Quantitation of residual trypsin in cell-based therapeutics using immobilized α-1-antitrypsin or SBTI in an ELISA format.

An Enzyme-Linked Immunosorbent Assay (ELISA) has been developed for the quantitation of porcine trypsin as a process residual in cell therapy products based on its capture by either of two immobilized anti-trypsins, α-1-antitrypsin (α1AT) or soybean trypsin inhibitor (SBTI) followed by detection with a polyclonal goat anti-porcine trypsin-IgG conjugated with peroxidase. It was demonstrated that an extended range of antigen quantitation could be achieved that covered nearly three orders of magnitude of trypsin concentration. The utility of the assay was demonstrated by its application to samples generated in a cell-based therapeutic manufacturing setting.

Therapeutic human cells: manufacture for cell therapy/regenerative medicine.

: Human primary cells (e.g. adult stem cells) as well as differentiated cells, including those of the immune system, have been found to be therapeutically useful and free of ethical concerns.

Managing particulates in cellular therapy.

he concept of particulates, while common to many in the pharmaceutical and blood transfusion disciplines, represents a distinct challenge in the field of cellular therapy. With newly discovered products advancing through clinical trials, the focus has shifted to ensuring products are manufactured in a reliable and safe manner. Given the unique manufacturing processes and resulting products (i.

Developing assays to address identity, potency, purity and safety: cell characterization in cell therapy process development.

A major challenge to commercializing cell-based therapies is developing scalable manufacturing processes while maintaining the critical quality parameters (identity, potency, purity, safety) of the final live cell product. Process development activities such as extended passaging and serum reduction/elimination can facilitate the streamlining of cell manufacturing process as long as the biological functions of the product remain intact. Best practices in process development will be dependent on cell characterization; a thorough understanding of the cell-based product.

Proteomic analysis for the assessment of different lots of fetal bovine serum as a raw material for cell culture. Part IV. Application of proteomics to the manufacture of biological drugs.

Fetal bovine serum (FBS) is the most widely used growth supplement for cell cultures, primarily because of its high levels of growth stimulatory factors and low levels of growth inhibitory factors. Maintaining successful and consistent cell fermentations can be difficult, as FBS is a complex natural product and may vary from lot to lot even from a single manufacturer. The quality and concentration of both bulk and specific proteins can affect cell growth.

Precise and comparative pegylation analysis by microfluidics and mass spectrometry.

Standard SDS-PAGE analysis of a pegylated protein was able to confirm an increase in its molecular size after reaction with an activated polyethylene glycol (PEG) but could do little to identify the extent of pegylation or to support characterization of the consistency of the modified protein. In this article, we demonstrate the utility of the capillary electrophoresis technology (using a microfluidic system) in analyzing the pegylation pattern of a recombinant protein over a range of 1-12 PEGs per polypeptide. Confirmatory data from mass spectrometry analysis of pegylated adducts are also presented.

A potency assay for a replication incompetent adenovirus type 5 carrying a human fgf-4 gene.

A bioassay that measures the potency of the FGF-4 transgene carried by a replication incompetent adenovirus type 5, Ad5FGF-4, was developed on ARPE-19 cells. The assay is carried out in a microtiter plate format and measures cellular proliferation following infection of ARPE-19 cells with a serial dilution of Ad5FGF-4. Proliferation is measured as a percentage increase in absorbance reading in relation to a mock-infected control.

Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system.

With the recent completion of the human genome sequencing project, scientists now face the daunting challenge of deciphering the function of these newly found genes quickly and efficiently. For biotechnology, it is equally important to identify the therapeutically relevant genes as quickly as possible. Mammalian expression systems provide many advantages to aid in this task.

Common structure of rare replication-deficient E1-positive particles in adenoviral vector batches.

The use of the PER.C6 adenovirus packaging cell line in combination with a designated vector plasmid system, whereby the cell line and vector with E1 deleted have no sequence overlap, eliminates the generation of replication-competent adenovirus during vector production. However, we have found cytopathic effect (CPE)-inducing particles in 2 out of more than 40 large-scale manufacturing lots produced in PER.

Brachyspira hyodysenteriae contains eight linked gene copies related to an expressed 39-kDa surface protein.

A tandemly linked set of four open reading frames (ORFs), identified as vspA-D (variable surface protein) had been identified from previous cloning and sequencing of clones from a genomic library constructed from Brachyspira hyodysenteriae strain B204. The predicted translation products of these closely related genes were homologous to (but not identical with) a characterized 39-kDa surface-exposed membrane protein from this animal pathogen. Additional screening of the genomic library has been performed to retrieve what are believed to be additional vsp genes including the one expected to encode this 39-kDa protein.

A single short stretch of homology between adenoviral vector and packaging cell line can give rise to cytopathic effect-inducing, helper-dependent E1-positive particles.

An undesirable byproduct from recombinant adenoviral vectors is the emergence of replication competent adenovirus (RCA) that result from rare homologous recombination events between the viral E1-containing (permissive) mammalian host cell genome and the virus itself, restoring the E1 gene to the viral genome. To reduce or eliminate the problem of RCA, we evaluated production of a first generation Ad5 vector (Ad5FGF4) in the cell line PER.C6.