Showing papers in "Pharmaceutical bioprocessing in 2013"

Developability assessment as an early de-risking tool for biopharmaceutical development

Abstract: Increasing attrition of therapeutic candidates during preclinical and clinical development affects productivity and causes spiraling costs, negatively impacting the development of new treatments. For biopharmaceuticals, product design, lead selection and manufacturing process development constitute significant areas of risk because of their decisive influence on product quality, biological activity and safety, as well as cost of goods. Risk-management developability assessments, introduced early on in development, can help identify and address potential causes of attrition in preclinical and clinical stages related to product manufacturing, safety, delivery and efficacy issues. This article discusses the utilization of in silico and in vitro surrogate assays early on in development as part of a comprehensive developability assessment for novel biotherapeutics, incorporating a closer interaction between discovery and development functions. It further suggests how such approaches can have a significant impa...

Production and purification of the multifunctional enzyme horseradish peroxidase.

Abstract: The oxidoreductase horseradish peroxidase (HRP) is used in numerous industrial and medical applications. In this review, we briefly describe this well-studied enzyme and focus on its promising use in targeted cancer treatment. In combination with a plant hormone, HRP can be used in specific enzyme–prodrug therapies. Despite this outstanding application, HRP has not found its way as a biopharmaceutical into targeted cancer therapy yet. The reasons therefore lie in the present low-yield production and cumbersome purification of this enzyme from its natural source. However, surface glycosylation renders the recombinant production of HRP difficult. Here, we compare different production hosts for HRP and summarize currently used production and purification strategies for this enzyme. We further present our own strategy of glycoengineering this powerful enzyme to allow recombinant high-yield production in Pichia pastoris and subsequent simple downstream processing.

The impact of continuous multicolumn chromatography on biomanufacturing efficiency

Abstract: Over the past few years, the capacity bottleneck in biomanufacturing has shifted from the upstream process to the purification process. In particular, capture chromatography steps have become a limiting factor in facility throughput. This is a direct result of the discrepancy between expression levels, which have increased by a factor of ten, and the capacity of biochromatography media, which has at best doubled. Continuous countercurrent multicolumn chromatography is a promising approach to deal with the capacity bottleneck, particularly if designed in a single-use format. BioSMB™ technology is a disposable bioprocess technology that allows standard capture chromatography steps, such as Protein A capture, to be executed with much less installed chromatographic media using a multicolumn chromatography process. This allows an economically favorable, single-use option where none has previously been available. Various case studies have demonstrated the advantages of this technology for various separations. I...

Generation of monoclonal antibody-producing mammalian cell lines

Abstract: Clinical effectiveness has driven the commercial success of monoclonal antibody (mAb) products. Mammalian cells are currently the preferred system for large-scale production as the mAbs produced are biochemically similar to human forms. The cell line generation process is tedious and time-consuming as clones with high productivity, stable long-term expression and good product quality are rare occurrences. Cell line generation efficiency and mAb quality can be improved through host cell engineering, vector optimization and high-throughput clone selection. Targeted integration into predetermined sites on the chromosome is a promising new area being explored. This review covers existing technology and recent progress made in improving various aspects of the stable cell line generation process for mAb production.

The use of glutamine synthetase as a selection marker: recent advances in Chinese hamster ovary cell line generation processes

Abstract: Central to the needs of the biopharmaceutical industry to support the development of innovative biologics and biosimilars are more effective and efficient manufacturing processes which require highly productive cell lines with desired quality attributes. New designs of molecules such as antibody humanization have greatly reduced immunogenicity concerns, and advances in cell culture technology including media optimization and process control have driven monoclonal antibody productivities in excess of 10 g/l with peak cell densities in bioreactors climbing to over 35 million per ml. However, over this same timeframe, the fundamental processes utilized for cell line generation have not changed significantly, especially in the selection step for top-producing clonal cell lines. Cell line generation continues to be time consuming and labor intensive and has become the timeline limiting step for the majority of the industry. In order to meet the ‘Fast-to-Proof-of-Principle’ strategy, multiple efforts including ...

Emerging technologies for the integration and intensification of downstream bioprocesses

Abstract: Downstream processing is currently the major bottleneck for bioproduct generation. In contrast to the advances in fermentation processes, the tools used for downstream processes have struggled to keep pace in the last 20 years. Purification bottlenecks are quite serious, as these processes can account for up to 80% of the total production cost. Coupled with the emergence of new classes of bioproducts, for example, virus-like particles or plasmidic DNA, this has created a great need for superior alternatives. In this review, improved downstream technologies, including aqueous two-phase systems, expanded bed adsorption chromatography, convective flow systems, and fibre-based adsorbent systems, have been discussed. These adaptive methods are more suited to the burgeoning downstream processing needs of the future, enabling the cost-efficient production of new classes biomaterials with a high degree of purity, and thereby hold the promise to become indispensable tools in the pharmaceutical and food industries.

Quality by design for biopharmaceuticals: a historical review and guide for implementation

Abstract: This article reviews the history of quality-by-design (QbD), how this concept has been applied to biopharmaceuticals, and what can be expected from implementation of QbD. Although QbD may lead to better design of products and manufacturing processes, and offers the potential for reduced regulatory compliance costs, it will likely increase development costs. Process developers will require additional skills and knowledge in the ‘quality disciplines’, which are not normally part of the training of those in biopharmaceutical process development. A model for implementing QbD in biopharmaceutical manufacture is proposed. The reader will gain an understanding of how QbD principles have been applied to the development of biopharmaceuticals, as well as learning of the potential drawbacks of applying QbD tools indiscriminately. Excellent examples of QbD applied to biopharmaceuticals in the literature will be highlighted and suggested as the direction for future development in this area.

The hidden potential of small synthetic molecules and peptides as affinity ligands for bioseparations

Abstract: While extensively used as drugs, small synthetic molecules have not yet been widely applied in the industry as affinity ligands for the purification of biopharmaceuticals. Yet, a substantial amount of published research indicates that synthetic ligands, such as triazine scaffolds, amino acids, and peptides show a great deal of promise for becoming the next generation affinity ligands for bioseparations. In this review, we present a comprehensive account on small synthetic ligands, from triazine dyes to the most recent polycyclic peptide ligands, selected for targeting high-value biopharmaceuticals, such as immunoglobulins, blood factors and therapeutic enzymes. These ligands could play a significant role in improving downstream processing and helping the bioprocessing industry overcome the urgent issues of costs and availability posed by growing economies on the global pharmaceutical market.

The use of Drosophila S2 cells in R&D and bioprocessing

Abstract: Drosophila Schneider 2 (S2) cells have been available for approximately 40 years. Their use has intensified over the past 15 years: resolution of the whole Drosophila melanogaster genome and the amenability of S2 cells for siRNA-based studies are some of the reasons for their growing use. This review covers recent publications on use of S2 cells for research and manufacturing and points to some possible future developments in their use in the vaccine field. Relatively few groups have systematically developed the system to enable expression of challenging proteins. They demonstrated that these cells can constitute a robust, efficient protein expression system, with specific advantages such as homogeneous glycosylation profile; reproducibility between production runs; options for cultivation modes, including perfusion; and no cell lysis, leading to relatively low levels of contaminating host cell proteins. The platform has shown to be particularly well adapted for the production of challenging viral, malaria and immunotherapy antigens.

Applying quality by design to glycoprotein therapeutics: experimental and computational efforts of process control

Abstract: Therapeutic glycoproteins represent one of the most important classes of products in the pharmaceutical industry, accounting for 77 high-value drugs out of 642 pharmaceuticals approved by the European Medicines Agency. Their therapeutic efficacy, serum half-life and immunogenicity depend on glycosylation, a complex and prominent post-translational event, which in turn is influenced by manufacturing process conditions. For this reason, protein glycosylation is a critical quality attribute for these drugs. Herein, we review the impact of glycosylation on product function, and the role of manufacturing conditions on the resulting glycoform distribution. We further present promising developments in terms of alternative, genetically engineered hosts, as well as advances in process operation that influence the glycan profile of the recombinant product. Finally, we review work on dynamic mathematical modeling for protein glycosylation that allows researchers to evaluate genetic engineering and process operation ...

3D scaffolds in tissue engineering and regenerative medicine: beyond structural templates?

Abstract: The objective of this article is to systematically present the emerging understanding that 3D porous scaffolds serve not only as structural templates for tissue fabrication but also provide complex signaling cues to cells and facilitate oxygen and therapeutic agent delivery. Strategies in the field of tissue engineering and regenerative medicine often rely on 3D scaffolds to mimic the natural extracellular matrix as structural templates that support cell adhesion, migration, differentiation and proliferation, and provide guidance for neo-tissue formation. In addition to providing a temporary support for tissue fabrication, 3D scaffolds have also been used to study cell signaling that best mimics physiological conditions, thereby expanding our understanding beyond 2D cell cultures. It is now understood that cell responses to 3D scaffolds are distinctively different from 2D surfaces. Recently, 3D scaffolds emerged as a vehicle for improved oxygen transport to seeded cells and also to deliver relevant therap...

Single-use bioreactors for microbial cultivation

Abstract: Single-use bioreactors are commonly used in the biopharmaceutical industry today, however, they are mostly limited to mammalian cell culture processes. For microbial processes, concepts including the CELL-tainer® technology provide comparable oxygen mass transfer such as in stirred tank reactors. Data obtained with 15 and 120 l working volumes indicate excellent performance with Escherichia coli and Corynebacterium glutamicum cultures. Therefore, this type of single-use bioreactor is applicable in biopharmaceutical processes, and also in a seed train for bulk chemicals production such as amino acid production. It is expected that single-use technologies will be applied ever more frequently in microbial-fed batch cultivation processes in combination with improved monitoring and control.

Innovation in biomanufacturing: the only way forward

Abstract: Innovations in the biomanufacturing industry are predominantly incremental in nature, but there are occasional major technological shifts that occur as existing technologies reach their limits and as additional constraints are imposed by market forces or regulatory bodies These disruptive innovations tend to have a seismic effect, forcing companies to adopt novel strategies and business models in order to remain competitive In this article, we discuss how technological innovation can address some of the current challenges in biopharmaceutical manufacturing by anticipating shifts in market dynamics and cost structures We consider bioprocessing standards, unit operations, best practices, enabling technologies and look at potential entry points for innovation where technologies are likely to evolve incrementally and where more disruptive, radical changes may flourish, including quality by design and process analytical technology We conclude that the biomanufacturing industry can only thrive in the changi

High expression of the aspartate–glutamate carrier Aralar1 favors lactate consumption in CHO cell culture

Abstract: Process performance of mammalian cell cultures can be strongly impacted by high lactate accumulation, which can be a clone or media-dependent characteristic. In this study, the expression of specific genes was measured in several Chinese hamster ovary cell lines under culture conditions leading to different lactate profiles. A reduced expression of two genes was observed under conditions of high lactate accumulation: AGC1/Aralar1, a member of the malate–aspartate shuttle (MAS) and Timm8a1. Overexpression of either of these two genes in the lactate-producing cell line diminished lactate accumulation. This was achieved by promoting a metabolic switch to lactate consumption after day 6, while maintaining a glycolytic rate similar to the parental cells. On the other hand, the biochemical inhibition of MAS activity increased lactate accumulation. All together, these results indicate MAS as a key factor to promote a shift to lactate consumption in cultivated Chinese hamster ovary cells.

Next-generation sequencing technologies and their potential impact on CHO cell-based biomanufacturing

Abstract: There is growing interest in the possibility of harnessing detailed, mechanistic understanding of the biology of CHO cells to enhance the use of these cells in the manufacturing of biologics. Among the important questions about CHO cells are issues related to productivity, product quality attributes and stability. The advent of next-generation DNA sequencing technologies provides an opportunity to characterize the genome of various host cells and to link genomic changes to phenotypes. In this review, we discuss some of the current and emerging technologies for genome sequencing, their initial applications to CHO bioprocessing, and provide context relative to other omic approaches.

Immunopharmacological Activity Of Saponin From Terminalia Arjuna And Prosopis Spicigera

Abstract: The aim of the present work was to examine the immunopharmacological (anti-microbial and anti-inflammatory) effect of saponin on human peripheral blood mononuclear cells (PBMC) that are isolated as well as purified from infected (virally) and non-infected (normal) human whole blood samples. However, there is little information available concerning the effect of crude saponin on human PBMC in case of infected and non-infected human PBMCs. Saponin, one of the members of secondary metabolites that are isolated from aqueous leaves extract of Terminalia arjuna and Prosopis spicigera. Our group evaluated the immunopharmacological activity of saponin (0.5–30 mg/ml, 100 μl) in human PBMC were determined and measured cell proliferation using Concanvalin (Con A, 2.5 μg/ml, 50 μl) and estimate Th1 (IFN-gamma and TNF alpha) type of cytokines from cell culture supernatant including nitric oxide production. The results showed that crude saponin from Terminalia arjuna and Prosopis spicigera showed its inhibitory activity at higher doses (30 mg/ml, 100 μl) in Con A induced PBMC (infected and non-infected) proliferation. In addition, these saponin also inhibits its Th1 (IFN-gamma and TNF alpha) type of cytokines from Con A stimulated cell culture supernatant of PBMC and also inhibits nitric oxide production in a dose dependent manner. In conclusion, saponin appears to have potential as an anti-microbial and anti-inflammatory agent.

Recent advances in biocatalyst development in the pharmaceutical industry

Abstract: Biocatalysts are increasingly employed as more efficient and environmentally safer alternatives to traditional chemical catalysts in the manufacturing of fine chemicals. This is driven by advances in recombinant DNA technology, protein engineering and bioinformatics, all of which are critical in the discovery, tailoring and optimization of enzymes for industrial processes. In this article we review these key technological innovations, as well as highlight the strategic application of these tools in the development of biocatalysts in the production of advanced pharmaceutical intermediates.

Deciphering O-glycomics for the development and production of biopharmaceuticals

Abstract: The functional impact of glycosylation on drug efficacy and safety profiles has been demonstrated in a wide range of biopharmaceuticals. Understanding of the N-glycosylation pathway and the advent of analytical technologies has enabled both detailed and rapid characterization of N-glycans, thus providing critical insights for regulatory compliance. In comparison, O-glycosylation encompasses several types of protein modifications by a more heterogeneous pool of sugars, and the understanding of its biology is less mature. Concomitantly, there exist several limitations in O-glycan analytical strategies, impacting both O-glycoprotein-based drug development and regulatory compliance. Here, we aim to provide a critical review of the biology and functional importance of O-glycosylation in the context of existing and potential O-glycoprotein drugs, as well as the corresponding analytical methods. The ultimate goal is to identify the gaps in current analytical methods and propose potential future directions for O-glycosylation analysis, to support the development and production of O-glycosylated biopharmaceuticals.

Comparability of biotherapeutics: characterization of protein vaccine antigens

Abstract: Advances in biotechnology and analytical tools now permit the application of extensive analytical characterization packages to purified recombinant proteins, a significant progression from the traditional characterization of complex biological products primarily by their manufacturing process. In this article, the authors focus on comparability assessment of biotherapeutics, specifically vaccine protein antigens. Regulatory drivers and analytical approaches used to examine product comparability are discussed and two case studies are described in detail to demonstrate the comparability of pre- and post-change product at the early and late stages of vaccine development. In coming years the number of comparability studies will likely increase due to the greater number of vaccine manufacturers, production at multiple sites and with external partners, and the introduction of innovative process technologies. Comparability studies may be focused on only a few changes, or may be more extensive, to address the impact of multiple process changes at various stages of manufacturing.

Scale-up and predictability in process development with suspension cultures of mammalian cells for recombinant protein manufacture: comments on a trend reversal

Abstract: In the past (1980–2000), ‘non-instrumented’ small-scale systems for the suspension culture of mammalian cells disappointed when used for the development of processes that needed to be applied in controlled stirred tanks. That is, cell growth and productivity of cells was usually better in the larger pH/oxygen-controlled reactors. In more recent years, data from cultures as small as 10 ml, in non-instrumented systems, are frequently better (higher yield, viability and cell density) than the ones seen from cultures in well-controlled bioreactors (volumetric yields for recombinant proteins from CHO cells in bioreactors in general have improved by 20–40-fold over the period of 25 years). Why is this? The commentary here tries to explain, at least partially, the observed trend reversal. Large-scale suspension cultures for the manufacture of recombinant protein therapeutics have been used since the early 1980s, with human recombinant tissue plasminogen activator being the first protein made at the 10,000 l scale by stably transfected and gene-amplified CHO [1]. A most important step towards largescale use of CHO cells was the generation of suspension-adapted subpopulations, derived from adherent cultures with serum. Development of media and generation of populations of cells in serum-free culture was executed in ‘spinner flasks’, unique glass bottles ranging in volume from 100 ml to up to 5 l [2–4]. The 500-ml spinner FLASK was the most popular, used with cell cultures of up to 200 ml. Spinner flasks exhibited a flat Teflon impellor with a magnet at its lower end. A ‘spinner-base’ transferred a magnetic force through the glass body of the spinner to the impellor [101]. Spinning rates of 30–50 rpm assured a gentle movement of the suspension culture. The bottles did not have baffles, for fear that too high a shear stress would be exerted. The headspace over the liquid was considered sufficient to supply oxygen to the cells. During the later phases of the culture, when higher densities were expected, the two access ports to the flask could be ‘cracked open’. Thus, diffusive entry and exit of gasses was provided along the space of the winding of the cap. In a warm room (37°C, no CO 2 ), available to one of the authors (Wurm), up to 40 flasks could be used for the screening and identification of favorable process conditions. Two aspects of these spinner bottles have held back efficient optimization of processes. First, running more than 30 spinners by a single operator was impossible, both in terms of generation of seed cultures (large volumes) and in terms of workload. Second, severe oxygen limitations for cell densities higher than 3 × 10 cells/ml was ‘overlooked’ or neglected (including by Wurm) for a long time. Attempts for ‘high-throughput’ cultures with disposable multiwell plates (6-/12-/24-well; static or slowly shaken) were unsatisfactory, mostly because of ‘edge Scale-up and predictability in process development with suspension cultures of mammalian cells for recombinant protein manufacture: comments on a trend reversal

Serum-free media: standardizing cell culture system

Abstract: The first attempts at culturing animal cells in vitro made use of biological fluids, such as serum or tissue extracts. It was in the 1950s that a scientific approach was adopted to determine the defined nutrients required for mammalian cell growth. The idea of a chemically defined media was pioneered by Eagle, who determined the minimum ingredients that were essential for the growth of a number of human cell lines. This led to the development of Eagle’s minimal essential media that consisted of 13 amino acids, 8 vitamins and 6 ionic species [1]. This formulation appeared to provide the requirements for the growth of a number of isolated cell lines if supplemented with animal-sourced serum. Higher cell densities were obtainable by increasing the component concentrations of Eagle’s minimal essential media and became established through basal formulations such as Dulbecco’s modification of Eagles medium (DMEM). Clonal cell growth of selected cell lines was obtained by enrichment with an enhanced range of nutritional components, largely through the early work of Ham to produce the well-known Ham’s F-12 medium, Sato had the ingenious idea of combining these two approaches to blend a basal media formulation – DMEM/F-12 – that has become widely used for the growth of multiple cell lines to high density [2]. However, despite the inclusion of up to 70 components in these well-defined basal media formulations, supplementation with dialyzed serum (~10%) is necessary to provide sustained growth of most cell lines.

Trends regarding viral barrier implementation in animal cell culture processes

Abstract: Over the past 20 years, a handful of large-scale viral contaminations have been reported along with various strategies, technologies and best practices for mitigation of adventitious viral and mycoplasma contaminations in upstream cell culture processes. Despite the available information, many uncertainties exist regarding implementation of viral barriers for upstream processes. Focusing primarily on high-temperature, short-time treatment of cell culture medium, this White Paper describes the current state of implementation of in-process, upstream viral barriers. It includes blinded data and insights from 12 leading biopharmaceutical companies, as well as six industry-leading consultants. Among 12 companies included in the study, a total of 24 high-temperature, short-time units are currently, or will soon be, in operation. The results generated are beneficial as a guide for viral risk-mitigation efforts, at both established and start-up biopharmaceutical companies.

Single-use technology supporting the comeback of continuous bioprocessing

Abstract: By many measures biotechnology recently celebrated its 30th anniversary. The current dominance of the fed-batch culture of suspension cells in manufacturing of biopharmaceuticals often obscures its robust history of process development. One interesting theme, for protein biologicals especially, is the waxing and waning of a variety of approaches to continuous processing. In fact, a number of implementations of this mode of production have long been in use by such well-known pharmaceutical manufacturers as Genzyme (Sanofi) and Centocor (Johnson & Johnson/ Janssen) to make highly successful products, such as Cerezyme® and Remicade®. Interest in continuous bioprocessing (CB) is growing of late due to a number of economic, technological and regulatory developments. Features provided by single-use bioproduction systems complement those provided by CB methods, and the number of operations supported by them is large and growing.

Plug and Play? Interconnected multifunctional chips for enhancing efficiency of biopharmaceutical R&D

Abstract: Gregory F Payne Fischell Department of Bioengineering & Institute for Bioscience & Biotechnology Research, University of Maryland, College Park, MD, USA “ one thinks of the entire drug discovery, development, production, testing and validation pipeline as an expanded bioprocess, one might consider next generation microfabricated devices as potential high content/high throughput vehicles that can change the paradigm – dramatically reduce cost and enhance ”

Virus-like particle bioprocessing: challenges and opportunities

Abstract: Rapid advances in the design and manufacture of structured materials will continue to see high levels of innovation, and the commercialization of new matrices tailored to virus-like particle purification.

Applications of genome-scale metabolic network models in the biopharmaceutical industry

Abstract: Biotechnology is currently evolving through the era of big data, thanks to advances in the high-throughput technologies for rapid and inexpensive genome sequencing and other genome-wide studies [1]. With the daunting amount of data, it has been possible to put them together into a coherently organized biological network that provides counterintuitive insights on biological systems [2]. Among such biological networks, a genome-scale metabolic network model is expected to play an increasingly important role in the biopharmaceutical industry [3]. Before enumerating their specific strengths, it is important to note that principles underlying genome-scale metabolic network models are consistent with the holistic perspective of systems biology, the aim of which is to unveil hidden factors causing diseases and to find relevant treatment strategies [4]. Despite the importance of metabolism in a biological system, studies on diseases in relation to metabolism were far fewer in number than those performed on signaling and transcriptional regulatory networks [5]. However, metabolism, highly linked with observable phenotypes, is a biological network that is more comprehensively characterized when compared with the other two types of networks [6]. Metabolism is, therefore, amenable to large-scale mathematical modeling and simulation. It is with this motivation that the genome-scale metabolic simulation deserves more attention in drug discovery campaigns and optimization of a host strain for the production of biopharmaceuticals. Reconstruction and application of genome-scale metabolic network models have been forged as a major research strategy of systems biology. Over the last decade, genome-scale metabolic models have been built for almost all biologically important organisms across the domains of archea, bacteria and eukaryotes [3]. They range from simple micro organisms such as Escherichia coli [7] and Saccharomyces cerevisiae [8] to higher organisms including Chinese hamster ovary (CHO) cells [9,10] and a generic human cell [11,12]. It should be noted that all these organisms that have been subjected to metabolic modeling are important cellular hosts for biopharmaceutical production or medically meaningful organisms that need to be cured (e.g., specific cancer cells) or destroyed (e.g., pathogens). A recent notable development of importance in the genomescale metabolic modeling would be the newly updated human metabolic network Recon 2 [12]. Recon 2 is a result of efforts from a group of researchers, going over a vast amount of literature and biochemical data and reconciling conflicting information. Scope of the hitherto reconstructed genome-scale metabolic models manifest high expectations for their potential contributions to biopharmaceutical industry. Genome-scale metabolic network models are not just a simple pileup of biochemical reactions, but allow mathematical simulation under precisely defined conditions of constraints [13]. Once the experimentally Applications of genome-scale metabolic network models in the biopharmaceutical industry

From the mold in Dr Florey’s coat to blockbuster drugs

Abstract: Michael Butler University of Manitoba, Department of Microbiology, 418 Buller Building, 66 Chancellors Circle, Winnipeg, Manitoba, R3T 2N2, Canada Tel.: +1 204 474 6543 Fax: +1 204 474 7603 E-mail: butler@cc.umanitoba.ca » launch of the journal Pharmaceutical Bioprocessing can be welcomed as a new forum for the dissemination of information and scientific advances in this area of biopharmaceutical «

Metabolic-sensing characteristics of absorption-photometry for mammalian cell cultures in biopharmaceutical processes

Abstract: Background: In mammalian cell culture processes, metabolite concentrations have to be routinely monitored to ensure the consistency of both process operation and product quality. The absorption photometric-based technology (APBT) has been introduced recently as a new option for characterizing metabolic profiles of the cell-culture processes. In order to understand the measurement technology, the benchmarking study was conducted under various conditions, including known standard samples and untreated cell-culture samples. Results: The standard samples, with known concentrations of the metabolites, were analyzed using APBT, and its repeatability, as well as its accuracy was compared with the other two common technologies used in the industry: membrane-based technology, and supplementary HPLC. The three technologies were further validated using the supernatant collected from batch cell-cultures from two different Chinese hamster ovary cell lines with extended culture duration, covering a broad range of exper...