The effect of osmolarity on metabolism and morphology in adhesion and suspension chinese hamster ovary cells producing tissue plasminogen activator

The effects of constant osmolarity, between 300 and500 mOsm/kg, on the metabolism of Chinese HamsterOvary (CHO) cells producing tissue plasminogenactivator (tPA) were compared between adhesion andsuspension cultures. In both suspension and adhesionculture, the specific rates of glucose consumption(_G), lactate production (q_L), and tPAproduction (q_tPA) increased as osmolarityincreased, while these rates decreased when osmolaritywas higher than the respective critical levels. However, specific growth rate () decreased withincrease in osmolarity and this slope grew steeper inthe osmolarity range higher than the critical level. The decrease in in the adhesion culture was morerapid than that in the suspension culture. Thecritical osmolarity for adhesion culture (400 mOsm/kg)was lower than that for suspension culture (450 mOsm/kg). These results indicated that the adhesionculture was more sensitive to increase of osmolaritythan the suspension culture, while the specific ratesobtained from the adhesion cultures were in general1.5- to 3-fold higher than those obtained from thesuspension cultures. Cell volume increased asosmolarity increased in both the suspension andadhesion cultures, as reported previously forsuspension culture of hybridoma cells, but there wasno morphological change in the suspension culture. Incontrast, cell height decreased and cell adhesion areamarkedly increased as osmolarity increased in theadhesion culture. This morphological change inadhesion cultures may be one reason for the highersensitivity of adherent cells to the increase ofosmolarity than suspended cells.     

Influence of alterations in culture condition and changes in perfusion parameters on the retention performance of a 20 μm spinfilter during a perfusion cultivation of a recombinant CHO cell line in pilot scale

Since 1969 much attention has been devoted to the useof spinfilter systems for retention of mammalian cellsin continuous perfusion cultivations. Previousinvestigations dealt with hydrodynamic conditions,fouling processes and upscaling. But hydrodynamicconditions and fouling processes seem to have asecondary importance in spinfilter performance duringauthentic perfusion cultivations. Obviously,alterations in culture condition are more relevantespecially during long-term processes. Therefore, ourpratical approach focussed on the performance qualityof a commercially available 20 m spinfilterduring a perfusion cultivation of a recombinant CHOcell line in pilot scale regarding the followingissues: 1) retention of viable cells in thebioreactor; 2) removal of dead cells and cell debrisfrom the bioreactor; 3) alterations in culturecondition; and 4) changes in perfusion mode.Furthermore, we tested the performance of 20 mspinfilters in 2 and 100 l pilot scale using solidmodel particles instead of cells. Our investigationsshowed that retention of viable cells in pilot scalewas independent of spinfilter rotation velocity andperfusion rate; the retention increased from 75 to 95%corresponding to operation time, enlarging celldiameter and enhanced formation of aggregates in theculture during the perfusion cultivation. By means ofthe Cell Counter and Analyzer System (CASY) anoperation cut off of 13 m was determined forthis spinfilter. Using solid model particles in 2 lscale, optimal retention was achieved at a tip speedof 0.43 m s^-1 (141 rpm) – furtherenhancement of spinfilter rotation velocity up to0.56 m s^-1 (185 rpm) decreased the retentionrapidly. In pilot scale best retention performance wasobtained with tip speeds of 0.37 m s^-1(35 rpm) and 1.26 m s^-1 (120 rpm). Hence,significant retention in pilot scale could already beachieved with low agitation. Therefore, the additionof shear force protectives could be avoided so thatthe purification of the target protein from thesupernatant would be facilitated.     

In vitro cytotoxicity of Acanthamoeba spp. isolated from contact lens containers in Korea by crystal violet staining and LDH release assay

In order to observe the cytotoxicity of Acanthamoeba spp., which were isolated from contact lens containers as ethiological agents for the probable amoebic keratitis in Korea, the crystal violet staining method and LDH release assay were carried out. In the crystal violet staining method, among eight contact lens container isolates, isolate 3 (Acanthamoeba KA/LS5) showed 83.6% and 81.8% of cytotoxicity, and isolate 7 (Acanthamoeba KA/LS37) showed 28.2% and 25.1% of cytotoxicity, in 1 mg/ml and 0.5 mg/ml lysate treatments, respectively. Acanthamoeba culbertsoni and A. healyi showed 84.0% and 82.8% of cytotoxicity. Similar results were observed in A. castellanii and A. hatchetti which showed 83.6% and 75.5% of cytotoxicity. Acanthamoeba royreba and A. polyphaga showed 9.0% and 1.7% of cytotoxicity. In the LDH release assay, isolate 3 (20.4%) showed higher cytotoxicity than other isolates in 1 mg/ml lysate treatment. The results provide that at least isolate 3 has the cytotoxic effect against CHO cells and seems to be the pathogenic strain.     

Nonuniform Microtubular Polarity Established by CHO1/MKLP1 Motor Protein Is Necessary for Process Formation of Podocytes

Podocytes are unique cells that are decisively involved in glomerular filtration. They are equipped with a complex process system consisting of major processes and foot processes whose function is insufficiently understood (Mundel, P., and W. Kriz. 1995. Anat. Embryol. 192:385–397). The major processes of podocytes contain a microtubular cytoskeleton. Taking advantage of a recently established cell culture system for podocytes with preserved ability to form processes (Mundel, P., J. Reiser, A. Zúñiga Mejía Borja, H. Pavenstädt, G.R. Davidson, W. Kriz, and R. Zeller. 1997b. Exp. Cell Res. 36:248–258), we studied the functional significance of the microtubular system in major processes. The following data were obtained: (a) Microtubules (MTs) in podocytes show a nonuniform polarity as revealed by hook-decoration. (b) CHO1/ MKLP1, a kinesin-like motor protein, is associated with MTs in podocytes. (c) Treatment of differentiating podocytes with CHO1/MKLP1 antisense oligonucleotides abolished the formation of processes and the nonuniform polarity of MTs. (d) During the recovery from taxol treatment, taxol-stabilized (nocodazole- resistant) MT fragments were distributed in the cell periphery along newly assembled nocodazole-sensitive MTs. A similar distribution pattern of CHO1/MKLP1 was found under these circumstances, indicating its association with MTs. (e) In the recovery phase after complete depolymerization, MTs reassembled exclusively at centrosomes. Taken together, these findings lead to the conclusion that the nonuniform MT polarity in podocytes established by CHO1/MKLP1 is necessary for process formation.     

Long-term stable production of monocyte-colony inhibition factor (M-CIF) from CHO microcarrier perfusion cultures

Monocyte-colony inhibition factor (M-CIF) was produced in microcarrier perfusion cultures from engineered Chinese hamster ovary (CHO) cells. Three and fifteen liter microcarrier perfusion bioreactors equipped with internal spin filters were operated for over two months. Approximately 60 L and 300 L of culture filtrate were harvested from the 3L and 15L microcarrier perfusion bioreactors respectively. During the perfusion operation, cell density reached 2–6 × 10⁶ cells/ml. Importantly, stable expression of M-CIF from the CHO cells under non-selection condition was maintained at a level of 4–10 mg/L. Specific productivity was maintained at 1.8–3.4 mg/billion cells/day. The ability of the recombinant CHO cells to migrate from microcarrier to microcarrier under our proprietary HGS-CHO-3 medium greatly facilitated microcarrier culture scale-up and microcarrier replenishment. Future directions for microcarrier perfusion system scale-up and process development are highlighted. This revised version was published online in August 2006 with corrections to the Cover Date.     

Development and manufacturability assessment of chemically‐defined medium for the production of protein therapeutics in CHO cells

Advantages of using internally developed chemically‐defined (CD) media for cell culture‐based therapeutic protein production over commercial media include better raw material control and medium vendor options, and most importantly, flexibility for process development and subsequent optimization needed for therapeutic protein production. Through several rounds of design of experiment (DOE) screening, and medium component supplementation and optimization studies, we successfully developed a CD basal medium (CDM) for CHO cell culture. The internally prepared liquid CDM demonstrated comparable cell culture performance to that from a commercially available control medium. However, when the same CDM formulation was transferred to two major commercial medium suppliers for manufacturing, cell culture performance utilizing these newly prepared media was significantly reduced compared with the in‐house prepared counterpart. An investigation was launched to assess whether key medium components were sensitive to large‐scale preparation of the final bulk media by the vendors. Further work necessitated the reformulation of the original CDM formulation into a core medium that was suitable for large‐scale media manufacturing. The modified preparation of the core medium with two separate supplements to generate the final CDM was able to recover the expected cell culture performance and monoclonal antibody (mAb) productivity. Confirmation of cell culture robustness in cell growth and production was corroborated in two additional mAb‐expressing cell lines. This work demonstrates that a robust CD medium is not only one that performs during the development stage, but also one that must be reproducible by commercial media vendors. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1163–1171, 2015     

Influence of protein and carbohydrate contents of soy protein hydrolysates on cell density and IgG production in animal cell cultures

The variety of compounds present in chemically defined media as well as media supplements makes it difficult to use a mechanistic approach to study the effect of supplement composition on culture functionality. Typical supplements, such as soy protein hydrolysates contain peptides, amino acids, carbohydrates, isoflavones, and saponins. To study the relative contribution of these compound classes, a set of hydrolysates were produced, containing 58‐83% proteinaceous material and 5‐21% carbohydrates. While the content of the different compounds classes varied, the composition (e.g., peptide profiles, carbohydrate composition) did not vary in hydrolysates. The hydrolysates were supplemented to a chemically defined medium in cell culture, based on equal weight and on equal protein levels. The latter showed that an increase in the carbohydrate concentration significantly (P value < 0.004) increased integral viable cell density (IVCD) (R = 0.7) and decreased total IgG (R = ?0.7) and specific IgG production (R = ?0.9). The extrapolation of effects of protein concentration showed that an increase in protein concentration increased total and specific IgG production and suppressed IVCD. In addition to proteins and carbohydrates, the functionality of soy protein hydrolysates may be modulated by the presence of other minor compounds. In the current study, the large differences in the balance between total proteins and total carbohydrates in the supplemented media seem to be a main factor influencing the balance between the viable cell density, total IgG, and specific IgG production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1396–1405, 2015     

Effect of Bcl‐xL overexpression on sialylation of Fc‐fusion protein in recombinant Chinese hamster ovary cell cultures

The sialic acid of glycoproteins secreted by recombinant Chinese hamster ovary (rCHO) cells can be impaired by sialidase under culture conditions which promote the extracellular accumulation of this enzyme. To investigate the effect of Bcl‐x_L overexpression on the sialylation of glycoproteins produced in rCHO cell culture, two rCHO cell lines producing the same Fc‐fusion protein, which were derived from DUKX‐B11 and DG44, respectively, were engineered to have regulated Bcl‐x_L overexpression using the Tet‐off system. For both cell lines, Bcl‐x_L overexpression improved cell viability and extended culture longevity in batch cultures. As a result, a maximum Fc‐fusion protein titer increased by Bcl‐x_L overexpression though the extent of titer enhancement differed between the two cell lines. With Bcl‐x_L overexpression, the sialylation of Fc‐fusion protein, which was assessed by isoelectric focusing gel and sialic acid content analyses, decreased more slowly toward the end of batch cultures. This was because Bcl‐x_L overexpression delayed the extracellular accumulation of sialidase activity by reducing cell lysis during batch cultures. Taken together, Bcl‐x_L overexpression in rCHO cell culture increased Fc‐fusion protein production and also reduced the impairment of sialylation of Fc‐fusion protein by maintaining high viability during batch cultures. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1133–1136, 2015     

Acylation and cholesterol binding are not required for targeting of influenza A virus M2 protein to the hemagglutinin-defined budozone

Influenza virus assembles in the budozone, a cholesterol-/sphingolipid-enriched (“raft”) domain at the apical plasma membrane, organized by hemagglutinin (HA). The viral protein M2 localizes to the budozone edge for virus particle scission. This was proposed to depend on acylation and cholesterol binding. We show that M2–GFP without these motifs is still transported apically in polarized cells. Employing FRET, we determined that clustering between HA and M2 is reduced upon disruption of HA’s raft-association features (acylation, transmembranous VIL motif), but remains unchanged with M2 lacking acylation and/or cholesterol-binding sites. The motifs are thus irrelevant for M2 targeting in cells.