Feasibility of spectral pH measurement during the low-pH virus inactivation step of continuous therapeutic antibody production

Acidic virus inactivation is commonly used during production of biotherapeutic products to provide virus safety in case of undetected virus contamination. Accurate pH measurement is required to ensure the product pH reaches a virus-inactivating level (typically 3.5–3.7), and a level post-inactivation that is appropriate for later purification steps (typically 5.5–7.5). During batch low-pH inactivation in discrete tanks, potentiometric glass probes are appropriate for measuring pH. During continuous inactivation for 2–3 weeks in an enclosed product stream, probe calibration drift and lag may lead to poor accuracy, and operational difficulties when compensating for drift. Monitoring the spectral response of compounds (indicators) in the product stream whose spectra are pH-sensitive offers a possible alternative way to measure pH without these drawbacks. Such indicators can already exist in the stream (intrinsic) or can be added (extrinsic). Herein are reported studies evaluating the feasibility of both.Promising ultraviolet screening results with the two extrinsics studied, thiamine and ascorbic acid, led to the addition of both to product stream samples titrated to different potentiometric pH values in the 3.3–4.5 range (a representative range encountered during continuous inactivation), and attempts to model pH using sample ultraviolet spectra. One model, based on variability in six spectral attributes, was able to predict pH of an independent sample set within ±0.07 units at the 95% confidence level. Since a typical inactivating pH tolerance is ±0.1 units, the results show that extrinsic indicators potentially can measure inactivation pH with sufficient accuracy. Suggested future steps and an alternative approach are presented.     

Interference of electron donors in the measurement of the proton gradient and membrane potential by flow dialysis

The three most commonly used electron donors for flow dialysis measurements of membrane potential lead to the development of an apparent but artifactual membrane potential with the interior negative in the presence or absence of membrane vesicles. The same three electron donors used in flow dialysis determinations of delta pH in the presence or absence of membrane vesicles lead to the development of an apparent but artifactual delta pH with the interior acidic. These artifacts have been evaluated using two probes for membrane potential, namely, TPP+ and rubidium in the presence of valinomycin and for two probes of delta pH, namely, acetate and DMO. Measurements were made over a range of ionic strengths.     

Use of ribozyme cleavage kinetics to measure salt-induced changes in solution pH

Several ribozymes are active in molar concentrations of monovalent salts, and pH rate curves under such conditions are consistent with mechanisms involving general acid–base catalysis. Interpreting the apparent pK_a values requires an accurate estimation of solution pH, which can be difficult to obtain using a typical glass pH electrode in the presence of high salt concentrations. In the current work we have used the VS ribozyme as a “biological pH meter” to evaluate the effects of molar concentrations of monovalent salts on changes in solution pH. We find that almost all of the measured change in pH observed in high concentrations of LiCl is due to a real change in solution pH. In contrast, high concentrations of NaCl cause errors in pH measurement in addition to changes in actual pH. Different buffer systems affect both the direction and the magnitude of pH changes: we observed changes in measured pH of up to 1 pH unit, which require proper interpretation to avoid adverse effects on the conclusions drawn from pH rate and other experiments that utilize very high salt concentrations.     

Comparisons of optical pH and dissolved oxygen sensors with traditional electrochemical probes during mammalian cell culture

Small-scale upstream bioprocess development often occurs in flasks and multi-well plates. These culturing platforms are often not equipped to accurately monitor and control critical process parameters; thus they may not yield conditions representative of manufacturing. In response, we and others have developed optical sensors that enable small-scale process monitoring. Here we have compared two parameters critical to control in industrial cell culture, pH and dissolved oxygen (DO), measured with our optical sensors versus industrially accepted electrochemical probes. For both optical sensors, agreement with the corresponding electrochemical probe was excellent. The Pearson Correlations between the optical sensors and electrochemical probes were 98.7% and 99.7%, for DO and pH, respectively. Also, we have compared optical pH sensor performance in regular (320 mOsm/kg) and high-osmolality (450 mOsm/kg) cell culture media to simulate the increase in osmolality in pH-controlled cultures. Over a pH range of 6.38-7.98 the average difference in pH readings in the two media was 0.04 pH units. In summary, we have demonstrated that these optical sensors agree well with standard electrochemical probes. The accuracy of the optical probes demonstrates their ability to detect potential parameter drift that could have significant impact on growth, production kinetics, and protein product quality. We have also shown that an increase in osmolality that could result from controlling pH or operating the reactor in fed-batch mode has an insignificant impact on the functionality of the pH patches.     

Advances in primary recovery: centrifugation and membrane technology

Significant and continual improvements in upstream processing for biologics have resulted in challenges for downstream processing, both primary recovery and purification. Given the high cell densities achievable in both microbial and mammalian cell culture processes, primary recovery can be a significant bottleneck in both clinical and commercial manufacturing. The combination of increased product titer and low viability leads to significant relative increases in the levels of process impurities such as lipids, intracellular proteins and nucleic acid versus the product. In addition, cell culture media components such as soy and yeast hydrolysates have been widely applied to achieve the cell culture densities needed for higher titers. Many of the process impurities can be negatively charged at harvest pH and can form colloids during the cell culture and harvest processes. The wide size distribution of these particles and the potential for additional particles to be generated by shear forces within a centrifuge may result in insufficient clarification to prevent fouling of subsequent filters. The other residual process impurities can lead to precipitation and increased turbidity during processing and even interference with the performance of the capturing chromatographic step. Primary recovery also poses significant challenges owing to the necessity to execute in an expedient manner to minimize both product degradation and bioburden concerns. Both microfiltration and centrifugation coupled with depth filtration have been employed successfully as primary recovery processing steps. Advances in the design and application of membrane technology for microfiltration and dead-end filtration have contributed to significant improvements in process performance and integration, in some cases allowing for a combination of multiple unit operations in a given step. Although these advances have increased productivity and reliability, the net result is that optimization of primary recovery processes has become substantially more complicated. Ironically, the application of classical chemical engineering approaches to overcome issues in primary recovery and purification (e.g., turbidity and trace impurity removal) are just recently gaining attention. Some of these techniques (e.g., membrane cascades, pretreatment, precipitation, and the use of affinity tags) are now seen almost as disruptive technologies. This paper will review the current and potential future state of research on primary recovery, including relevant papers presented at the 234th American Chemical Society (ACS) National Meeting in Boston.     

Rapid pH change due to bacteriorhodopsin measured with a tin-oxide electrode.

The photocurrent transient generated by bacteriorhodopsin (bR) on a tin-oxide electrode is due to pH change and not to charge displacement as previously assumed. Films of either randomly oriented or highly oriented purple membranes were deposited on transparent electrodes made of tin-oxide-coated glass. The membranes contained either wild-type or D96N-mutant bR. When excited with yellow light through the glass, the bR pumps protons across the membrane. The result is a rapid local pH change as well as a charge displacement. Experiments with these films show that it is the pH change rather than the displacement that produces the current transient. The calibration for the transient pH measurement is given. The sensitivity of a tin-oxide electrode to a transient pH change is very much larger than its sensitivity to a steady-state pH change.     

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported¹. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases². Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells³. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis^3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.     

Energy transduction in vesicles of the methanogenic strain Gö1

Abstract Everted vesicles of the methanogenic strain Gö1 synthesized ATP in response to methanogenesis from methyl-coenzyme M and H₂. Simultaneously, a transmembrane pH gradient (ΔpH) was generated as evident from fluorescence quenching of acridine orange. Protonophorous uncouplers prevented ΔpH generation and ATP synthesis, but did not affect methanogenesis. The ATP synthase inhibitor diethylstilbestrol (DES) inhibited ATP synthesis but had no effect on methanogenesis and on ΔpH formation, indicating the essential role of the transmembrane proton potential in ATP synthesis. Progress has also been made in assigning specific functions to membrane components in methanogenesis from methyl-CoM and H₂. Separation of cell extracts into cytoplasmic and membrane fraction revealed an essential role of membrane-bound components in electron transfer: methanogenesis catalyzed by the cytoplasmic fraction from strain Gö1 was stimulated several fold by membranes from various methanogens. This stimulation was prevented if the membranes had been treated with oxidants (O₂, K₃[Fe(CN)₆]) or SH reagents (Ag⁺, p -chloromercuribenzoate, iodoacetamide) pointing to the involvement of functional SH groups in methanogenesis from methyl-CoM and H₂.     

Two-color fluorescent probes for imaging the dipole potential of cell plasma membranes

The dipole potential (Psi(d)) constitutes a large and functionally important part of the electrostatic potential of cell plasma membranes. However, its direct measurement is not possible. Herein, new 3-hydroxyflavone fluorescent probes were developed that respond strongly to Psi(d) changes by a variation of the intensity ratio of their two well-separated fluorescence bands. Using fluorescence spectroscopy with cell suspensions and confocal microscopy with adherent cells, we showed, for the first time, two-color fluorescence ratiometric measurement and visualization of Psi(d) in cell plasma membranes. Using this new tool, a heterogeneous distribution of this potential within the membrane was evidenced.     

In situ pH maintenance for mammalian cell cultures in shake flasks and tissue culture flasks

pH in animal cell cultures decreases due to production of metabolites like lactate. pH control via measurement and base addition is not easily possible in small‐scale culture formats like tissue‐culture flasks and shake flasks. A hydrogel‐based system is reported for in situ pH maintenance without pH measurement in such formats, and is demonstrated to maintain pH between 6.8 and 7.2 for a suspension CHO cell line in CD CHO medium and between 7.3 and 7.5 for adherent A549 cells in DMEM:F12 containing 10% FBS. This system for pH maintenance, along with our previous report of hydrogels for controlled nutrient delivery in shake flasks can allow shake flasks to better mimic bioreactor‐based fed batch operation for initial screening during cell line and process development for recombinant protein production in mammalian cells. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Reaction of the purple membrane with a carbodiimide.

Purple membrane was reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at pH 4.5 and 8.0. At pH 4.5, the reaction yields cross-linked bacteriorhodopsin. The cross-linking is inhibited by pretreatment of the membrane with papain, or by the presence of carbohydrazide or glycine ethyl ester in the reaction mixture. The product of the pH 8.0 reaction is not cross-linked, but it displays altered properties. Two measures of photochemical activity (light-induced change in proton binding (delta h) and decay of photointermediate M) show changes indicative of slowed proton uptake. The delta h is increased by ethyl dimethylaminopropylcarbodiimide. This increase is unaffected by pretreatment of the membrane with papain, and it is not reversed by NH2OH. However, the reaction is inhibited by millimolar concentrations of CaCl2. The altered delta h is not apparent in detergent-solubilized membranes. Ethyl dimethylaminopropylcarbodiimide does not appear to cause a large alteration in the membrane surface charge, as measured by Ca2+ binding. We conclude that (1) at acid pH, ethyl dimethylaminopropylcarbodiimide can be used for cross-linking or for attachment of specific probes to the C-terminal region of bacteriorhodopsin, and hence to the cytoplasmic side of the purple membrane, and (2) at alkaline pH, ethyl dimethylaminopropylcarbodiimide reacts at a diffent type of site and appears to inhibit the proton pump.     

Resolution of phospholipid conformational heterogeneity in model membranes by spin-label EPR and frequency-domain fluorescence spectroscopy.

We have utilized both fluorescent and nitroxide derivatives of stearic acid as probes of membrane structural heterogeneity in phospholipid vesicles under physiological conditions, as well as conditions of varying ionic strengths and temperatures where spectral heterogeneity has been previously observed and attributed to multiple ionization states of the probes. To identify the source of this spectral heterogeneity, we have utilized complimentary measurements of the relaxation properties (lifetimes) and motion of both (a) spin labeled and anthroyloxy derivatives of stearic acid (i.e., SASL and AS) and (b) a diphenylhexatriene derivative of phosphatidylcholine (DPH-PC) in single component membranes containing dimyristoylphosphatidylcholine (DMPC). We use an 15N stearic-acid spin label for optimal sensitivity to membrane heterogeneity. The lifetime and dynamics of the fluorescent phospholipid analogue DPH-PC (with no ionizable groups over this pH range) were compared with those of AS, allowing us to discriminate between changes in membrane structure and the ionization of the label. The quantum yield and rotational dynamics of DPH-PC are independent of pH, indicating that changes in pH do not affect the conformation of the host phospholipids. However, both EPR spectra of SASL and the lifetime or dynamics of AS are affected profoundly by changes in solution pH. The apparent pKa's of these two probes in DMPC membranes were determined to be near pH 6.3, implying that at physiological pH and ionic strength these stearic-acid labels exist predominantly as a single ionized population in membranes. Therefore, the observed temperature- and ionic-strength-dependent alterations in the spectra of SASL as well as the lifetime or dynamics of AS in DMPC membranes at neutral pH are due to changes in membrane structure rather than the ionization of the probes. The possibility that ionic gradients across biological membranes induce alterations in phospholipid structures, thereby modulating lipid-protein interactions is discussed.     

Boron induces hyperpolarization of sunflower root cell membranes and increases membrane permeability to k

Although many studies have alluded to a role for boron (B) in membrane function, there is little evidence for a direct effect of B on the plasmalemma of higher plant cells. These studies were conducted to demonstrate, by electrophysiological techniques, a direct effect of B on the membrane potential (E_m) of sunflower (Helianthus annuus [L.], cv Mammoth Grey Stripe) root tip cells and to determine if the response to B occurs rapidly enough to account for the previously observed effects of B on ion uptake. By inserting a glass microelectrode into an individual cell in the root tip, the E_m of the cell was determined in basal salt medium (BSM), pH 6.0. The perfusion solution surrounding the root tissue was then changed to BSM + 50 micromolar H₃BO₃, pH 6.0. The exposure to B induced a significant plasmalemma hyperpolarization in sunflower root cells within 20 minutes. After just 3 minutes of exposure to B, the change in E_m was already significantly different from the negligible change in E_m observed over time in root cells never exposed to B. Membrane hyperpolarization could be caused by a stimulation of the proton pump or by a change in the conductance of one or more permeable ions. Since B has been shown to affect K⁺ uptake by plants, the electrophysiological techniques described above were used to determine if B has an effect on membrane permeability to K⁺, and could thereby lead to an increased diffusion potential. When sunflower root tips were pretreated in 50 micromolar B for 2 hours, cell membranes exhibited a significantly greater depolarization with each 10-fold increase in external [K⁺] than minus-B cells. Subsequent studies demonstrated that the depolarization due to increased external [K⁺] was also significantly greater when tissue was exposed to B at the same time as the 10-fold increase in [K⁺], indicating that the effect of B on K⁺ permeability was immediate. Analysis of sunflower root tips demonstrated that treatment in 50 micromolar B caused a significantly greater accumulation of K⁺ after 48 hours. The B-induced increase in K⁺ uptake may cause a subsequent stimulation of the H⁺-ATPase (proton pump) and lead to the observed hyperpolarization of root cell membranes. Alternatively, B may stimulate the proton pump, with the subsequent hyperpolarization resulting in an increased driving force for K⁺ influx.     

On the steady-state electrical potential difference across the thylakoid membranes of chloroplasts in illuminated plant cells.

The potential defference across the thyladoid membranes under steady-state saturating light conditions, measured with microcapillary glass electrodes, was found to be small as compared to the potential initially generated at the onset of illunimation. This result is discussed to be in agreement with quantitative estimates on the approximate magnitudes of the potential generating electron flux through the photo-synthetic electron transport chain and of the potential dissipating ion fluxes across the thylakoid membrane under steady-state conditions. It is concluded that a pH gradient of approx. 3-3.4 units is built up in the light across the membrane. The negative diffusion potential associated with this gradient is suggested to cause the transient negative potential observed in the dark after illumination.     

Reaction of the purple membrane with a carbodimide

Purple membrane was reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at pH 4.5 and 8.0. At pH 4.5, the reaction yields cross-linked bacteriorhodopsin. The cross-linking is inhibited by pretreatment of the membrane with papain, or by the presence of carbohydrazide or glycine ethyl ester in the reaction mixture. The product of the pH 8.0 reaction is not cross-linked, but it displays altered properties. Two measures of photochemical activity (light-induced change in proton binding (Δh?) and decay of photointermediate M) show changes indicative of slowed proton uptake. The Δh? is increased by ethyl dimethylaminopropylcarbodiimide. This increase is unaffected by pretreatment of the membrane with papain, and it is not reversed by NH₂OH. However, the reaction is inhibited by millimolar concentrations of CaCl₂. The altered Δh? is not apparent in detergent-solubilized membranes. Ethyl dimethylaminopropyl-carbodiimide does not appear to cause a large alteration in the membrane surface charge, as measured by Ca²⁺ binding.We conclude that (1) at acid pH, ethyl dimethylaminopropylcarbodiimide can be used for cross-linking or for attachment of specific probes to the C-terminal region of bacteriorhodopsin, and hence to the cytoplasmic side of the purple membrane, and (2) at alkaline pH, ethyl dimethylaminopropylcarbodiimide reacts at a different type of site and appears to inhibit the proton pump.     

Oxonol dyes as monitors of membrane potential. Their behavior in photosynthetic bacteria.

The reponses of oxonol dyes to single and multiple single turnovers of the photosynthetic apparatus of photosynthetic bacteria have been studied, and compared with the responses of the endogenous carotenoid pigments. The absorbance changes of the oxonols can be conveniently measured at 587 nm, because this is an isosbestic point in the 'light-minus-dark' difference spectrum of the chromatophores. The oxonols appear to respond to the light-induced 'energization' by shifting their absorption maxima. In the presence of K+, valinomycin abolished and nigericin enhanced such shifts, suggesting that the dyes, respond to the light-induced membrane potential. Since the dyes are anions at neutral pH values, they probably distribute across the membrane in accordance with the potential, which is positive inside the chromatophores. The accumulation of dye, which is indicated by a decrease in the carotenoid bandshift, poises the dye-membrane equilibrium in favor of increased dye binding and this might be the cause of the spectral shift. The dye response has an apparent second-order rate constant of approx. 2 . 10(6) M-1 . s-1 and so is always slower than the carotenoid bandshift. Thus the dyes cannot be used to monitor membrane potential on submillisecond timescales. Nevertheless, on a timescale of seconds the logarithm of the absorbance change at 587 nm is linear with respect to the membrane potential calibrated with the carotenoid bandshift. This suggests that under appropriate conditions the dyes can be used with confidence as indicators of membrane potential in energy-transducing membranes that do not possess intrinsic probes of potential.     

Effect of pH and iron content of transferrin on its binding to reticulocyte receptors

The effect of pH on the binding of apotransferrin and diferric transferrin to reticulocyte membrane receptors was investigated using rabbit transferrin and rabbit reticulocyte ghosts, intact cells and a detergent-solubilized extract of reticulocyte membranes. The studies were performed within the pH range 4.5–8.0. The binding of apotransferrin to ghosts and membrane extracts and its uptake by intact reticulocytes was high at pH levels below 6.5 but decreased to very low values as the pH was raised above 6.5. By contrast, diferric transferrin showed a high level of binding and uptake between pH 7.0 and 8.0 in addition to binding only slightly less than did apotransferrin at pH values below 6.5. It is proposed that the high affinity of apotransferrin for its receptor at lower pH values and low affinity at pH 7.0 or above allow transferrin to remain bound to the receptor when it is within acidic intracellular vesicles, even after loss of its iron, but also allow ready release from the cell membrane when it is exteriorized by exocytosis after iron uptake. The binding of transferrin to the receptor throughout the endocytosis-exocytosis cycle may protect it from proteolytic breakdown and aid in its recycling to the outer cell membrane     

On the steady-state electrical potential difference across the thylakoid membranes of chloroplasts in illuminated plant cells

The potential difference across the thylakoid membranes under steady-state saturating light conditions, measured with microcapillary glass electrodes, was found to be small as compared to the potential initially generated at the onset of illumination. This result is discussed to be in agreement with quantitative estimates on the approximate magnitudes of the potential generating electron flux through the photo-synthetic electron transport chain and of the potential dissipating ion fluxes across the thylakoid membrane under steady-state conditions. It is concluded that a pH gradient of approx. 3–3.4 units is built up in the light across the membrane. The negative diffusion potential associated with this gradient is suggested to cause the transient negative potential observed in the dark after illumination.     

Study of thymocytes resistance to ionising irradiation in vitro

The influence of ionising irradiation with doses of 0.5, 1, 2 Gy upon the suspension of mice thymocytes was studied. The state of the system of lipid peroxidation-antioxidation activity and structural and functional state of cell membrane was studied. It was found that, 15 minutes' after irradiation POL processes are within the norm. But the content of vitamins A and E decreases (the former after 0.5 Gy, the latter after 1 Gy). Changes in structural and functional state of thymocyte membranes can be observed by using fluorescent probes. Particularly, it turned out that after irradiation accessibility of incorporated proteins increases, the polarity of lipids increases and the lipophility of membrane lipids decreases. These changes can lead to short-time increase in specific functional activity of cells, but they can cause rapid death of cells as well.     

Barfly: sculpting membranes at the Drosophila neuromuscular junction

The ability of a cell to change the shape of its membranes is intrinsic to many cellular functions. Proteins that can alter or recognize curved membrane structures and those that can act to recruit other proteins which stabilize the membrane curvature are likely to be essential in cell functions. The BAR (Bin, amphiphysin, RVS167 homology) domain is a protein domain that can either induce lipidic membranes to curve or can sense curved membranes. BAR domains are found in several proteins at neuronal synapses. We will review BAR domain structure and the role that BAR domain containing proteins play in regulating the morphology and function of the Drosophila neuromuscular junction. In flies the BAR domain containing proteins, endophilin and syndapin affect synaptic vesicle endocytosis, whereas CIP4, dRich, nervous wreck and syndapin affect synaptic morphology. We will review the growing evidence implicating mutations in BAR domain containing proteins being the cause of human pathologies.