Near‐infrared spectroscopy is considered to be one of the most promising spectroscopic techniques for upstream bioprocess monitoring and control. Traditionally the nature of near‐infrared spectroscopy has demanded multivariate calibration models to relate spectral variance to analyte concentrations. The resulting analytical measurements have proven unreliable for the measurement of metabolic substrates for bioprocess batches performed outside the calibration process. This paper presents results of an innovative near‐infrared spectroscopic monitor designed to follow the concentrations of glycerol and methanol, as well as biomass, in real time and continuously during the production of a monoclonal antibody by a Pichia pastoris high cell density process. A solid state instrumental design overcomes the ruggedness limitations of conventional interferometer‐based spectrometers. Accurate monitoring of glycerol, methanol, and biomass is demonstrated over 274 days postcalibration. In addition, the first example of feedback control to maintain constant methanol concentrations, as low as 1 g/L, is presented. Postcalibration measurements over a 9‐month period illustrate a level of reliability and robustness that promises its adoption for online bioprocess monitoring throughout product development, from early laboratory research and development to pilot and manufacturing scale operation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:749–759, 2014

17time from pool acetate to expired C02 as 60 minutes which is slightly shorter than the half life from the carboxyl carbon of acetate to C02 obtained in this investigation of 76.5 minutes. This result following equilibration during an infusion period is probably a better figure than that obtained following a single injection. The longer half life (135 min) of conversion from the methyl carbon of acetate to pool C02 found when 2-C14 acetate was infused, emphasizes the difference in the pathway of oxidation of the methyl carbon and of the carboxyl carbon of acetate.The biological half lives, as calculated from the fall of specific activity of body pool bicarbonate and from expired C02, are in excellent agreement. Thus, blood data may be used for studying the oxidative rates of acetate, eliminating the turnover time of the bicarbonate pool which otherwise masks the picture of oxidation in the animal body.Summary. The rates of oxidation of acetate to COa were estimated by using l-C14 and 2-C14 labeled acetate and measuring both blood and expired C1402. Following 1-C14 labeled acetate infusion, the half life of blood and expired C140a was about 76 minutes indicating a mean turnover time of acetate carboxyl to pool C02 of about 110 minutes.The half lives of 2-C14 labeled acetate were 135 minutes and 195 minutes respectively. The half life of the pool acetate was 1.2 minutes; turnover time 1.7 minutes; turnover rate 3.4 meq per hour and pool size 6.0 meq per sheep.

A pilot-scale fermentation was developed for an antifungal compound produced by a filamentous fungus. Replacement of galactose with lactose (20-fold cost savings) and a threefold phosphate reduction (15 to 5 g/L) improved productivity 2.5-fold. Addition of supplements--glycine, cobalt chloride, and trace elements--resulted in a further twofold productivity increase, greater process robustness, and less foaming which reduced antifoam addition tenfold (30 to <3 mL/L). Mid-cycle lactose limitations were addressed by raising initial lactose levels (40 to 120 g/L) resulting in another twofold productivity increase. Overall, peak titers increased tenfold from 45 +/- 9 to 448 +/- 39 mg/L, and productivities improved from 3 to 25 mg/L day. Despite its high productivity, process scale up was challenged by high broth viscosity (5,000-6,000 cP at 16.8 s(-1)). Gassed power requirements at the 600 L scale (4.7 kW/1,000 L) exceeded available power at the 15,000 L scale (3.0 kW/1,000 L), and broth transfer to the downstream isolation facility was hindered. Mid-cycle broth dilution with up to five 10 vol% additions of 12 wt% lactose solution or whole medium-reduced viscosity three- to fivefold (1,000-1,500 cP at 16.8 s(-1)), gassed power within scale-up limits (2.5 kW/1,000 L), and peak titer by up to 45%. The process was scaled up to the 15,000 L working volume based on constant aeration rate (vvm) and peak impeller tip speed, raising superficial velocities at similar shear. This strategy maximized mass transfer rates at target gassed power per unit volume levels, and along with controlled broth viscosity, precluded multiple dilution additions. A final titer of 333 mg/L with one dilution addition was achieved, somewhat lower than expected, likely owing to inhibition from some unmeasured volatile compound (not believed to be carbon dioxide) during an extended period of high back-pressure in the early production phase.

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