A highly productive chemically defined fed-batch process was developed to maximize titer and volumetric productivity for Chinese hamster ovary cell-based recombinant protein manufacturing. Two cell lines producing a recombinant antibody (cell line A) and an Fc-fusion protein (cell line B) were used for development. Both processes achieved product titers of 10 g/L on day 18 under chemically defined conditions. For cell line B, the use of plant derived hydrolysates combined with the optimized chemically defined medium increased the titer to 13 g/L. Volumetric productivities were increased from a base line of about 200 mg/L/d to about 500 mg/L/d under chemically defined conditions and as high as 700 mg/L/d with cell line B using plant derived hydrolysates. Peak cell densities reached greater than 20E6 vc/mL, and cell viabilities were maintained above 80% on day 18 without the use of antiapoptotic genes or temperature shift. A rapid compound screening method was developed to effectively test positive factors within 72 h. Peak volumetric oxygen uptake rates (OUR) more than tripled from the baseline condition. Oxygen demand continued to increase after maximum cell density was reached with a maximal OUR of 3.7 mmol/L/h. The new process format was scaled up and verified at 100 L pilot scale using reactor equipment of similar configuration as used at manufacturing scale.

The effect of ammonium on the glycosylation pattern of the recombinant immunoadhesin tumor necrosis factor–IgG (TNFR‐IgG) produced by Chinese hamster ovary cells is elucidated in this study. TNFR‐IgG is a chimeric IgG fusion protein bearing one N‐linked glycosylation site in the Fc region and three complex‐type N‐glycans in the TNF‐receptor portion of each monomer. The ammonium concentration of batch suspension cultures was adjusted with glutamine and/or NH4Cl. The amount of galactose (Gal) and N‐acetylneuraminic acid (NANA) residues on TNFR‐IgG correlated in a dose‐dependent manner with the ammonium concentration under which the N‐linked oligosaccharides were synthesized. As ammonium increased from 1 to 15 mM, a concomitant decrease of up to 40% was observed in terminal galactosylation and sialylation of the molecule. Cell culture supernatants contained measurable β‐galactosidase and sialidase activity, which increased throughout the culture. The β‐galactosidase, but not the sialidase, level was proportional to the ammonium concentration. No loss of N‐glycans was observed in incubation studies using β‐galactosidase and sialidase containing cell culture supernatants, suggesting that the ammonium effect was biosynthetic and not degradative. Several biosynthetic mechanisms were investigated. Ammonium (a weak base) is known to affect the pH of acidic intracellular compartments (e.g., trans‐Golgi) as well as intracellular nucleotide sugar pools (increases UDP‐N‐acetylglucosamine and UDP‐N‐acetylgalactosamine). Ammonium might also affect the expression rates of β1,4‐galactosyltransferase (β1,4‐GT) and α2,3‐sialyltransferase (α2,3‐ST). To separate these mechanisms, experiments were designed using chloroquine (changes intracellular pH) and glucosamine (increases UDP‐GNAc pool [sum of UDP‐GlcNAc and UDP‐GalNAc]). The ammonium effect on TNFR‐IgG oligosaccharide structures could be mimicked only by chloroquine, another weak base. No differences in N‐glycosylation were found in the product synthesized in the presence of glucosamine. No differences in β1,4‐galactosyltransferase (β1,4‐GT) and α2,3‐sialyltransferase (α2,3‐ST) messenger RNA (mRNA) and enzyme levels were observed in cells cultivated in the presence or absence of 13 mM NH4Cl. pH titration of endogenous CHO α2,3‐ST and β‐1,4‐GT revealed a sharp optimum at pH 6.5, the reported trans‐Golgi pH. Thus, at pH 7.0 to 7.2, a likely trans‐Golgi pH range in the presence of 10 to 15 mM ammonium, activities for both enzymes are reduced to 50% to 60%. Consequently, ammonium seems to alter the carbohydrate biosynthesis of TNFR‐IgG by a pH‐mediated effect on glycosyltransferase activity. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 637–646, 2000.

The intracellular pool of UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine has been shown to act as a central target during the inhibitory action of ammonium ions in vitro cultivated mammalian cell cultures. This pool has been demonstrated to be elevated at the end of a batch cultivation and very quickly as a response to exogenously applied ammonium chloride by using four different cell lines (hybridoma, BHK, CHO, and Ltk(-)929). The amount of enlarged UDP aminohexoses is correlated to the inhibitor concentration and additionally dependent on the cell line. The formation of the UDP sugars is associated with a transient reduction of the UTP pool. Moreover, the quick formation of UDP-GNAc is strictly dependent on the presence of glucose and ammonium. Both metabolites act as biochemical precursors. Additionally, the formation of UDP-GNAc after ammonium application has been shown to increase with an elevated cultivation pH and to be independent of the inhibition of transcription and translation processes. The intracellular amount of UDP-GNAc correlates with the level of growth inhibition in mammalian cell lines. (c) 1994 John Wiley & Sons, Inc.

Agglomerated Pt thin films have been proposed as electrodes for electrochemical devices like micro‐solid oxide fuel cells (μ‐SOFCs) operating at low temperatures. However, comprehensive studies elucidating the interplay between agglomeration state and electrochemical properties are lacking. In this contribution the electrochemical performance of agglomerated and “dense” Pt thin film electrodes on yttria‐stabilized‐zirconia (YSZ) is correlated with their microstructural characteristics. Besides the microscopically measurable triple‐phase‐boundary (tpb) where Pt, YSZ and air are in contact, a considerable contribution of “nanoscopic” tpbs to the electrode conductivity resulting from oxygen permeable grain boundaries is identified. It is demonstrated that “dense” Pt thin films are excellent electrodes provided their grain size and thickness are in the nanometer range. The results disprove the prevailing idea that the performance of Pt thin film electrodes results from microscopic and geometrically measurable tpbs only.

Mitigating risks to biotherapeutic protein production processes and products has driven the development of targeted process analytical technology (PAT); however implementing PAT during development without significantly increasing program timelines can be difficult. The development of a monoclonal antibody expressed in a Chinese hamster ovary (CHO) cell line via fed-batch processing presented an opportunity to demonstrate capabilities of altering percent glycated protein product. Glycation is caused by pseudo-first order, non-enzymatic reaction of a reducing sugar with an amino group. Glucose is the highest concentration reducing sugar in the chemically defined media (CDM), thus a strategy controlling glucose in the production bioreactor was developed utilizing Raman spectroscopy for feedback control. Raman regions for glucose were determined by spiking studies in water and CDM. Calibration spectra were collected during 8 bench scale batches designed to capture a wide glucose concentration space. Finally, a PLS model capable of translating Raman spectra to glucose concentration was built using the calibration spectra and spiking study regions. Bolus feeding in mammalian cell culture results in wide glucose concentration ranges. Here we describe the development of process automation enabling glucose setpoint control. Glucose-free nutrient feed was fed daily, however glucose stock solution was fed as needed according to online Raman measurements. Two feedback control conditions were executed where glucose was controlled at constant low concentration or decreased stepwise throughout. Glycation was reduced from ∼9% to 4% using a low target concentration but was not reduced in the stepwise condition as compared to the historical bolus glucose feeding regimen.