High-throughput screening of antibody-expressing CHO clones using an automated shaken deep-well system.

Biopharmaceutical protein manufacturing requires the highest producing cell lines to satisfy current multiple grams per liter requirements. Screening more clones increases the probability of identifying the high producers within the pool of available transfectant candidate cell lines. For the predominant industry mammalian host cell line, Chinese hamster ovary (CHO), traditional static-batch culture screening does not correlate with the suspension fed-batch culture used in manufacturing, and thus has little predictive utility.

Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxin in Escherichia coli.

Secretion of heterologous proteins into Escherichia coli cell culture medium offers significant advantages for downstream processing over production as inclusion bodies; including cost and time savings, and reduction of endotoxin. Signal peptides play an important role in targeting proteins for translocation across the cytoplasmic membrane to the periplasmic space and release into culture medium during the secretion process. Alpha toxin (AT) was selected as an antigen for vaccine development against Staphylococcus aureus infections.

The Bacillus subtilis TatAdCd system exhibits an extreme level of substrate selectivity.

The Tat system preferentially transports correctly folded proteins across the bacterial membrane although little is known of the proofreading mechanism. Most research has focused on TatABC systems from Gram-negative bacteria, especially Escherichia coli, and much less is known of the TatAC-type systems from Gram-positive organisms. We have previously shown that the Bacillus subtilis TatAdCd system is functional in an E.

Comprehensive analysis of host cell impurities in monoclonal antibodies with improved sensitivity by capillary zone electrophoresis mass spectrometry.

Four methods were compared for analysis of host-cell protein (HCP) impurities in a recombinant mAb. First, CZE-MS/MS was used to analyze the digest of an HCP sample following extraction of the mAb with proteins A and L affinity columns; 220 protein groups and 976 peptides were identified from the depleted HCP digest. Second, a nanoACQUITY UltraPerformance LCH system was also used to analyze the depleted HCP digest; 34 protein groups and 53 peptides from 50 ng of the depleted HCP digest and 290 protein groups and 1011 peptides were identified from 1 μg of the depleted HCP digest.

Quantitative analysis of the supernatant from host and transfected CHO cells using iTRAQ 8-plex technique.

We employed UPLC-MS/MS with iTRAQ 8-plex labeling to quantitatively analyze the supernatant produced by two Chinese hamster ovary (CHO) cell lines (CHO K1SV and CHO CAT-S). In each case, the supernatant from the host and three transfected clones were analyzed at days 5, 7, and 10 of culture. A total of eight iTRAQ 8-plex experiments were performed.

Assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging.

Regulatory authorities require that cell lines used in commercial production of recombinant proteins must be derived from a single cell progenitor or clone. The limiting dilution method of cell cloning required multiple rounds of low-density cell plating and microscopic observation of a single cell in order to provide evidence of monoclonality. Other cloning methods rely on calculating statistical probability of monoclonality rather than visual microscopic observation of cells.