Intensification of a multi-product perfusion platform through medium and process development

Integrated Continuous Biomanufacturing (ICB) provides many important strategic advantages for therapeutic protein production through process intensification, simplification and integration. The success of this technology will be significantly enhanced by the platform’s ability to push towards high productivity in conjunction with minimizing the associated perfusion rate, resulting in dramatic reductions in cost of good manufactured. We have previously demonstrated that an in-house chemically defined medium can support cell densities exceeding 100 million viable cells/mL in 10L perfusion bioreactors with an average productivity of 2 g/L/day. Further optimization utilizing high throughput technology specifically tailored to improve cell specific productivity (SPR) resulted in an intensified medium that is capable of achieving greater than 2X increase in SPR while maintaining low cell specific perfusion rate (CSPR). When combined with process knowledge and efforts to improve shear protection in a high oxygen demand environment, we were able to achieve 4 g/L/day volumetric productivity of an IgG for over 30 days in a state of control. In this talk, recent case studies on the application of this intensified perfusion platform to cell lines producing different classes of biologics will be described, effects on product quality will be illustrated, and engineering and economic considerations for commercial scale will be discusse

Scale-down high-throughput perfusion development with ambr 250

The ambr® 250 bioreactor system (Sartorius Stedim) has become a useful tool for CHO cell culture process development, increasing throughput and decreasing development timelines. The ambr® 250 provides the ability to independently run up to 24 single-use fed-batch bioreactors through the use of an automated liquid handling platform. Growing interest in intensified perfusion processes for continuous biomanufacturing has created a need for an equivalent high throughput small scale system for perfusion. Thus far, only discrete models and semi-continuous approaches have been available to mimic perfusion processes at small scale. With the development of the ambr® 250 perfusion system, truly continuous medium exchange and cell retention at the 200mL scale has been achieved. Primary criteria for evaluating the applicability of the ambr® 250 perfusion system to modeling larger scale intensified perfusion processes include viable cell density and volumetric productivity. We have demonstrated the development system can support growth of CHO cell cultures in excess of 90 million cells per milliliter with IgG volumetric productivity greater than 3 g/L/day. These results are comparable to data obtained at the 10L scale. Sustainability of culture, impact on product quality, and potential use for applications ranging from clone selection to scale-down modeling will be discussed

Isolation and binding properties of methanobactin from the facultative methanotroph Methylocystis strain SB2

Methanotrophs, also called aerobic methane oxidizing bacteria (AMOB), are a group of bacteria that use methane as their sole source of carbon and energy. AMOB share a general pathway for the metabolism of methane to carbon dioxide. The first step in AMOB metabolism is the oxidation of methane to methanol. The enzyme methane monooxygenase is responsible for the first step in methane metabolism. Methanotrophs have a large demand for copper and have been shown to produce an extracellular copper binding compound (cbc) now termed methanobactin (mb). Methanobactin is a small, The second part of the dissertation presents evidence that less structurally complex forms of methanobactin have similar copper binding properties to previously isolated forms of methanobactin. Mb-SB2 is shown to have transition metal binding properties that areM similar to those found in the complex form of mb from Methylosinus trichosporium OB3b (mb-OB3b). Attention is given to the ability of mb-SB2 to form gold nanoparticles.</p

Cerium regulates expression of alternative methanol dehydrogenases in Methylosinus trichosporium OB3b

Methanotrophs have multiple methane monooxygenases that are well known to be regulated by copper, i.e., a “copper switch.” At low copper/biomass ratios the soluble methane monooxygenase (sMMO) is expressed while expression and activity of the particulate methane monooxygenase (pMMO) increases with increasing availability of copper. In many methanotrophs there are also multiple methanol dehydrogenases (MeDHs), one based on Mxa and another based on Xox. Mxa-MeDH is known to have calcium in its active site, while Xox-MeDHs have been shown to have rare earth elements in their active site. We show here that the expression levels of Mxa-MeDH and Xox-MeDH in Methylosinus trichosporium OB3b significantly decreased and increased, respectively, when grown in the presence of cerium but the absence of copper compared to the absence of both metals. Expression of sMMO and pMMO was not affected. In the presence of copper, the effect of cerium on gene expression was less significant, i.e., expression of Mxa-MeDH in the presence of copper and cerium was slightly lower than in the presence of copper alone, but Xox-MeDH was again found to increase significantly. As expected, the addition of copper caused sMMO and pMMO expression levels to significantly decrease and increase, respectively, but the simultaneous addition of cerium had no discernible effect on MMO expression. As a result, it appears Mxa-MeDH can be uncoupled from methane oxidation by sMMO in M. trichosporium OB3b but not from pMMO

A TonB-dependent Transporter is Responsible for Methanobactin Uptake by Methylosinus trichosporium OB3b

Methanobactin, a small modified polypeptide synthesized by methanotrophs for copper uptake, has been found to be chromosomally encoded. The gene encoding the polypeptide precursor of methanobactin, mbnA, is part of a gene cluster that also includes several genes encoding proteins of unknown function (but speculated to be involved in methanobactin formation) as well as mbnT, which encodes a TonB-dependent transporter hypothesized to be responsible for methanobactin uptake. To determine if mbnT is truly responsible for methanobactin uptake, a knockout was constructed in Methylosinus trichosporium OB3b using marker exchange mutagenesis. The resulting M. trichosporium mbnT::Gmr mutant was found to be able to produce methanobactin but was unable to internalize it. Further, if this mutant was grown in the presence of copper and exogenous methanobactin, copper uptake was significantly reduced. Expression of mmoX and pmoA, encoding polypeptides of the soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), respectively, also changed significantly when methanobactin was added, which indicates that the mutant was unable to collect copper under these conditions. Copper uptake and gene expression, however, were not affected in wild-type M. trichosporium OB3b, indicating that the TonB-dependent transporter encoded by mbnT is responsible for methanobactin uptake and that methanobactin is a key mechanism used by methanotrophs for copper uptake. When the mbnT::Gmr mutant was grown under a range of copper concentrations in the absence of methanobactin, however, the phenotype of the mutant was indistinguishable from that of wild-type M. trichosporium OB3b, indicating that this methanotroph has multiple mechanisms for copper uptake

A comparison of methanobactins from Methylosinus trichosporium OB3b and Methylocystis strain SB2 predicts methanobactins are synthesized from diverse peptide precursors modified to create a common core for binding and reducing copper ions

Methanobactins (mb) are low-molecular mass, copper-binding molecules secreted by most methanotrophic bacteria. These molecules have been identified for a number of methanotrophs, but only the one produced by Methylosinus trichosporium OB3b (mb-OB3b) has to date been chemically characterized. Here we report the chemical characterization and copper binding properties of a second methanobactin, which is produced by Methylocystis strain SB2 (mb-SB2). mb-SB2 shows some significant similarities to mb-OB3b, including its spectral and metal binding properties, and its ability to bind and reduce Cu(II) to Cu(I). Like mb-OB3b, mb-SB2 contains two five-member heterocyclic rings with associated enethiol groups, which together form the copper ion binding site. mb-SB2 also displays some significant differences compared to mb-OB3b, including the number and types of amino acids used to complete the structure of the molecule, the presence of an imidazolone ring in place of one of the oxazolone rings found in mb-OB3b, and the presence of a sulfate group not found in mb-OB3b. The sulfate is bonded to a threonine-like side chain that is associated with one of the heterocyclic rings and may represent the first example of this type of sulfate group found in a bacterially derived peptide. Acid-catalyzed hydrolysis and decarboxylation of the oxazolone rings found in mb-OB3b and mb-SB2 produce pairs of amino acid residues and suggest that both mb-OB3b and mb-SB2 are derived from peptides. In support of this, the gene for a ribosomally produced peptide precursor for mb-OB3b has been identified in the genome of M. trichosporium OB3b. The gene sequence indicates that the oxazolone rings in mb-OB3b are derived from the combination of a cysteine residue and the carbonyl from the preceding residue in the peptide sequence. Taken together, the results suggest methanobactins make up a structurally diverse group of ribosomally produced, peptide-derived molecules, which share a common pair of five-member rings with associated enethiol groups that are able to bind, reduce, and stabilize copper ions in an aqueous environment

Liver mitochondrial membrane crosslinking and destruction in a rat model of Wilson disease

Wilson disease (WD) is a rare hereditary condition that is caused by a genetic defect in the copper-transporting ATPase ATP7B that results in hepatic copper accumulation and lethal liver failure. The present study focuses on the structural mitochondrial alterations that precede clinical symptoms in the livers of rats lacking Atp7b, an animal model for WD. Liver mitochondria from these Atp7b–/– rats contained enlarged cristae and widened intermembrane spaces, which coincided with a massive mitochondrial accumulation of copper. These changes, however, preceded detectable deficits in oxidative phosphorylation and biochemical signs of oxidative damage, suggesting that the ultrastructural modifications were not the result of oxidative stress imposed by copper-dependent Fenton chemistry. In a cell-free system containing a reducing dithiol agent, isolated mitochondria exposed to copper underwent modifications that were closely related to those observed in vivo. In this cell-free system, copper induced thiol modifications of three abundant mitochondrial membrane proteins, and this correlated with reversible intramitochondrial membrane crosslinking, which was also observed in liver mitochondria from Atp7b–/– rats. In vivo, copper-chelating agents reversed mitochondrial accumulation of copper, as well as signs of intra-mitochondrial membrane crosslinking, thereby preserving the functional and structural integrity of mitochondria. Together, these findings suggest that the mitochondrion constitutes a pivotal target of copper in WD