PP3 enhanced growth of CHO line 4 in shake flask cultures and 24DW plates in a dose dependent manner. Production was enhanced in presence of 1 g/L of PP3 peptone

compared to no peptone in both shake flasks and 24DW plates. Higher concentrations of PP3 did not show further enhancement in protein production in either culture system. Correlation analysis of data from both systems gave a Pearson coefficient value of 0.986 for growth and 0.900 for production with a P value <0.05. This indicates that there is a positive linear relationship between the data sets obtained from the two culture systems and they are highly correlated. To compare the performance of 24DW plates and shake flasks in a fed batch culture process, CHO line 1 was grown in a basal medium in both culture systems, fed with a CD supplement (5%, v/v) on days 0, 2, 4, and 6, and sampled on various days of culture. As shown in Fig. 5, the CD supplement Protease Inhibitor Library cell assay enhanced the growth of cells in both 24DW plates and

shake flasks, Apoptosis inhibitor however somewhat higher growth was observed in shake flask cultures. Despite lower growth in 24DW plates, both systems showed equivalent protein production. In a separate study (data not shown), six different feeds were tested in fed batch process on CHO line 1 in both culture systems and protein production was determined on various days of culture. A high and significant correlation was obtained between 24DW plates and shake flask for protein production on three different days of culture (Pearson correlation coefficient 0.94 with P = 0.00). Results obtained from these fed batch studies indicate that while the overall cell growth patterns show some differences,

the production response is highly correlated between two systems. The premise of our approach was that the miniaturized cell culture system (shaking 24DW plates) can be used for cell culture process development, if the system shows significant correlation with conventional shake flask system. To assess this approach, concurrent studies were performed in 24DW plates with the Duetz sandwich-covers and conventional shake flask systems. Feasibility studies included screening of multiple CHO cell lines in 24DW plates concurrently with shake 4��8C flasks to understand cell line dependent variability. Other studies included assessment of well-to-well and plate-to-plate variation for CHO cell growth and mAb production. Regardless of the medium and cell line, growth kinetics of the cells grown in 24DW plates showed similar patterns to cells grown in shake flask. Moreover, the production levels in 24DW plates were equivalent to shake flasks. Determination of inter- and intra-plate variability is important for data consistency and accuracy in any plate based assay. Edge effect is a very common phenomenon observed in a multi well plate assays caused by differential evaporation across the plate.

These numbers check details underline the potential of solution-state techniques to study flexible assemblies and at the same time suggest that this

potential has not yet been fully exploited for RNP complexes. NMR-based structural studies of RNPs have so far addressed small to medium-size complexes (briefly reviewed in the next session). However, most molecular machines involved in RNA metabolism and in regulatory RNA pathways are multi-component assemblies of more than 50 kDa. Due to their modular architecture, the divide-and-conquer approach is useful to decipher the atomic details of RNA–protein interfaces. On the other hand, since only the full complex retains functionality, the architecture of high-molecular-weight RNPs in solution is relevant to understand structure–function relationships. This perspective article discusses recent advances in NMR methodologies to investigate large proteins and nuclei acids and proposes ways to exploit these developments, possibly in combination with complementary techniques in structural biology, to study

high-molecular-weight RNP complexes in their functional forms. The structure of RNA–protein complexes with molecular weight (MW) < 50 kDa can be solved by standard NMR techniques, taking advantage of 13C/15N labeling of either the Trichostatin A solubility dmso protein or the RNA component of the complex. 13C/15N edited, 12C/14N filtered NOESY experiments [9] and [10] are instrumental for the detection of intermolecular NOEs. Structural studies in

solution are particularly relevant for proteins in complex with single-stranded short RNA sequences, which maintain some extent of disorder in the complex. Many RNA-binding domains are quite tolerant in terms Pyruvate dehydrogenase lipoamide kinase isozyme 1 of the RNA sequences they bind to; therefore, prior to the structural investigation, it is important to find the RNA sequence with the highest affinity for the cognate protein, which is likely to yield the best quality intermolecular NOEs. To this end, an NMR based method has been developed that uses the magnitude of the protein chemical shifts deviations upon titration of RNA to derive the sequence specificity of an RNA-binding domain [11]. The nucleotide type is varied systematically at each position within the RNA target, where the nucleotides at positions other than the one under analysis have a random identity. Analysis of the deviations of the chemical shifts of the target protein allows identifying patterns of sequence specific recognition at each nucleotide position, in a manner that is independent of the RNA structural and sequence context. The method works for target RNAs as long as 6–8 nucleotides, which in most cases covers the length of the RNAs recognized by the widespread RRM (RNA recognition motif), KH (K-homology), PAZ (Piwi/Argonaute/Zwille) and Zn-finger domains.