Molecular design and downstream processing of turoctocog alfa (NovoEight), a B-domain truncated factor VIII molecule.

Turoctocog alfa (NovoEight) is a third-generation recombinant factor VIII (rFVIII) with a truncated B-domain that is manufactured in Chinese hamster ovary cells. No human or animal-derived materials are used in the process. The aim of this study is to describe the molecular design and purification process for turoctocog alfa. A five-step purification process is applied to turoctocog alfa: protein capture on mixed-mode resin; immunoaffinity chromatography using a unique, recombinantly produced anti-FVIII mAb; anion exchange chromatography; nanofiltration and size exclusion chromatography. This process enabled reduction of impurities such as host cell proteins (HCPs) and high molecular weight proteins (HMWPs) to a very low level. The immunoaffinity step is very important for the removal of FVIII-related degradation products. Manufacturing scale data shown in this article confirmed the robustness of the purification process and a reliable and consistent reduction of the impurities. The contribution of each step to the final product purity is described and shown for three manufacturing batches. Turoctocog alfa, a third-generation B-domain truncated rFVIII product is manufactured in Chinese hamster ovary cells without the use of animal or human-derived proteins. The five-step purification process results in a homogenous, highly purified rFVIII product.

Deep sequencing reveals different compositions of mRNA transcribed from the F8 gene in a panel of FVIII-producing CHO cell lines.

Coagulation factor VIII (FVIII) is one of the most complex biopharmaceuticals due to the large size, poor protein stability and extensive post-translational modifications. As a consequence, efficient production of FVIII in mammalian cells poses a major challenge, with typical yields two to three orders of magnitude lower than for antibodies. In the present study we investigated CHO DXB11 cells transfected with a plasmid encoding human coagulation factor VIII. Single cell clones were isolated from the pool of transfectants and a panel of 14 clones representing a dynamic range of FVIII productivities was selected for RNA sequencing analysis. The analysis showed distinct differences in F8 RNA composition between the clones. The exogenous F8-dhfr transcript was found to make up the most abundant transcript in the present clones. No correlation was seen between F8 mRNA levels and the measured FVIII productivity. It was found that three MTX resistant, nonproducing clones had different truncations of the F8 transcripts. We find that by using deep sequencing, in contrast to microarray technology, for determining the transcriptome from CHO transfectants, we are able to accurately deduce the mature mRNA composition of the transgene and identify significant truncations that would probably otherwise have remained undetected.

Genomic structure of the human mitochondrial chaperonin genes: HSP60 and HSP10 are localised head to head on chromosome 2 separated by a bidirectional promoter.

Although the mitochondrial chaperonin Hsp60 and its co-chaperonin Hsp10 have received great attention in the last decade, and it has been proposed that mutations and variations in these genes may be implicated in genetic diseases, the genome structure of the human HSP60 and HSP10 genes (also known as HSPD1 and HSPE1, respectively) has not been firmly established. The picture has been confused by the presence of many pseudogenes of both HSP60 and HSP10 and the long surviving assumption that the HSP60 gene is intron-less. An earlier report on the partial sequence of the human HSP60 gene and the presence of introns has largely been overlooked. We present the full sequence of the human HSP60 and HSP10 genes. The two genes are linked head to head comprising approximately 17 kb and consist of 12 and 4 exons, respectively. The first exon of the human HSP60 gene is non-coding and the first exon of the human HSP10 gene ends with the start codon. Analysis of human and mouse expressed sequence tag sequences in GenBank indicates that alternative splicing occurs resulting in HSP60 gene transcripts with different exon-1 sequences. By sequencing of the exons, the exon/intron boundaries and the region between the two genes in 10 Danish individuals (five couples), nine nucleotide variations and one intronic deletion have been detected that, by subsequent typing of one child from each couple, have been assigned to five haplotypes. The human HSP60 gene has been localised, by radiation hybrid mapping, between markers AFMA121YH1 and WI-10756 on chromosome 2. This location and the position of two homologous fragments in the Human Genome Assembly are consistent with cytogenetic position 2q33.1. Using a luciferase-reporter assay, we demonstrate that the region between the two genes functions as a bi-directional promoter. The transcriptional activity of the promoter fragment in the HSP60 direction is approximately twice that in the HSP10 direction under normal growth conditions and, upon heat-shock, promoter activity in either direction increased by a factor of approximately 12. One of the nucleotide variations detected is localised in a putative SP1-transcription-factor-binding site in the bidirectional promoter region and analysis of the transcriptional activity of the promoter fragment with this variation has shown that it does not affect transcription levels both with and without heat-shock.