[et_pb_section fb_built=”1″ _builder_version=”4.24.2″ _module_preset=”default” custom_css_free_form=”@media (max-width: 375px) {|| .et-db #et-boc .et-l .et_pb_post_content_0_tb_body {|| width:100% !important;|| }}” locked=”off” global_colors_info=”{}” theme_builder_area=”post_content”][et_pb_row _builder_version=”4.27.4″ _module_preset=”default” custom_padding=”2em||0px||false|false” global_colors_info=”{}” theme_builder_area=”post_content”][et_pb_column type=”4_4″ _builder_version=”4.27.4″ _module_preset=”default” global_colors_info=”{}” theme_builder_area=”post_content”][et_pb_heading title=”Four Methods to Control pH in Bioreactors” _builder_version=”4.27.4″ _module_preset=”default” custom_css_free_form=”selector h1 {|| text-wrap: pretty;||}” global_colors_info=”{}” theme_builder_area=”post_content”][\/et_pb_heading][et_pb_image src=”https:\/\/www.alicat.com\/wp-content\/uploads\/2022\/01\/article-ph-control-bioreactor.webp” align=”center” force_fullwidth=”on” _builder_version=”4.27.5″ _module_preset=”default” custom_margin=”1.5rem||||false|false” hover_enabled=”0″ custom_css_free_form=”selector img {|| object-fit: cover;||}” locked=”off” global_colors_info=”{}” theme_builder_area=”post_content” module_id=”article-hero-image” sticky_enabled=”0″][\/et_pb_image][et_pb_divider color=”#2A3B47″ divider_weight=”2.5px” _builder_version=”4.27.4″ _module_preset=”default” height=”0px” custom_margin=”3rem||3rem||true|false” global_colors_info=”{}” theme_builder_area=”post_content”][\/et_pb_divider][\/et_pb_column][\/et_pb_row][et_pb_row _builder_version=”4.27.4″ _module_preset=”default” custom_padding=”0px||0px||true|false” locked=”off” global_colors_info=”{}” theme_builder_area=”post_content”][et_pb_column type=”4_4″ _builder_version=”4.27.4″ _module_preset=”default” global_colors_info=”{}” theme_builder_area=”post_content”][et_pb_text _builder_version=”4.27.4″ _module_preset=”default” custom_margin=”||2rem||false|false” global_colors_info=”{}” theme_builder_area=”post_content”]Keeping pH at healthy biological levels is potentially the most powerful tool in upstream bioprocessing to increase product titers. Bioreactions are expensive and run on long timelines, making it critical to maximize each run.<\/p>\n
Mammalian cells are only viable in environments with pH between 7.1 and 7.4. But the cells naturally produce carbon dioxide as a byproduct of respiration, driving pH down. Cells naturally have mechanisms to remove carbon dioxide and prevent toxic buildups, and buffering systems to keep their pH at healthy levels. Bioreactors need to mimic these features to ensure the cells can thrive.[\/et_pb_text][et_pb_heading title=”What influences culture pH?” _builder_version=”4.27.4″ _module_preset=”default” title_level=”h3″ global_colors_info=”{}” theme_builder_area=”post_content”][\/et_pb_heading][et_pb_text _builder_version=”4.27.4″ _module_preset=”default” custom_margin=”||2rem||false|false” global_colors_info=”{}” theme_builder_area=”post_content”]At the beginning of a bioreactor run, the buffer in the culture medium keeps the pH at the desired levels. Carbon dioxide may even be sparged in to keep the pH low. But during the exponential growth phase of the culture, the cells generate large amounts of carbon dioxide as a metabolic waste product. The buffer breaks, and the pH rapidly falls.<\/p>\n
At that point, the pH of the culture needs to be raised. Historically, base was pumped into the culture medium. Now, newer bioreactor setups use macro spargers to introduce large bubbles of air or nitrogen. These bubbles don\u2019t dissolve easily, so they strip the carbon dioxide from the culture, bringing it to the headspace of the bioreactor where it is removed.<\/p>\n
Recent literature has looked closely at the most effective means of controlling pH in a bioreactor. Here, we rank four approaches to pH control, from least to most effective.[\/et_pb_text][et_pb_text _builder_version=”4.27.4″ _module_preset=”default” global_colors_info=”{}” theme_builder_area=”post_content”]<\/p>\n
[\/et_pb_text][et_pb_text _builder_version=”4.27.4″ _module_preset=”default” custom_margin=”||2rem||false|false” global_colors_info=”{}” theme_builder_area=”post_content”]The partial pressure of dissolved carbon dioxide (pCO2<\/sub>) in a culture is a critical process parameter because of its effect on intra- and extracellular pH. pCO2<\/sub> determines the concentration gradient through which intracellular carbon dioxide (generated as a byproduct of cell growth) leaves the cells and enters the culture medium. If pCO2<\/sub> is too high, CO2<\/sub> can\u2019t leave the cells, so the intracellular pH will drop and the cells will die. If pCO2<\/sub> is too low, the cells lose will CO2<\/sub> too quickly, their pH will rise, and the intracellular environment will become too basic for the cells to be viable.[\/et_pb_text][et_pb_text _builder_version=”4.27.4″ _module_preset=”default” global_colors_info=”{}” theme_builder_area=”post_content”]<\/p>\n [\/et_pb_text][et_pb_text _builder_version=”4.27.4″ _module_preset=”default” custom_margin=”||2rem||false|false” global_colors_info=”{}” theme_builder_area=”post_content”]Particularly within the buffered pH range, pCO2<\/sub> and pH aren\u2019t directly correlated. Therefore, pCO2<\/sub> gives much less information about the health of the culture than the pH measurement.<\/p>\n The main reason for this is that with small changes to pH within the realm of cell viability (pH 7.1 – 7.4), bicarbonate buffer in the (extracellular) medium<\/a> will effect pCO2<\/sub> according to Le Chatelier’s principle.<\/p>\n Also, mammalian cells generate lactate, in addition to carbon dioxide, as a byproduct of respiration<\/a>. Lactate and CO2<\/sub> both make the culture more acidic, but lactate’s effect on pH isn’t reflected in the pCO2<\/sub> measurement.<\/p>\n After the buffer breaks and the pH starts to drop significantly, pCO2<\/sub> and pH are much more closely related. But it’s still not a strong means of monitoring and controlling pH — which is the variable that actually matters to increasing cell titers.[\/et_pb_text][et_pb_heading title=”Less effective: Pumping base into the culture” _builder_version=”4.27.4″ _module_preset=”default” title_level=”h3″ global_colors_info=”{}” theme_builder_area=”post_content”][\/et_pb_heading][et_pb_text _builder_version=”4.27.4″ _module_preset=”default” custom_margin=”||2rem||false|false” global_colors_info=”{}” theme_builder_area=”post_content”]Cells produce large amounts of carbon dioxide during the exponential growth phase, resulting in significant pH drops. A simple solution is to pump base into the bioreactor using direct feedback from pH sensors.<\/p>\n This method is most effective in:<\/p>\n [\/et_pb_text][et_pb_text _builder_version=”4.27.4″ _module_preset=”default” global_colors_info=”{}” theme_builder_area=”post_content”]<\/p>\n [\/et_pb_text][et_pb_text _builder_version=”4.27.4″ _module_preset=”default” custom_margin=”||2rem||false|false” global_colors_info=”{}” theme_builder_area=”post_content”]For a high product titer, the pH needs to be constant in the entire bioreactor, which requires thorough mixing. Otherwise, there will be localized pockets of particularly high or low pH. But mixing leads to foam and increases shear stress, both of which hurt cell viability.<\/p>\n These problems compound as bioreactor size increases, meaning that pumping base simply doesn\u2019t scale up well. Mixing time and agitation both need to increase with bioreactor size to avoid worsening localization, but this comes at the expense of increased foam and shear stress.<\/p>\n Pumping in base also increases the osmolality<\/a> (solute per kilogram of culture medium) of the culture. In larger bioreactors, this can decrease cell viability and product titer.[\/et_pb_text][et_pb_heading title=”More effective: Combining pumping base and sparging gases” _builder_version=”4.27.4″ _module_preset=”default” title_level=”h3″ global_colors_info=”{}” theme_builder_area=”post_content”][\/et_pb_heading][et_pb_text _builder_version=”4.27.4″ _module_preset=”default” custom_margin=”||2rem||false|false” global_colors_info=”{}” theme_builder_area=”post_content”]The traditional method of controlling pH involves pumping base into a bioreactor<\/a> during the early stages of a cell culture and then using sparging gases during the exponential growth phase. Base is particularly useful immediately after the buffer breaks, and sparging gases give tight control, for maximum yields from a culture.[\/et_pb_text][et_pb_text _builder_version=”4.27.4″ _module_preset=”default” global_colors_info=”{}” theme_builder_area=”post_content”]<\/p>\n [\/et_pb_text][et_pb_text _builder_version=”4.27.4″ _module_preset=”default” custom_margin=”||2rem||false|false” global_colors_info=”{}” theme_builder_area=”post_content”]The most up-to-date setups primarily use sparging gases to control pH. Recent literature has moved away from other methods to focus on optimizing the control loop for sparging gases using feedback from pH and other critical process parameters — including pCO2<\/sub>.[\/et_pb_text][et_pb_heading title=”Most effective: Using only sparging gases” _builder_version=”4.27.4″ _module_preset=”default” title_level=”h3″ global_colors_info=”{}” theme_builder_area=”post_content”][\/et_pb_heading][et_pb_text _builder_version=”4.27.4″ _module_preset=”default” custom_margin=”||2rem||false|false” global_colors_info=”{}” theme_builder_area=”post_content”]Sparging gases can control pH more tightly than the other methods discussed, primarily because they can react fastest to changes in other critical process parameters. The mix of sparging gases also gives the most precise control and the ability to respond to the largest amount of information from the various sensors in the bioreactor.<\/p>\n Specifically, several factors make this method most effective:<\/p>\n [\/et_pb_text][et_pb_text _builder_version=”4.27.4″ _module_preset=”default” global_colors_info=”{}” theme_builder_area=”post_content”]<\/p>\n [\/et_pb_text][et_pb_text _builder_version=”4.27.4″ _module_preset=”default” custom_margin=”||2rem||false|false” global_colors_info=”{}” theme_builder_area=”post_content”]If gases flow in too quickly, they introduce shear stress. If gases flow in too slowly, the cells won\u2019t reproduce as quickly as they would otherwise be able, and will not achieve maximum yield.<\/p>\n Well-designed spargers, in contrast, will scale well and produce consistently high titers.[\/et_pb_text][et_pb_text _builder_version=”4.27.4″ _module_preset=”default” text_font=”|700|||||||” custom_margin=”||2rem||false|false” locked=”off” global_colors_info=”{}” theme_builder_area=”post_content”]Learn more: Comparing stirred tank, shaker, and rocker bioreactors pCO2<\/sub> effects pH, but they aren\u2019t tightly correlated<\/h4>\n
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Pumping base doesn\u2019t guarantee a constant pH throughout the bioreactor<\/h4>\n
Pumping base has significant downsides, and sparging gases have significant advantages<\/h4>\n
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Intelligent sparger engineering and control loop design are critical<\/h4>\n