Process development of a New Haemophilus influenzae type b conjugate vaccine and the use of mathematical modeling to identify process optimization possibilities

Vaccination is one of the most successful public health interventions being a cost‐effective tool in preventing deaths among young children. The earliest vaccines were developed following empirical methods, creating vaccines by trial and error. New process development tools, for example mathematical modeling, as well as new regulatory initiatives requiring better understanding of both the product and the process are being applied to well‐characterized biopharmaceuticals (for example recombinant proteins). The vaccine industry is still running behind in comparison to these industries. A production process for a new Haemophilus influenzae type b (Hib) conjugate vaccine, including related quality control (QC) tests, was developed and transferred to a number of emerging vaccine manufacturers. This contributed to a sustainable global supply of affordable Hib conjugate vaccines, as illustrated by the market launch of the first Hib vaccine based on this technology in 2007 and concomitant price reduction of Hib vaccines. This paper describes the development approach followed for this Hib conjugate vaccine as well as the mathematical modeling tool applied recently in order to indicate options for further improvements of the initial Hib process. The strategy followed during the process development of this Hib conjugate vaccine was a targeted and integrated approach based on prior knowledge and experience with similar products using multi‐disciplinary expertise. Mathematical modeling was used to develop a predictive model for the initial Hib process (the ‘baseline’ model) as well as an ‘optimized’ model, by proposing a number of process changes which could lead to further reduction in price. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:568–580, 2016     

Temperature-induced alterations of in vivo chlorophyll a fluorescence induction in cucumber as affected by DCMU

Induction of chlorophyll a fluorescence and photosynthesis as affected by temperature were measured in cucumber leaf discs. Abrupt changes of the maximal variable fluorescence, Fv(p), and photosynthesis were observed around 9° and 21°C when the temperature was decreased from 30° to 0°C. The temperature-dependent maximal fluorescence of DCMU-treated leaf discs showed a single change around 21°C. Temperature-induced chlorophyll a fluorescence alterations are discussed in relation to electron transport activity of the two photosystems and photosynthetic activity of the cucumber leaf discs.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Fm maximal fluorescence - Fv(p) maximal variable fluorescence - qE energy-dependent fluorescence quenching - qQ Qa-dependent fluorescence quenching     

Development of a low pH fermentation strategy for fumaric acid production by Rhizopus oryzae

Dicarboxylic acids that are produced from renewable resources are becoming attractive building blocks for the polymers industry. In this respect, fumaric acid is very interesting. Its low aqueous solubility facilitates product recovery. To avoid excessive waste salt production during downstream processing, a low pH for fumaric acid fermentation will be beneficial. Studying the influence of pH, working volume and shaking frequency on cell cultivation helped us to identify the best conditions to obtain appropriate pellet morphologies of a wild type strain of Rhizopus oryzae. Using these pellets, the effects of pH and CO(2) addition were studied to determine the best conditions to produce fumaric acid in batch fermentations under nitrogen-limited conditions with glucose as carbon source. Decreasing either the fermentation pH below 5 or increasing the CO(2) content of the inlet air above 10% was unfavourable for the cell-specific productivity, fumaric acid yield, and fumaric acid titer. However, switching off the pH control late in the batch phase did not affect these performance parameters and allowed achieving pH of 3.6. A concentration of 20 gL(-1) of fumaric acid was obtained at pH 3.6 while the average cell mass specific productivity and fumaric acid yield were the same as at pH 5.0. Consequently, relatively modest amounts of inorganic base were required for pH control, while recovery of the acid should be relatively easy at pH 3.6.     

A two-step,one-pot enzymatic synthesis of cephalexin from D-phenylglycine nitrile

A cascade of two enzymatic transformations is employed in a one-pot synthesis of cephalexin. The nitrile hydratase (from R. rhodochrous MAWE)-catalyzed hydration of D-phenylglycine nitrile to the corresponding amide was combined with the penicillin G acylase (penicillin amidohydrolase, E.C. 3.5.1.11)-catalyzed acylation of 7-ADCA with the in situ-formed amide to afford a two-step, one-pot synthesis of cephalexin. D-Phenylglycine nitrile appeared to have a remarkable selective inhibitory effect on the penicillin G acylase, resulting in a threefold increase in the synthesis/hydrolysis (S/H) ratio. 1,5-Dihydroxynaphthalene, when added to the reaction mixture, cocrystallized with cephalexin. The resulting low cephalexin concentration prevented its chemical as well as enzymatic degradation; cephalexin was obtained at 79% yield with an S/H ratio of 7.7.     

Initial steps of signal generation in photoactive yellow protein revealed with femtosecond mid-infrared spectroscopy

Photoactive yellow protein (PYP) is a bacterial blue light sensor that induces Halorhodospira halophila to swim away from intense blue light. Light absorption by PYP's intrinsic chromophore, p-coumaric acid, leads to the initiation of a photocycle that comprises several distinct intermediates. Here we describe the initial structural changes of the chromophore and its nearby amino acids, using visible pump/mid-infrared probe spectroscopy. Upon photoexcitation, the trans bands of the chromophore are bleached, and shifts of the phenol ring bands occur. The latter are ascribed to charge translocation, which probably plays an essential role in driving the trans to cis isomerization process. We conclude that breaking of the hydrogen bond of the chromophore's C=O group with amino acid Cys69 and formation of a stable cis ground state occur in approximately 2 ps. Dynamic changes also include rearrangements of the hydrogen-bonding network of the amino acids around the chromophore. Relaxation of the coumaryl tail of the chromophore occurs in 0.9-1 ns, which event we identify with the I(0) to I(1) transition observed in visible spectroscopy.     

Time-resolved methods in biophysics. 5. Femtosecond time-resolved and dispersed infrared spectroscopy on proteins.

In this contribution we describe how femtosecond time-resolved infrared spectroscopy provides insight into the function and dynamics of pigment-protein complexes, and what the technical requirements are to perform such experiments. We further discuss a few examples of experiments performed on the photoactive yellow protein and photosynthetic complexes in more detail.     

A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates

Cross-linked enzyme aggregates (CLEAs) were prepared from several enzymes (penicillin G acylase, hydroxynitrile lyase, alcohol dehydrogenase, and two different nitrilases) by precipitation and subsequent cross-linking using dextran polyaldehyde. In most cases, higher immobilization yields were obtained using the latter cross-linker as compared with the commonly used glutaraldehyde. Active site titration of penicillin acylase CLEAs showed that the higher activity originated from a significantly lower loss in active sites using dextran polyaldehyde as a cross-linking agent. It is proposed that macromolecular cross-linkers are too large to penetrate the protein active site and react with catalytically essential amino acid residues.     

Quantitative determination of glucose transfer between cocurrent laminar water streams in a H‐shaped microchannel

To explore the applicability of a laminar fluid diffusion interface (LFDI) for the controlled feeding of microbioreactors, glucose diffusion experiments were carried out in a rounded H‐shaped microstructure etched in a glass substrate. The diffusion channel of the microstructure had a length of 4 mm and a depth of 50 μm with a trapezoidal cross section with a width of 100 μm at the bottom and 200 μm at the surface of the channel. The microchannel was operated at residence times of less than 1 s ensuring high‐mass‐transfer rates. It was confirmed, both by microscopic observations as well as computational fluid dynamics (CFD) studies that the flow characteristics in the microchannel were fully laminar. Special attention was paid to flow splitting at the end of the channel, because the CFD simulations indicated that the performance of the device was sensitive to unequal flow splitting. The difference in outflow volume of the two streams was measured to be small (1.25% ± 0.6%). The measured glucose concentration in both exit ports at a fixed residence time was found to be stable in time and reproducible in multiple experiments. CFD simulation was shown to be a powerful tool for estimating the mass transfer in the LFDI, even at very short residence times. The results obtained in this work show the applicability of LFDI for the controlled diffusive supply of a solute to a water stream, with as possible application substrate and/or precursor feeding to microreactors. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

TUT7 controls the fate of precursor microRNAs by using three different uridylation mechanisms

Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis. Using biochemistry, single-molecule, and deep sequencing techniques, we here investigate the mechanism by which human TUT7 (also known as ZCCHC6) recognizes and uridylates precursor miRNAs (pre-miRNAs) in the absence of Lin28. We find that the overhang of a pre-miRNA is the key structural element that is recognized by TUT7 and its paralogues, TUT4 (ZCCHC11) and TUT2 (GLD2/PAPD4). For group II pre-miRNAs, which have a 1-nt 3′ overhang, TUT7 restores the canonical end structure (2-nt 3′ overhang) through mono-uridylation, thereby promoting miRNA biogenesis. For pre-miRNAs where the 3′ end is further recessed into the stem (as in 3′ trimmed pre-miRNAs), TUT7 generates an oligo-U tail that leads to degradation. In contrast to Lin28-stimulated oligo-uridylation, which is processive, a distributive mode is employed by TUT7 for both mono- and oligo-uridylation in the absence of Lin28. The overhang length dictates the frequency (but not duration) of the TUT7-RNA interaction, thus explaining how TUT7 differentiates pre-miRNA species with different overhangs. Our study reveals dual roles and mechanisms of uridylation in repair and removal of defective pre-miRNAs.