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.

Adaptation of mammalian cells to growth in serum-free media.

A three-step protocol is described for adapting an anchorage-dependent, serum-dependent recombinant mammalian cell lineage to high density serum-free suspension culture. The objective is a cell lineage that is well-suited for the manufacture of a recombinant protein. The first step of the protocol generates an anchorage-independent cell lineage by culturing trypsin-treated cells in spinner flasks using serum-containing medium. The second step adapts the lineage to serum-free medium through a series of serum reduction steps in the presence of defined growth-promoting additives. The third step adapts the lineage to high-cell-density conditions by culturing the cells in a bioreactor in a manner that allows development of tolerance to growth-inhibiting substances released by the cells. Examples are presented for the use of this protocol for recombinant CHO cells.

Experiences of virus, retrovirus and retrovirus-like particles in Chinese hamster ovary (CHO) and hybridoma cells used for production of protein therapeutics.

Garnick and coworkers indicated that they experienced two independent MVM outbreaks in a period where approximately 2000 fermentations were performed, hypothesizing that such events were rare but inevitable consequences of very large scale operations. In GIs experience over the last 12 years we have seen no incidence of MVM (or any other virus) in close to 3000 fermentations, albeit at lower volumes than produced at Genentech; GI has used 250-2500L bioreactors for manufacturing whereas Genentech have reported using 100-10,000L bioreactors. Nonetheless, volumes of complex media in the same range as used at Genentech have been used at GI with no observations of viral contamination events. The reason for this is not clear. However, GI's experience in combination with experience from sub-contract testing agencies who service the majority of the biotechnology industry may call the inevitability of an MVM outbreak into question. It would appear that very few adventitious viral contaminations of cell cultures have occurred in industry in the last decade. Interestingly, the frequency of contamination events appear to be lower in CHO cells than in hybridoma cells. It should be noted, however, that these conclusions are not statistically based and the scope of the above survey was somewhat limited. RVLPs are present in both CHO and hybridoma cells. The characteristics of both are compared in Table 4. C-type particles from hybridoma cells are more abundant as a rule than those from CHO cells. Although the majority of C-type particles produced by hybridoma cells appear to be non-infective (in S+L- assays), approximately one in a million particles are competent to replicate in S+L- cells. The evidence that C-type particles can replicate in human cells has proved difficult to reproduce consistently. It is likely that replication of xenotropic hybridoma C-type particles in human cells is inefficient or restricted to only a small number of specific cell lines. C-type RVLPs from CHO cells are produced less abundantly than those from hybridoma cells and are not competent to replicate due to a defective endonuclease gene. However, over the last two decades the use of hybridoma cells and products derived from these cells has not provided any evidence of transmission of these viruses to humans; in addition they can be readily removed or inactivated. Thus, neither agent would appear to constitute a significant risk to pharmaceutical products made from their respective host cells. Nonetheless, given the difference in relative safety profiles between RVLPs from CHO and hybridoma cells it is not unreasonable to propose that safety factors (clearance factors in removal/inactivation studies in excess of the reduction of virus loads to zero) required should be less for a CHO process than for a hybridoma process.

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.

Extracellular insulin degrading activity creates instability in a CHO-based batch-refeed continuous process.

In a batch-refeed continuous process involving a recombinant Chinese hamster ovary cell line, a brief upset was occasionally observed during which cell growth halted and cell viability dropped. This was found to be associated with depletion of insulin from the medium early during the affected passage. Insulin depletion was found to be primarily the result of insulin degrading activity released by the cells during the preceding passage.

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.

Acetylatable lipoic acid residues interact directly with lipoamide dehydrogenase in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The proposal that the lipoate acetyltransferase component (E2) of the pyruvate dehydrogenase multienzyme (PD) complex from Escherichia coli contains three covalently bound lipoyl residues, one of which acts to pass reducing equivalents to lipoamide dehydrogenase (E3), has been tested. The PD complex was incubated with pyruvate and N-ethylmaleimide, to yield an inactive PD complex containing lipoyl groups on E2 with the S6 acetylated and the S8H irreversibly alkylated with N-ethylmaleimide. This chemically modified form would be expected to exist only on two of the three proposed lipoyl groups. The third nonacetylatable lipoyl group, which is proposed to interact with E3, would remain in its oxidized form. Reaction of the N-ethylmaleimide-modified PD complex with excess NADH should generate the reduced form of the proposed third nonacetylatable lipoyl group and thereby make it susceptible to cyclic dithioarsinite formation with bifunctional arsenicals (BrCH2CONHPhAsCl2; BrCH2[14C]CONHPhAsO). Once "anchored" to the reduced third lipoyl group via the--AsO moiety, these reagents would be delivered into the active site of E3 by the normal catalytic process of the PD complex where the BrCH2CONH--group inactivates E3. Whereas the E3 component of native PD complex is inactivated by the bifunctional reagents in the presence of excess NADH (owing to the above delivery process), the E3 component of the PD complex modified with N-ethylmaleimide in the presence of pyruvate is not inhibited. The results indicate that acetylatable lipoyl residues interact directly with E3 and do not support a functional role for a proposed third lipoyl residue.

Inhibition of pyruvate dehydrogenase multienzyme complex from Escherichia coli with a radiolabeled bifunctional arsenoxide: evidence for an essential histidine residue at the active site of lipoamide dehydrogenase.

Incubation of pyruvate dehydrogenase multienzyme complex (PD complex) from Escherichia coli with thiamin pyrophosphate, pyruvate, coenzyme A, Mg2+, and the radiolabeled bifunctional arsenoxide p-[(bromoacetyl)-amino]phenyl arsenoxide (BrCH214CONHPhAsO) led to the irreversible loss of lipoamide dehydrogenase (E3) activity. The mode of inactivation occurred by initial "anchoring" of the reagent via its -AsO group to reduced lipoyl residues on lipoate acetyltransferase (E2) (generated by substrates) followed by the delivery of the BrCH214CO- moiety into the active site of E3 where an irreversible alkylation ensued [Stevenson, K. J., Hale, G., & Perham, R. N. (1978) Biochemistry 17, 2189]. To account for nonspecific alkylations, not mediated by this delivery process, control experiments were conducted in which the radiolabeled bifunctional reagent was incubated with PD complex in the absence of substrates. E3 subunits were isolated from inhibited and control PD complexes by chromatography on hydroxylapatite in the presence of 8 M urea. Acid hydrolysis of the alkylated E3 and control E3 samples produced radiolabeled carboxymethylated amino acids that were identified and quantitated by high-voltage electrophoresis and amino acid/radiochemical analysis. The inhibited sample contained N3-(carboxymethyl)histidine and a small amount of S-(carboxymethyl)cysteine. These residues were not present in significant amounts in the controls. The loss of 81% of E3 activity correlated with the alkylation of about 0.7 residue of histidine and 0.1 residue of cysteine per mol of E3.

Inhibition of pyruvate dehydrogenase multienzyme complex from Escherichia coli with a bifunctional arsenoxide: selective inactivation of lipoamide dehydrogenase.

The bifunctional reagent p-[(bromoacetyl)-amino]phenyl arsenoxide (BrCH2CONHPhAsO) in the presence of excess reduced nicotinamide adenine dinucleotide has been shown to cause the irreversible active site directed inactivation of the lipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase multienzyme (PD) complex from Escherichia coli. The ability of the lipoate acetyltransferase (E2) component to bind coenzyme A was decreased by about 50% in this system. In the presence of thiamine pyrophosphate, pyruvate, coenzyme A, and Mg2+, E3 inactivation by BrCH2CONHPhAsO was selective (coenzyme A binding was unaffected) and stoichiometrically related to PD complex inactivation, indicating that a complement of E3 is necessary for full complex activity. The activity of the pyruvate dehydrogenase (E1) component was unaltered by BrCH2-CONHPhAsO in both systems. On inhibition of the PD complex with BrCH2CONHPhAsO, the reagent mediated interchain cross-linking between E2 and about half of the E3 subunits. A marked change occurred in the quaternary structure of the PD complex, with some E1 and E3 subunits being dissociated from the E2 core. The mechanism outlined by Stevenson et al. [Stevenson, K. J., Hale, G., & Perham, R. N. (1978) Biochemistry 17, 2189] for the inhibition of the PD complex by BrCH2CONHPhAsO must be revised on the basis of these findings. E3 is only partially modified by delivery of the bromoacetyl moiety of the bifunctional reagent (covalently attached to lipoyl residues of E2 through dithioarsinite bonds) into the active site of bound E3. The inhibition of E3, dissociated from the PD complex during cross-linking, likely occurs via direct interaction of the free enzyme with BrCH2CONHPhAsO by initial dithioarsinite modification of the reduced active-site disulfide followed by alkylation of a nearby residue.