduced manufacturing costs, and enhanced product quality when compared with the traditional batch mode of operation. The system uses new automation strategies that robustly link unit operations. We present the optimized process fit, sterility and bioburden control strategy, and automation features (such as pH feedback control and in-line detergent addition), which enabled continuous operation of a 14-day end-to-end monoclonal antibody purification process at the clinical manufacturing scale.     

Pellet manufacturing by extrusion-spheronization using process analytical technology

The aim of this study was to investigate the phase transitions occurring in nitrofurantoin and theophylline formulations during pelletization by extrusion-spheronization. An at-line process analytical technology (PAT) approach was used to increase the understanding of the solid-state behavior of the active pharmaceutical ingredients (APIs) during pelletization. Raman spectroscopy, near-infrared (NIR) spectroscopy, and X-ray powder diffraction (XRPD) were used in the characterization of polymorphic changes during the process. Samples were collected at the end of each processing stage (blending, granulation, extrusion, spheronization, and drying). Batches were dried at 3 temperature levels (60°C, 100°C, and 135°C). Water induced a hydrate formation in both model formulations during processing. NIR spectroscopy gave valuable real-time data about the state of water in the system, but it was not able to detect the hydrate formation in the theophylline and nitrofurantoin formulations during the granulation, extrusion, and spheronization stages because of the saturation of the water signal. Raman and XRPD measurement results confirmed the expected pseudopolymorphic changes of the APIs in the wet process stages. The relatively low level of Raman signal with the theophylline formulation complicated the interpretation. The drying temperature had a significant effect on dehydration. For a channel hydrate (theophylline), dehydration occurred at lower drying temperatures. In the case of isolated site hydrate (nitrofurantoin), dehydration was observed at higher temperatures. To reach an understanding of the process and to find the critical process parameters, the use of complementary analytical techniques are absolutely necessary when signals from APIs and different excipients overlap each other. Published: September 30, 2005     

Monitoring cellular state transitions in a production-scale CHO-cell process using an electronic nose

An electronic nose is used to monitor the bioreactor off-gas composition in perfused cultivations of a CHO-cell line producing recombinant human blood coagulation factor VIII. The applicability of the electronic nose for monitoring cellular state transitions and process control is explained. It is shown that the instrument can reveal characteristic process states related to product and lactate formation, and detect microbial infections in a very early stage of the infection. The visualization of ideal process conditions is realized by using principal component analysis (PCA) and the on-line applicability of this method is outlined. The results illustrate the potential of the electronic nose as on-line sensor for ensuring product and process quality in production-scale bioprocesses.     

Developing lisocabtagene maraleucel chimeric antigen receptor T-cell manufacturing for improved process,product quality and consistency across CD19+ hematologic indications

Background aimsAutologous chimeric antigen receptor (CAR) T-cell therapies have demonstrated substantial clinical benefit across several hematologic malignancies. However, patient-to-patient variability and heterogeneity of starting cellular material across patient populations and disease indications pose challenges to manufacturing consistency. Lisocabtagene maraleucel (liso-cel) is an autologous, CD19-directed, defined-composition, 4-1BB CAR T-cell product administered at equal target doses of CD8⁺ and CD4⁺ CAR⁺ T cells. Here the authors describe the optimization of the liso-cel manufacturing platform for product quality and consistency.MethodsLeukapheresis starting materials were collected from patients with large B-cell lymphoma, mantle cell lymphoma or chronic lymphocytic leukemia treated with liso-cel in clinical trials (NCT02631044 and NCT03331198). The liso-cel manufacturing process involves selection of CD8⁺ and CD4⁺ T cells from leukapheresis material followed by independent CD8⁺ and CD4⁺ T-cell activation, transduction, expansion, formulation and cryopreservation. Multivariate design of experimental approaches was utilized to optimize process conditions at both specific unit operations and across the process. Flow cytometry methods were used to assess cellular composition, memory phenotypes and cell proliferation. Antigen-specific functions, including cytokine secretion, cytolytic activity and proliferation, were assessed using endpoint assays after independent stimulation of CD8⁺ and CD4⁺ CAR⁺ T-cell product components.ResultsReductions in process duration time, optimization of drug product container and formulation and activation signal optimization led to significantly increased CAR⁺ T-cell product viability. The heterogeneity of patient-derived starting material, including low absolute lymphocyte counts in some samples, was reduced through early T-cell purification, leading to median T-cell frequencies >95% in selected materials across disease indications and limited non-T-cell impurities. These changes further increased lineage purity in CD8⁺ and CD4⁺ CAR⁺ T-cell drug products. CD8⁺ and CD4⁺ CAR⁺ T-cell component lot functional profiles demonstrated multifunctional mechanisms of action, including differential cytokine release, differential cytolytic kinetics and high frequencies of proliferating cells. Correlative analyses demonstrated strong underlying associations between starting material attributes and final CAR⁺ T-cell product phenotype.ConclusionsDespite substantial heterogeneity of starting leukapheresis material quality/composition between individual patients and across disease indications/histologies, the liso-cel manufacturing platform is robust and capable of generating a consistent drug product from diverse starting materials with a single manufacturing platform.     

An optimized process for manufacturing anAzospirillum inoculant for crops

Summary An optimized process for manufacturing a crop moculant was developed with anAzospirillum lipoferum strain. This process involves the entrapment of living cells in alginate beads and dehydration. The influence of several parameters, alginate concentration, additions of adjuvants at different stages, dilution of culture broth, water activity and dehydration method on bacterial survival is presented. The highest survival was obtained by addition of skim milk and controlled air-dehydration of the alginate beads. Finally, a powdered inoculant was obtained, containing more than 10 billion cells/g, easy to store and to handle, which can be used in the field as a microgranule or as a seed coating. Furthermore, the biodegradability insures that there is no environmental pollution.     

Liposomes: an overview of manufacturing techniques

During the last few decades liposomes have attracted great interest as ideal models for biological membranes as well as efficient carriers for drugs, diagnostics, vaccines, nutrients and other bioactive agents. The extensive and ever increasing literature covering the field of liposomology written by researchers with diverse backgrounds is an indication of increasing interest in this field. Many techniques and methodologies have evolved for the manufacture of liposomes, on small and large scales, since their introduction to the scientific community around 40 years ago. This article intends to provide an overview of the advantages and disadvantages of liposome preparation methods in general with particular emphasis on the heating method, developed in our laboratory, as a model technique for fast production of the lipid vesicles.     

Leukapheresis guidance and best practices for optimal chimeric antigen receptor T-cell manufacturing

Chimeric antigen receptor (CAR) T-cell therapy is an individualized immunotherapy that genetically reprograms a patient's T cells to target and eliminate cancer cells. Tisagenlecleucel is a US Food and Drug Administration-approved CD19-directed CAR T-cell therapy for patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia and r/r diffuse large B-cell lymphoma. Manufacturing CAR T cells is an intricate process that begins with leukapheresis to obtain T cells from the patient's peripheral blood. An optimal leukapheresis product is essential to the success of CAR T-cell therapy; therefore, understanding factors that may affect the quality or T-cell content is imperative. CAR T-cell therapy requires detailed organization throughout the entire multistep process, including appropriate training of a multidisciplinary team in leukapheresis collection, cell processing, timing and coordination with manufacturing and administration to achieve suitable patient care. Consideration of logistical parameters, including leukapheresis timing, location and patient availability, when clinically evaluating the patient and the trajectory of their disease progression must be reflected in the overall collection strategy. Challenges of obtaining optimal leukapheresis product for CAR T-cell manufacturing include vascular access for smaller patients, achieving sufficient T-cell yield, eliminating contaminating cell types in the leukapheresis product, determining appropriate washout periods for medication and managing adverse events at collection. In this review, the authors provide recommendations on navigating CAR T-cell therapy and leukapheresis based on experience and data from tisagenlecleucel manufacturing in clinical trials and the real-world setting.     

Enzymatic production of glycerol carbonate from by-product after biodiesel manufacturing process

Glycerol carbonate is one of the higher value-added products derived from glycerol. In this study, glycerol carbonate (GC) was synthesized by transesterification of glycerol and dimethyl carbonate (DMC) using Novozym 435 (Candida antarctica Lipase B) at various conditions. For the enzymatic production of GC, the optimum conditions were the amount of enzyme (75 g/L), DMC/glycerol molar ratio (2.00), reaction temperature (60°C) and organic solvent (acetonitrile). Experimental investigation of the effect of water content revealed that the conversion of GC was maximized with no added water. The addition of surfactant such as Tween 80 increased the GC conversion, which finally reached 96.25% under the optimum condition and with surfactant addition.     

Identification of an endosome-specific antigen.

We have used successive density gradient centrifugation with vesicles prepared from a human hepatoma Hep G2 post nuclear supernatant to obtain a highly enriched preparation of early endosomes. A monoclonal antibody (8E4) raised against this early endosome preparation recognizes a single polypeptide highly enriched in light vesicle membranes. The antigen has a molecular weight of 195 kDa by SDS-PAGE in the presence or absence of a reducing agent. Western blot analysis shows that the 8E4 antigen is detectable only in light vesicle membranes and not among heavy membranes, whole cytosol, or nuclear pellet proteins. The 8E4 antigen appears to be an integral membrane protein as it is precipitated by Triton X-114. The distribution of the 8E4 antigen in a Nycodenz density gradient fractionation of light vesicle membranes is identical to the distribution of 125I-ASOR-labeled early endosomes but distinct from the distribution of the plasma membrane enzyme, alkaline phosphodiesterase. In addition, incubation of cells with a horseradish peroxidase-transferrin conjugate followed by 3,3'-diaminobenzidine cytochemistry specifically quenches 8E4 antigen detection by protein dot blot analysis. These data strongly suggest that the 8E4 antigen is an integral membrane protein primarily located in endocytic vesicles.     

Monitoring of staphylococcal starters in two French processing plants manufacturing dry fermented sausages

AIMS: The growth and survival of Staphylococcus xylosus and Staphylococcus carnosus were monitored during sausage manufacture in two processing plants. METHODS AND RESULTS: The gram-positive, catalase-positive cocci isolated from the processing plants F10 and F11 were identified by Staphylococcus-specific PCR and species-specific oligonucleotide array. In the inoculated products with starter cultures, 90% of staphylococcal strains isolated in F10 were identified as S. xylosus and 10% as S. carnosus. In F11, 77% were identified as S. xylosus and 20% as S. carnosus. Staphylococcus xylosus dominated the staphylococcal microbiota while S. carnosus survived during the process. The pulse-field gel electrophoresis analysis revealed that all S. xylosus and S. carnosus strains isolated corresponded to the starter strains inoculated. The two starter strains of S. xylosus co-dominated in the isolates from sausages of F11, whereas the strain with pattern A1 was dominant in the isolates from sausages of F10. In the environments, no S. carnosus and S. xylosus were found, whereas Staphylococcus equorum and Staphylococcus saprophyticus were the main species isolated. CONCLUSIONS: This work highlighted the domination of S. xylosus starter strains, which showed a strong capacity to grow during sausage process, while S. carnosus survived during the process. SIGNIFICANCE AND IMPACT OF THE STUDY: Successful implantation of starter cultures is obviously a prerequisite for their contribution to sensorial qualities. Thus, the monitoring of the growth and the survival of S. xylosus and S. carnosus are required to guarantee a well-adapted starter culture. This study revealed that the two Staphylococcus species are suitable for manufacturing sausages in processing plants with very different capacities of production.