BioProcessing Journal

Characterization of a Lyophilized Immunoaffinity Chromatography Matrix Employed to Purify Hepatitis B Surface Antigen for Pharmaceutical Use

Immunoaffinity chromatography is an indispensable purification tool. However, its use has been limited by cost, purification cycle numbers, and storage requirements. Therefore, authors speculated that a possible solution to these problems could be CB.Hep-1 monoclonal antibody (mAb)-immunosorbent lyophilization. This study sought to assess the impact of the CB.Hep-1 mAb quantification by enzyme-linked immunoadsorbent assay and the CB.Hep-1 mAb-immunosorbent lyophilization process for its impact on hepatitis B virus surface antigen purification for pharmaceutical use. Study results found that CB.Hep-1 mAb lyophilization did not affect mAb purity and antigen recognition capacity. CB.Hep-1 mAb-immunosorbent lyophilization did not modify volume-weight factor, infrared spectrum, particle-size distribution, particle density and viscosity, antigen adsorption capacity, antigen elution capacity, antigen recovery, antigen purity, gamma immunoglobulin (IgG) leakage, and purification cycle number. Therefore, the lyophilized CB.Hep-1 mAb and CB.Hep-1 mAb-immunosorbents can be successfully used for hepatitis B vaccine production…

Analytics Biologics Production

cb.hep-1 gamma immunoglobulin hepatitis b vaccine igg immunoadsorbent assay immunoaffinity chromatography lyophilization mab mab monoclonal antibody

Statistical Verification that One Round of Fluorescence-Activated Cell Sorting (FACS) Can Effectively Generate a Clonally-Derived Cell Line

Mammalian cell line generation is foundational to protein-based therapeutic product development. Ensuring production cell line “clonality” (i.e., clonal derivation of the production cell line) is a requirement for product registration. Many single-cell deposition approaches have been developed since the inception of traditional limiting-dilution. However, regulatory expectations, in line with the capability of this original cloning technique, still lead many institutions to either execute multiple rounds of single-cell plating or conduct extensive screening immediately post-cloning to demonstrate the single-cell origin of a production cell line. Using fluorescence-activated cell sorting (FACS), we have developed a strategy to verify single-cell deposition that, when combined with statistical analysis, creates a highly-effective and efficient process for generating clonally-derived production cell lines in just one round of plating…

Analytics Biologics Production

clonality cloning techniques facs fluorescence-activated cell mammalian cells protein-based therapeutics statistical analysis

Use of Interactive Laser-Scanning Imaging Cytometry (ILIC) for Real-Time Cytotoxicity Assessment of Bacteriocins Against Cultured Mammalian Cells

The evaluation of bacteriocin cytotoxicity is a critical first step in guaranteeing its safe use in food and therapeutic applications. In this study, the bacteriocins nisin, pediocin, and colicin ([Col] E1, E3, E6, E7, and K) were evaluated for cytotoxicity against cultured mammalian cells. Cellular membrane potential (delta psi) changes of bacteriocin-treated cells were used as an index of cytotoxicity. Simian virus 40-transfected human colon (SV40-HC) cells and African green monkey kidney epithelial cells (Vero) cells were cultured (35°C, 10% CO2, humidified air) as monolayers on tissue culture plates. Log phase cells (~104 cells/mL) were treated with partially purified, individual bacteriocin preparations at 170, 350, and 700 activity units (AU)/mL…

Analytics Biologics Production

analytics bacteriocin cytotoxicity bacteriocins biologics production colicin cytometry cytotoxic events ILIC interactive laser cytometry mammalian cell culture pediocin SV40 Vero cells

Counting and Processing Methods Impact Accuracy of Adipose Stem Cell Doses

Cell therapy products derived from adipose tissue have some unique processing issues with regard to obtaining accurate cell counts. This is because processing methods may not only show us the nucleated stromal vascular fraction (SVF) cells but also the micellular and microvesicle particles. This is true for both veterinary and human clinical products, and poses special concerns for in-clinic processing where the cell therapy dose is correlated with cell numbers and other QC data is not especially useful. In this study, multiple cell counting methods were compared for SVF cell preparation that were derived from canine adipose tissue using commercially-available processing kits. The data clearly showed that many non-nucleated particles appear cell-like by size and shape, and can lead to counting errors with automated counters. In addition, certain reagents important to processing can have properties wherein the reagents alone (e.g., lecithin) may be counted as cells. The most accurate cell numbers were from hemocytometer-counting of cells stained with 4´,6-diamidino-2-phenylindole (DAPI) which shows the nuclei in concert with a viability stain such as trypan blue. The data clearly showed that care must be taken when counting cells used as a therapeutic dose…

Analytics Biologics Production

adipose tissue cells analytics biologics production cell counting cell gene therapy cell therapy dose cellular therapies dapi hemocytometer counting micellular micellular particles microvesicle microvesicle particle stromal vascular fraction SVF

Large-Scale Production and Purification of a Lentiviral Vector Reference Material

Lentiviral vectors (LVV) are widely used in an increasing number of approved cell and gene therapies, and benchmarking is important in evaluating production processes and products. We have produced a lentiviral vector reference material (LVV-RM) that expresses the common marker gene (green fluorescent protein [GFP]) to enable data comparisons and support LVV research programs. To generate that reference material, we have applied our previously developed fed-batch process to a 100 L production using an inducible LVV-producer cell line. The LVV material was harvested three days post-induction from a 200 L single-use bioreactor. A downstream purification process was also developed and scaled-up to meet production requirements: It consisted of nuclease digestion, clarification by depth filtration, chromatography capture using CIM-QA monolith anion exchangers, and ultrafiltration/diafiltration using a hollow fiber membrane prior to bulk-filling the final product. A total of 4.4 L of diafiltered and concentrated LVV product was obtained (9.2 E10 particles/mL, 3.6 E7 TU/mL) and stored at -80°C. Nearly 8000 vials are now available to the LVV community via the American Type Culture Collection (ATCC).

Analytics Biologics Biologics Production Cell & Gene Therapy Chromatography Manufacturing

bioprocessing bioreactor downstream purification fed-batch process HEK293 suspension cell-line lentiviral vector LVV-RM LVVRM reference material scale-up stable producer clone

Breaking the DoE Bottleneck: How Definitive Screening Designs Enable Faster, Cheaper Biologics Process Development

Efficient bioprocess characterization is essential for both regulatory compliance and commercial viability of biologics. Traditional approaches using resolution III/IV screening designs followed by response surface methodology are time-consuming, costly, and not always effective in identifying the important experimental effects. Definitive screening designs (DSDs) represent a novel class of three-level screening designs that can simultaneously evaluate main effects and quadratic relationships. While DSDs are increasingly used in bioprocess development, practical implementation guidelines remain limited. This case study bridges this gap by introducing a model-based framework to identify critical process parameters (CPPs) and optimize operating ranges for robust biologics production using plasmid DNA (pDNA). Minimal 14-run DSDs evaluated six input parameters and successfully identified CPPs and optimal operating ranges. This approach reduces experimental requirement by >50% compared to traditional designs, providing an efficient and economical strategy for bioprocess characterization and optimization.

Analytics Biologics Biologics Production Manufacturing

analytics biologics production boston institute technology cell gene therapy design-of-experiments doe e coli e. coli fermentation pDNA process characterization process development process optimization upstream development

In Situ Hybridization: The Importance of Ultrapure Water for RNA Technologies

In situ hybridization (ISH) for localization of DNA/RNA hybrids in cytological preparations was first described in 1969 by Gall and Pardue. This method enables mRNA transcripts to be detected in tissue sections. Unlike expression analyses based on polymerase chain reactions, the exact localization of the target transcripts can be identified within the tissue…

Biologics Cell & Gene Therapy Manufacturing

arium pro vf in situ hybridization laboratories rna technologies sartorius target transcripts ultrapure water

Biopharmaceutical Manufacturing: Historical and Future Trends in Titers, Yields, and Efficiency in Commercial-Scale Bioprocessing

This article documents the progress, current state, and projected future trends in titer and yield as industrial and technological benchmarks for commercial-scale biopharmaceutical manufacture. Biopharmaceutical product commercial-scale manufacturing (bioprocessing) was benchmarked by tracking titers and yields over time, from the 1980s to the present, and further out ten years. This study compiled commercial-scale titer and yield data for a set of 39 major biopharmaceuticals, nearly all mammalian-expressed proteins, particularly, monoclonal antibody products. This included extensive searches of many potential data sources, including contacting knowledgeable bioprocessing professionals. In the 1980s and early 1990s, average titers at commercial scale started out at < 0.5 g/L. The current average reported commercial-scale titer is 2.56 g/L. We also confirmed that the manufacture of commercial products has, over the years, undergone repeated cycles of technical production upgrades, with titers and yields increasing incrementally, even for the oldest products. BioPlan estimates that ?3 g/L is now the industry standard titer for new bioprocesses being developed, with ?7 g/L now presumed to be the general industry top-end titer level that, while not unusual, is not often achieved. In terms of yields, we found a 70% yield to be the current industry average yield, not the often-cited 75%. Improvements in downstream purification technologies (e.g., as demonstrated by higher yields) have been fewer and adopted more slowly than upstream production.

Biologics

biomanufacturing biopharmaceuticals titer levels yields

Characterization and Optimization of Biologics Manufacturing Using Space-Filling Designs and Machine Learning

This study assessed a novel statistical approach using space-filling designs (SFDs) and self-validating ensemble modeling (SVEM) machine learning to efficiently identify key process factors using recombinant adeno-associated virus type 9 (rAAV9) gene therapy manufacturing as a case study. Based on risk assessment of parameters that may impact rAAV9 production, we have evaluated six process parameters using 24-run SFDs generated by the JMP statistical software. SFDs are a new class of design of experiment (DoE) created with the objective of covering the entire design space as completely as possible; this in turn allows more accurate modeling of complex response surface behavior typically found in bioprocesses.

Analytics Biologics Biologics Production HEK293 Manufacturing Process Automation

ambr 15 benchtop bioreactor mimic boston institute technology design-of-experiments doe machine learning microbioreactor process development sartorius space-filling designs

Streamline Your Cell Line Screening: With the Automated, Microscale, Stirred Tank, Single-Use Bioreactor

The price per patient for protein-based and monoclonal antibody (mAb) therapies runs into thousands of dollars per patient each year. These therapies cost considerably more to manufacture than small molecules. Hence, if mammalian or insect cell lines expressing high protein titres can be selected and optimized for protein expression using microscale bioreactor models early in development, then manufacturing costs can be reduced significantly…

Analytics Biologics Biologics Production Cell Lines Manufacturing Process Automation Regulatory

ambr15 benchtop bioreactor mimic cell line development cell line screening mab media development microbioreactor monoclonal antibody Process Automation sartorius stirred tank

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