Quantitative definition and monitoring of the host cell protein proteome using iTRAQ: a study of an industrial mAb producing CHO-S cell line

There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO-S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRAQ experiment to analyze technical replicates of three samples, which were culture harvest (HCCF), Protein A flow through and Protein A eluate and an 8 sample format to analyze technical replicates of four sample types; HCCF compared to Protein A eluate and subsequent cation and anion exchange purification. In the 6 sample iTRAQ experiment, 8781 spectra were confidently matched to peptides from 819 proteins (including the mAb chains). Across both the 6 and 8 sample experiments 936 proteins were identified. In the 8 sample comparison, 4187 spectra were confidently matched to peptides from 219 proteins. We then used the iTRAQ data to enable estimation of the relative change of individual proteins across the purification steps. These data provide the basis for application of iTRAQ for process development based upon knowledge of critical HCPs

Identification of copper as a cell culture media component causing metabolite depletion and product sequence variants

The level of peptide sequence variants in a biologic drug substance batch is a critical product quality attribute that should be monitored and controlled. These sequence variants are typically caused by DNA single nucleotide variants that arise in cloning and amplification, mistranscription due to unstable vector DNA or cell age/production stresses, or mistranslation via tRNA wobble or mischarging. In this work, a low frequency of monoclonal antibody sequence variants was detected by mass spectrometry in a drug substance batch. The variants were distributed throughout the heavy and light chains at average levels of under 1% per site with no apparent codon bias. No product-coding DNA mutations were detected via deep sequencing data. This pattern of low level, widely-distributed variation strongly suggested a misincorporation mechanism via mischarging of aminoacyl-tRNA, presumably due to amino acid depletion during the process. Copper is a critical cell culture media component that can be modulated in fed-batch processes to induce lactate consumption via its role as a cofactor for mitochondrial function and respiration. However, complete consumption of lactate can also trigger reduced levels of other metabolites required for recombinant protein assembly, which can lead to product sequence variants. To investigate the potential relationship between media copper supplementation and sequence variants, various levels of copper were supplemented into the basal media for fed-batch cultures at the 250 mL bioreactor scale. Mass spectrometry analysis of the partially purified antibody indicated a positive correlation between the amount of copper supplemented and the level of detected sequence variants as well as a mechanism for sequence variant reduction via targeted nutrient feeding. This work has identified a potential mechanism of sequence variant generation related to cell culture media copper levels as well as process alterations to prevent such variation in future batches, highlighting the importance of carefully controlling trace metal levels. Additional studies may be required to validate the potential mechanism

Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3

The eukaryotic initiation factor 3 (eIF3) plays an important role in translation initiation, acting as a docking site for several eIFs that assemble on the 40S ribosomal subunit. Here, we use mass spectrometry to probe the subunit interactions within the human eIF3 complex. Our results show that the 13-subunit complex can be maintained intact in the gas phase, enabling us to establish unambiguously its stoichiometry and its overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Dissociation takes place as a function of ionic strength to form three stable modules eIF3(c:d:e:l:k), eIF3(f:h:m), and eIF3(a:b:i:g). These modules are linked by interactions between subunits eIF3b:c and eIF3c:h. We confirmed our interaction map with the homologous yeast eIF3 complex that contains the five core subunits found in the human eIF3 and supplemented our data with results from immunoprecipitation. These results, together with the 27 subcomplexes identified with increasing ionic strength, enable us to define a comprehensive interaction map for this 800-kDa species. Our interaction map allows comparison of free eIF3 with that bound to the hepatitis C virus internal ribosome entry site (HCV-IRES) RNA. We also compare our eIF3 interaction map with related complexes, containing evolutionarily conserved protein domains, and reveal the location of subunits containing RNA recognition motifs proximal to the decoding center of the 40S subunit of the ribosome