Viral safety of B-domain deleted recombinant factor VIII.

The possible transmission of blood-borne pathogens has been the impetus behind the development of recombinant products formulated in the absence of human-derived components. The viral safety of Chinese hamster ovary (CHO)-cell-based pharmaceuticals is well established. Over 100 million infusions have been administered without a single known incident of CHO-related viral transmission. The manufacturing process for B-domain deleted recombinant factor VIII (BDDrFVIII) builds on this safety record by using a state-of-the-art multitiered approach to viral safety. This approach includes: (1) extensive testing of the CHO cells used to produce BDDrFVIII; (2) routine viral monitoring of the cell culture production process; (3) a purification process in which a specific viral inactivation procedure has been included; (4) a final formulation that does not incorporate human albumin as the stabilizer; and (5) a thorough validation of the viral inactivation and removal capacity of the purification process. This multifaceted viral safety program offers the hemophilia community a factor VIII product with an exceptional degree of viral safety.

Genetic and phenotypic markers and their relationship to product quality and consistency.

Manufacturers of products derived from biological systems have long sought to identify relevant genotypic and phenotypic markers displayed by production strains and cell lines which could be employed as in-process monitors to predict product quality. Ideally, changes in these markers would signal possible changes in product quality and could be used to ensure batch to batch product consistency. In mammalian cell culture-based manufacturing processes, individual cell lines can exhibit varying genotypes and phenotypes, not all of which are relevant to cellular protein synthesis. In this paper we present data illustrating that two key phenotypic markers, thought to be relevant to protein biosynthesis (specific growth rate and cellular productivity), can vary significantly without causing obvious changes in product characteristics. Additionally, we outline our approach to genotypic characterization at the cell bank and post-process stages and our rationale for this approach.

Differential cytokeratin gene expression reveals early dorsal-ventral regionalization in chick mesoderm.

The induction and spatial patterning of early mesoderm are known to be critical events in the establishment of the vertebrate body plan. However, it has been difficult to define precisely the steps by which mesoderm is initially subdivided into functionally discrete regions. Here we present evidence for a sharply defined distinction between presumptive dorsal and presumptive ventral regions in early chick mesoderm. Northern blot and in situ hybridization analyses reveal that transcripts corresponding to CKse1, a cytokeratin gene expressed during early development, are present at high levels in the presumptive ventral mesoderm, but are greatly reduced or undetectable in the future dorsal region of mesoderm, where the formation of axial structures occurs later in development. This distinction is present even while the mesoderm layer is being formed, and persists during the extensive cellular movements and tissue remodelling associated with morphogenesis. These results point to an early step in which two fundamentally distinct states are established along the presumptive dorsal-ventral axis in the mesoderm, and suggest that determination in this germ layer occurs in a hierarchical manner, rather than by direct specification of individual types of histological differentiation. The differential expression of CKse1 represents the earliest molecular index of dorsoventral regionalization detected thus far in the mesoderm.

Isolation of a chick cytokeratin cDNA clone indicative of regional specialization in early embryonic ectoderm.

During early vertebrate development, a series of inductive tissue interactions appear to be involved in establishing regional specializations that are eventually elaborated in the basic body plan of the embryo. These early inductive interactions are particularly difficult to study because they often occur in the absence of any associated morphological changes. In the chick embryo, the regional subdivision of the early ectoderm is evidenced by a marked lens-forming bias in the head ectoderm, which is absent from the presumptive dorsal epidermis of the trunk region. This striking divergence in developmental state is present long before any differentiation into lens or epidermal phenotypes can be detected. As a strategy for isolating genes whose differential expression might be a reflection of this regional subdivision, a cDNA library was prepared from early embryos and screened for differential hybridization to radiolabelled probes prepared from head ectoderm and trunk ectoderm. Two related cDNA clones were isolated that hybridize to transcripts present at much higher levels in trunk ectoderm than in head ectoderm. Sequence analysis of one of these clones revealed a high degree of similarity to members of the type II subfamily of intermediate filament cytokeratins. This clone (pCKse1) was used to examine cytokeratin gene expression in ectodermal tissues. A large increase in the level of CKse1 transcripts was found to take place in trunk ectoderm, approximately coordinate with neurulation, contrasting sharply with the much lower levels detected in head ectoderm and neural ectoderm at all stages tested. These results indicate that differential cytokeratin gene expression can occur within a contiguous layer of simple embryonic epithelia, and that this expression pattern coincides closely to the subdivision of the early ectoderm into regions with distinct developmental potencies. This type of regulation has not been described previously for members of the cytokeratin gene family.

CHO DUKX cell lineages preadapted to growth in serum-free suspension culture enable rapid development of cell culture processes for the manufacture of recombinant proteins.

Using an adaptive strategy, Chinese hamster ovary (CHO) cell lines were developed that are capable of robust growth in serum-free suspension culture. These preadapted derivatives of the commonly used strain of CHO cells (CHO DUKX), termed PA-DUKX, were used for the introduction and stable expression of several heterologous human genes. A significant advantage of recombinant PA-DUKX cells was their ability to readily resume growth in serum-free suspension culture after transfection and amplification of heterologous genes. Expression of recombinant human proteins in PA-DUKX cells was quantitatively similar to that of lineages generated using conventional CHO DUKX cells. In addition, recombinant human proteins expressed by transfected PA-DUKX lineages were shown to be biochemically and structurally similar to those expressed in CHO DUKX cells, PA-DUKX host cell technology provides an opportunity for reducing the time and resources required to develop large-scale, suspension culture-based manufacturing processes employing serum-free medium. (c) 1996 John Wiley & Sons, Inc.