No abstract

The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures.

Isolated equine chondrocytes, from juveniles and adults, were cultured in resorbable polyglycolic acid meshes for up to 5 weeks with semicontinuous feeding using a custom‐made system to intermittently compress the regenerating tissue. Assays of the tissue constructs indicate that intermittent compression at 500 and 1000 psi (3.44 and 6.87 MPa, respectively) stimulated the production of extracellular matrix, enhancing the rate of de novo chondrogenesis. Constructs derived from juvenile cells contained concentrations of extracellular matrix components at levels more like that of native tissue than did constructs derived from adult cells. With intermittent pressurization, however, even adult cells were induced to increase the production of extracellular matrix. At both levels of intermittent pressure, the concentration of sulfated glycosaminoglycan in constructs from juvenile cells was found to be up to ten times greater than concentrations in control (nonpressurized) and adult cell‐derived constructs. Whereas collagen concentrations in the 500 psi and control constructs were not significantly different for either juvenile or adult cell‐derived constructs, intermittent pressurization at 1000 psi enhanced the production of collagen, suggesting that there may be a minimum level of pressure necessary to stimulate collagen formation. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 166–174, 1999.

A promising alternative for the repair of peripheral nerve injuries is the bioartificial nerve graft, or BNG, comprised of a tubular conduit preseeded with Schwann cells, which are an effective substrate for enhancing nerve regeneration. The physical properties of the conduit, porosity and wall thickness, as well as the Schwann cell seeding density, were tested for their effect on axon growth using rat dorsal root ganglia. These parameters can influence the amount of nutrients and growth factors made available to the neural tissue. Results show that a greater wall thickness and lower porosities have a detrimental effect on the growth of the axons. Over a four week period, axons extended 3.2 mm for the optimum case (DeltaR = 0.82 mm, epsilon = 0.75) compared to 1.8 and 1.6 mm for a lower porosity (0.55) and a greater wall thickness (1.4 mm), respectively. A maximum in the growth rate occurs at a porosity of 75% for Schwann cell seeded conduits but not for unseeded ones. When compared to mass transfer predictions, the results suggest that, at higher porosities, more growth factors diffuse out of the conduit, while at low porosities there is competition for nutrients. Increasing the Schwann cell seeding density enhances growth but also leads to an increase in the number of axons along the length of the conduit. This is indicative of branching of the axons, which requires additional resources to maintain and can lead to painful neuroma formation. Wall thickness and porosity were found not to have any significant effect on the axon number sprouting from the dorsal root ganglia and the mean diameter (p > 0.05). Considerations need to be made, not just on the polymer used, but also on its porosity, wall thickness, and Schwann cell seeding density. These parameters can be adjusted to create a bioartificial nerve graft that provides the optimal environment for nerve growth.

A semi-continuous compression/perfusion system has been custom made to allow the application of intermittent hydrostatic pressure, at physiological levels, to regenerating tissues over the long term. To test the system, isolated foal chondrocytes were seeded in resorbable polyglycolic acid meshes and cultured in the system for 5 weeks. The cell/polymer constructs were subjected to an intermittent hydrostatic pressure of 500 psi and were fed semi-continuously. Assays of the resulting tissue constructs indicate that the reactor supports cartilage development and that physiological intermittent compression enhances the production of extracellular matrix by the chondrocytes. The concentrations of sulfated glycosaminoglycan were found to be at least twice as high as those in control (unpressurized) samples. A correlation between the sulfated glycosaminoglycan content and the compressive modulus in pressurized, but not control, samples suggests that physiological intermittent pressurization not only enhances the production of extracellular matrix but may also influence matrix organization resulting in a stronger construct.