Case study and application of process analytical technology (PAT) towards bioprocessing: II. Use of ultra-performance liquid chromatography (UPLC) for making real-time pooling decisions for process chromatography

Process analytical technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community due to the potential for continuous real-time quality assurance resulting in improved operational control and compliance. Two of the key goals that have been outlined for PAT are "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions". Recently, we have been examining the feasibility of applying different analytical tools for designing PAT applications for bioprocessing. We have previously shown that a commercially available online high performance liquid chromatography (HPLC) system can be used for analysis that can facilitate real-time decisions for column pooling based on product quality attributes (Rathore et al., 2008). In this article we test the feasibility of using a commercially available ultra- performance liquid chromatography (UPLC) system for real-time pooling of process chromatography columns. It is demonstrated that the UPLC system offers a feasible approach and meets the requirements of a PAT application. While the application presented here is of a reversed phase assay, the approach and the hardware can be easily applied to other modes of liquid chromatography.     

Defining process design space for biotech products: case study of Pichia pastoris fermentation

The concept of "design space" has been proposed in the ICH Q8 guideline and is gaining momentum in its application in the biotech industry. It has been defined as "the multidimensional combination and interaction of input variables (e.g., material attributes) and process parameters that have been demonstrated to provide assurance of quality." This paper presents a stepwise approach for defining process design space for a biologic product. A case study, involving P. pastoris fermentation, is presented to facilitate this. First, risk analysis via Failure Modes and Effects Analysis (FMEA) is performed to identify parameters for process characterization. Second, small-scale models are created and qualified prior to their use in these experimental studies. Third, studies are designed using Design of Experiments (DOE) in order for the data to be amenable for use in defining the process design space. Fourth, the studies are executed and the results analyzed for decisions on the criticality of the parameters as well as on establishing process design space. For the application under consideration, it is shown that the fermentation unit operation is very robust with a wide design space and no critical operating parameters. The approach presented here is not specific to the illustrated case study. It can be extended to other biotech unit operations and processes that can be scaled down and characterized at small scale.     

Autoregulation of regulatory proteins is key for dynamic operation of GAL switch in Saccharomyces cerevisiae

Autoregulation and nucleocytoplasmic shuttling play important roles in the operation of the GAL regulatory system. However, the significance of these mechanisms in the overall operation of the switch is unclear. In this work, we develop a dynamic model for the GAL system and further validate the same using steady-state and dynamic experimental expression data. Next, the model is used to delineate the relevance of shuttling and autoregulation in response to inducing, repressing, and non-inducing-non-repressing media. The analysis indicates that autoregulation of the repressor, Gal80p, is key in obtaining three distinct steady states in response to the three media. In particular, the analysis rationalizes the intuitively paradoxical observation that the concentration of repressor, Gal80p, actually increases in response to an increase in the inducer concentration. On the other hand, although nucleocytoplasmic shuttling does not affect the dynamics of the system, it plays a dominant role in obtaining a sensitive response to galactose. The dynamic model was also used to obtain insights on the preculturing effect on the system behavior.     

Preparation and in vitro evaluation of xanthan gum facilitated superabsorbent polymeric microspheres

Interpenetrating polymer network (IPN) hydrogel microspheres of xanthan gum (XG) based superabsorbent polymer (SAP) and poly(vinyl alcohol) (PVA) were prepared by water-in-oil (w/o) emulsion crosslinking method for sustained release of ciprofloxacin hydrochloride (CIPRO). The microspheres were prepared with various ratios of hydrolyzed SAP to PVA and extent of crosslinking density. The prepared microspheres with loose and rigid surfaces were evidenced by scanning electron microscope (SEM). Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis confirmed the IPN formation. Differential scanning calorimetry (DSC) study was performed to understand the dispersion nature of drug after encapsulation. The in vitro drug release study was extensively evaluated depending on the process variables in both acidic and alkaline media. All the formulations exhibited satisfactory physicochemical and in vitro release characteristics. Release data indicated a non-Fickian trend of drug release from the formulations. Based on the results, this study suggest that CIPRO loaded IPN microspheres were suitable for sustained release application.     

Application of near‐infrared (NIR) spectroscopy for screening of raw materials used in the cell culture medium for the production of a recombinant therapeutic protein

Control of raw materials based on an understanding of their impact on product attributes has been identified as a key aspect of developing a control strategy in the Quality by Design (QbD) paradigm. This article presents a case study involving use of a combined approach of Near‐infrared (NIR) spectroscopy and Multivariate Data Analysis (MVDA) for screening of lots of basal medium powders based on their impact on process performance and product attributes. These lots had identical composition as per the supplier and were manufactured at different scales using an identical process. The NIR/MVDA analysis, combined with further investigation at the supplier site, concluded that grouping of medium components during the milling and blending process varied with the scale of production and media type. As a result, uniformity of blending, impurity levels, chemical compatibility, and/or heat sensitivity during the milling process for batches of large‐scale media powder were deemed to be the source of variation as detected by NIR spectra. This variability in the raw materials was enough to cause unacceptably large variability in the performance of the cell culture step and impact the attributes of the resulting product. A combined NIR/MVDA approach made it possible to finger print the raw materials and distinguish between good and poor performing media lots. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions

Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.     

RiDs db: Repeats in diseases database

The non-coding fraction of the human genome, which is approximately 98%, is mainly constituted by repeats. Transpositions, expansions and deletions of these repeat elements contribute to a number of diseases. None of the available databases consolidates information on both tandem and interspersed repeats with the flexibility of FASTA based homology search with reference to disease genes. Repeats in diseases database (RiDs db) is a web accessible relational database, which aids analysis of repeats associated with Mendelian disorders. It is a repository of disease genes, which can be searched by FASTA program or by limitedor free- text keywords. Unlike other databases, RiDs db contains the sequences of these genes with access to corresponding information on both interspersed and tandem repeats contained within them, on a unified platform. Comparative analysis of novel or patient sequences with the reference sequences in RiDs db using FASTA search will indicate change in structure of repeats, if any, with a particular disorder. This database also provides links to orthologs in model organisms such as zebrafish, mouse and Drosophila. AVAILABILITY: The database is available for free at http://115.111.90.196/ridsdb/index.php.     

Purification and characterization of a thermally stable yellow laccase from <Emphasis Type="Italic">Daedalea flavida</Emphasis> MTCC-145 with higher catalytic performance towards selective synthesis of substituted benzaldehydes

A laccase from the culture filtrate of white rot fungus Daedalea flavida MTCC-145 has been purified and characterized. The method involved concentration of the culture filtrate by ultrafiltration and an anion exchange chromatography on diethylaminoethyl (DEAE) cellulose. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (native PAGE) both gave single protein bands indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 75.0 kDa. Purification fold was 21.5 while recovery of the enzyme activity was 11.52%. Using 2,6-dimethoxyphenol, diammonium salt of 2,2'-[azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)] and 3,5-dimethoxy-4-hydroxybenzaldehyde azine as substrates, the Km, kcat, and k _cat/K _m values of the laccase were found to be 440 µM, 6.45 s^–1, 1.47 × 10⁴ M^–1 s^–1; 366 µM, 6.45 s^–1, 1.76 × 10⁴ M^–1 s^–1; and 226 µM, 6.45 s^–1, 2.85 × 10⁴ M^–1 s^–1, respectively. The pH and temperature optima were 4.5 and 50°C, respectively. The enzyme was most stable at pH 5.0 when exposed for 1 h. The purified laccase has yellow color and shows no absorption band around 610 nm characteristic of blue laccases. The enzyme transforms toluene and substituted toluenes to corresponding benzaldehyde and substituted benzaldehydes in the absence of mediator molecules with higher catalytic efficiency as compared to other known laccases.