BIOLOGICS APPLICATION SNAPSHOT

At-line spent media analysis is now a reality – don’t get left behind.

BACKGROUND There is a need for advanced process analytic technologies that can monitor cell media nutrients and cellular metabolites alongside bioreactors in process development labs. Timely analysis of media components during a process contributes to the understanding of critical environmental or nutrient influences on cell health and productivity. Biochemical analyzers based on enzymatic assays deliver useful data, but they do not give a complete picture of the bioreactor state as they are limited to a small number of analytes. Comprehensive spent media analysis of amino acids and other media components is bottlenecked by core lab turn-around times which slow media optimization and process decisions severely. To elevate process development decisions to real-time, researchers require new analytical platforms that simplify analysis and accelerate delivery of actionable intelligence.

THE EXPERIMENT DISCUSSION Traditional spent media analysis requires at least two Spent media analysis should no longer be a lengthy separate HPLC runs for the coverage of both amino process for any researcher. As highlighted here, a simple acids, vitamins, and other components. The RebelTM 7 minute daily run on the Rebel can provide quantitation eliminates this complexity by analyzing each sample for for a broad set of media components like the amino amino acids, vitamins, amines and dipeptides in a single acids shown here. In this fed-batch process, there were run. In this example, tiny bioreactor samples (approx. amino acids like Asn, Gln, His, Met, Pro, Ser and Trp which 10 µL) were diluted 100X with Rebel diluent and run on all were lower in concentration at the end of the process the Rebel. For simplicity, the average from four replicate compared to their initial concentrations. Conversely, runs per sample was plotted for the amino acids present there was a buildup of the amino acids like Ala, Arg, in the media only (Figure 1). Asp, Glu, Gly, Phe, Thr, Tyr and Val. In particular, there was a substantial increase in the amount of Gly in the culture highlighting that the media contained excessive amounts of it in the feed. With the Rebel alongside both bioreactors and conventional biochemical analyzers, process development researchers can now reveal deeper process insight via media nutrient levels in near real-time.

Figure 1: Selected media components from a time-lapse spent media analysis of a 14-day fed-batch process of Chinese-hamster ovary cells producing an IgG. The color scale is the normalized amount of each component relative to the first concentration it was detected at. Black squares represent time points when a component was not detected.

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Select the best chemically defined CHO media from the start with the Rebel.

BACKGROUND The selection of chemically defined cell media and feed systems is one of the initial steps in process development. Regardless of the cell line, industrial process or bioreactor type, all development teams must wade through the fog of spent media analysis and model building to define a media optimization strategy. A key aspect of that journey is the relationship with cell culture media providers, both outside vendors and specialized teams within your organization. Performing your own quick assays of the media you receive ensures consistency in the cell media fed to your cells.

THE EXPERIMENT Below is a rapid snapshot of eight chemically defined of the media once received is appropriate. In this cell media optimized for CHO cells (Figure 1). All experiment, certain amino acids like His, Met, Phe media tested did not include glutamine. Samples were and Tyr were present at very similar levels regardless taken from freshly delivered media and diluted 100X of the formulation. Other amino acids like Ala, prior to analysis. The concentrations reported by the Asn, Asp, Glu, Gly and Trp were not detected in all RebelTM were averaged across five replicates for each formulations. The dipeptide cystine was only present formulation. in one formulation, and there was a 12.5X difference between the highest (formulation D) and lowest DISCUSSION (formulation B) concentration of Arg. The vitamins B6- It is vital for researchers to work with established OH (pyridoxine) and NAM (nicotinamide) were present cell media providers to ensure that their cell media at high enough concentrations to be detected at this choice is right for their application, and that handling dilution in certain formulations whereas the nutrient choline was present in all the media tested.

Figure 1: Comparison of eight commercially available chemically defined CHO medias run on the Rebel. Components that were absent or not detected are shown with white boxes.

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Analyze media without removing your mAb on the Rebel.

BACKGROUND Engineering a high producing process is one of the major goals in biotherapeutic programs. The productivity correlates to the commercial viability of a biotherapeutic, and it can be attained through a combination of appropriate cell line selection and achieving process development milestones. Media optimization is an integral step towards achieving high concentrations of a desired monoclonal antibody (mAb), but challenges arise when performing media analysis in the presence of high concentrations of a protein. Protein precipitation or protein A purifications from harvested cell media may be required to separate the mAb from the media to make the samples amenable for traditional chromatography-based strategies for media analysis. The Rebel is not a chromatographic technique so dedicated clean up steps are not required as demonstrated below.

THE EXPERIMENT DISCUSSION Media analysis was performed on a commercially Improved cell line selection strategies and high cell available chemically-defined media (without Gln) density perfusion cultures have allowed for high titers with varying levels of an IgG1 in solution. The samples in many bioprocesses. However, the high protein were diluted 100X with the Rebel diluent, placed into concentrations typical of these productive processes vials and run immediately. No protein precipitation or may interfere with traditional media analysis without purification step was performed prior to analysis, and additional sample preparation and clean up steps that all samples were run for five replicates. (Figure 1) are required to remove the excess protein from the harvest. The Rebel has been optimized to run media analysis both with and without high levels of protein in solution, so no additional sample preparation steps are needed. As shown here, there is no interference from the increasing amounts of the mAb that were present in the cell media samples. The three highest concentration analytes measured were Arg, Asn, and Asp with concentrations of 17.44 ± 1.21 mM, 6.08 ± 0.21 mM and 8.91 ± 0.55 mM, respectively. The three lowest concentration media components were choline, Met and Trp with average concentrations of 0.64 ± 0.09 mM, 0.57 ± 0.06 mM and 0.46 ± 0.04 mM, respectively. The Rebel is a great at-line tool for groups on the cutting edge of bioprocessing who are cultivating high titers from their bioreactors and need rapid analytics to monitor nutrient levels from their media samples. Figure 1: Concentrations of media components analyzed from chemically-defined CHO media with different concentrations of a mAb present.

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Screening of chemically-defined media for suspension cultures of HEK293 cells.

BACKGROUND With a history of stable growth and easy transfection, HEK293 cells have been a workhorse cell line in cell biology and bioprocessing labs. Originally derived from human embryonic kidney cells grown in tissue culture, HEK293 cell lines are commonly used to produce both therapeutic proteins due to high efficiencies of transfectability and viral vectors such as lentivirus and adeno-associated virus (AAV). HEK293 was derived from adherent cells that prefer a compatible surface for growth, but this had limited the scalability of the platform. Therefore, there has been a desire to improve both process productivities and consistencies of the adherent HEK293 cells grown in serum-supplemented media. As a result, processes have been adapted for culturing of HEK293 cells in suspension and with serum-free, chemically-defined cell media.

THE EXPERIMENT DISCUSSION Commercially available chemically-defined cell media Across the four vendors, there was a high variation from four vendors were tested in this study. Individual in the fresh media composition. For instance, the samples of each fresh serum-free media (without Gln) dipeptide Ala-Gln was only detected in vendor 2, optimized for suspension cultures of HEK293 were whereas hydroxyproline (Hyp) was only detected in diluted 100X with the RebelTM diluent, placed into a vendor 3. Asparagine (Asn) was 25x higher in vendor vial, and run for five replicates each with no additional 3’s media compared to the next highest composition. sample preparation. (Figure 1) Vendor 3 had the highest concentration in roughly two-thirds of all the detected components in the study. In contrast, vendor 4 had the lowest concentration in over half of the components. In its entirety, researchers can utilize this type of quick media screen to survey their cell media options before investing in a media platform for their HEK293 suspension processes. Carrying this type of analysis forward whenever a fresh media batch is received or periodically during storage, would establish a historical record to refer to in case any unexpected variations arise in the culture’s health and productivity.

Figure 1: Media component concentrations from chemically-defined HEK293 media from four different vendors. Error bars are from the standard deviation of n = 5 replicates.

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Assessing the differences in fresh commercially available hybridoma media.

BACKGROUND Hybridomas are hybrid cell lines used to produce monoclonal antibodies. They are made by fusing B cells with myelomas to make immortalized and productive cell lines. Early in the field of hybridoma development, researchers used base media with serum-supplementation to support the growth and viability of the cell lines. An issue with serum use was the lot-to-lot variability associated with using this undefined component. As the desire to minimize run-to-run variability emerged, the focus shifted to culturing hybridomas in chemically-defined media. A common approach was to mix base media like DMEM/F12 with recombinant supplements. An encountered drawback from this approach was lower antibody production. To increase output, teams formulated chemically-defined media specifically for hybridoma manufacturing with higher levels of some media components (e.g., amino acids) compared to DMEM/F12. The higher levels of the media components supported better hybridoma growth and productivities, but the exact formulations were proprietary to the vendors.

THE EXPERIMENT DISCUSSION Two different chemically-defined cell medias intended The concentrations of media components are essential for hybridoma culturing were tested. Both media to consider when doing media optimization studies. samples were handled following the manufacturers’ Here, the nutrient levels in two chemically-defined instructions. The samples were diluted 50x before media were significantly higher than in the DMEM/ analysis on the Rebel with no additional sample F12 base media, which is typically used with serum- preparation. Each vendor’s media was run five times, supplementation. The levels of the amino acids Ala and levels were compared to the known formulation (73x and 71x higher), Asn (38x and 31x higher), Glu values of DMEM/F12 components. (Figure 1) (13x and 11x higher), Pro (13x and 15x higher) and Ser (20x and 12x higher) were significantly higher than the base media formulation levels in vendor 1 and 2, respectively. All amino acids were at least 50% higher in the chemically-defined media compared to the base media except for Gly, which was not detected in vendor 2’s media. Other media components like choline were 49x and 5x higher than the base media in vendor 1 and vendor 2, respectively. Vitamins (B1, B6-OH, NAM) were part of vendor 1’s media and were at least double the levels of DMEM/F12. When transitioning from a general base media to a chemically-defined media, run a quick Figure 1: The concentrations of the amino acids, vitamins, and other media analysis on the Rebel to give you the insight to nutrients in chemically-defined hybridoma media from two vendors help boost hybridoma growth and productivity. compared to the formulation values of DMEM/F12. Error bars are from the standard deviation of n = 5 replicates.

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Chemically-defined CHO media variations in dry and liquid media are identified with the Rebel.

BACKGROUND Chemically-defined CHO media comes in two forms: premixed liquids and dry powders. When deciding which type of media to use, a researcher must decide on the amount of media needed for the project. Liquid media allows for direct use in small format bioreactors, flasks and microplates. Powdered media must be reconstituted into solution, however the shipment and storage of powders is easier and cheaper, so it is often used for larger studies and volumes. Many organizations have dedicated media prep teams and labs to mix formulations of powdered media to simplify the workflows of cell culture and upstream process development labs. Running a quick fresh media assay prior to feeding the culture ensures consistency of the media components when scaling up or during the transition between liquid-to-dry media blends.

THE EXPERIMENT DISCUSSION The concentrations reported below are from the analysis The twenty shared media components tracked in this of a dry and liquid chemically-defined CHO media study highlight how there can be differences between the formulation (Figure 1). Both media blends came without initial forms of media despite the claims that the blends glutamine. For the dry formulation, the manufacturer’s are identical. Sixteen of the components were all within directions were followed to prepare the media. The ±10% of one another in concentration. However, there samples were diluted 10X with Rebel diluent and the were four amino acids that differed by more than 20%, reported concentrations were averages from five replicate and in all cases their concentrations were higher in the tests. liquid media compared to the dry media. His, Lys, Ser and Thr were all lower by 22%, 49%, 62% and 21%, respectively, in the dry media compared to the liquid media. The Rebel affords the ability to quickly screen media prior to adding it to your bioreactor so that variations in the nutrient levels like these can be detected ahead of time. Although the original formulation may be the same, one should not assume that transitioning from the liquid to dry formulation of a chemically-defined media will result in identical values. Storage conditions and mixing may cause shifts in the concentrations of media components so verify the media.

Figure 1: Comparison of media component concentrations between dry and liquid formulations of a commercial chemically-defined CHO media.

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Be fast and accurate with at-line cell media analysis with the Rebel.

BACKGROUND The selection of chemically-defined cell media for your CHO cell line is a critical step in process development. The media composition typically undergoes several modifications to ensure it supports cell productivity and the desired critical quality attributes of the biotherapeutic. The amino acid content of the media must be monitored during this process, and this test is routinely performed with liquid chromatography instruments with absorbance detection (e.g. HPLC-UV) which are located in centralized laboratories. While this approach yields accurate data, researchers must withstand delays of up to several weeks before getting data which hampers any real-time decision making.

THE EXPERIMENT DISCUSSION The amino acid composition of a commercially For the 17 detected amino acids and dipeptides, all available chemically-defined CHO media (without processed values were closely comparable (±5%) Gln) was assayed with both a HPLC-UV system and between the Rebel and HPLC. Of note, both Ala and the Rebel. In contrast to the third-party derivatization Gly were not detected by either approach. The lowest strategy required for the HPLC amino acid analysis, the concentration component was the dipeptide cystine media was simply diluted 100X with diluent, placed with reported concentrations of 0.22 ± 0.03 mM into a vial and run immediately with the Rebel. By and 0.24 mM by the Rebel and HPLC, respectively. eliminating the derivatization step with the Rebel, all The highest concentration amino acid was Asn with samples were run for five replicates. (Figure 1) reported concentrations of 5.85 ± 0.37 mM and 6.12 mM by the Rebel and HPLC, respectively. As demonstrated here, at-line amino acid analysis of chemically-defined cell media by the Rebel facilitates faster analytic turnaround times without the complexity of traditional core lab approaches. Additionally, the Rebel does not tradeoff accuracy for this improvement, and its results are comparable to other routine approaches. With these advantages, the Rebel shines in environments utilizing parallel, small volume microbioreactors, shake flasks and microwell plates where the volume requirements and slow response of conventional analytics approaches bottleneck the speed that process development groups strive to maintain.

Figure 1: Comparison of the Rebel and HPLC-UV amino acid analysis of a commercially available chemically-defined cell culture media for CHO cells.

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Comparison of various conventional and chemically-defined T cell medias.

BACKGROUND T cells are lymphocytes that have special cell surface receptors that recognize antigens, which is vital for an immune response. Recently, chimeric antigen receptor (CAR) T cells have been created to target particular antigens on tumor cells as a type of immunotherapy. CAR-T cell therapies are currently involved in numerous clinical trials as a potential breakthrough therapy across a wide variety of malignancies. CAR-T cells are commonly grown and expanded in RPMI (Roswell Park Memorial Institute) 1640 basal media supplemented with up to 10% fetal bovine serum (FBS). For clinical applications, there is a desire to use cell media platforms that allow for higher expansion rates, have more lot-to-lot consistency between batches, and exclude FBS and other animal-derived components due to concerns over contamination. Chemically-defined media provides improved batch-to-batch results and removes some regulatory concerns since it excludes serum products. However, serum-free media may yield lower CAR-T efficacies, so understanding how to optimize the media to improve the therapy is a necessary step.

THE EXPERIMENT DISCUSSION In this study, the Rebel was used to probe the essential There were significant differences in the essential amino amino acid content of RPMI + FBS, combo (both base acid content between the traditional RPMI with 10% and expansion) media with and without human serum FBS media and the chemically-defined media for T cell albumin (HSA), base media alone, and expansion media growth. The serum-free combination media (used as alone. All media samples were handled following the both a base and expansion media, blue) had the highest manufacturers’ instructions. The samples were diluted levels of most essential amino acids. The serum-free 25x before analysis on the Rebel with no additional expansion media (yellow) had the second-highest levels sample preparation. Each media sample was run five of all the media and had the highest levels of Trp. In fact, times. (Figure 1) other than the serum-free expansion media, only the RPMI + 10% media had Trp detected in the formulations. RPMI + 10 % serum media (black) and the serum-free chemically-defined base media (pink) had the lowest levels of the essential amino acids. A combination of media that included HSA (green) had essential amino acid levels that were moderate when compared to the highest and lowest media. With this quick insight from the Rebel, researchers can make informed decisions about which media platform is best for growing CAR T-cells.

Figure 1: The concentrations of the essential amino acids in five cell media platforms used for T-cell culturing. Error bars are from the standard deviation of n = 5 replicates.

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Comparing mesenchymal stem cell media with supplements on the Rebel.

BACKGROUND Mesenchymal stem cells (MSCs) are multipotent adult stem cells, which can differentiate into specialized cell types such as bone (osteoblasts), cartilage (chondrocytes), and fat (adipocytes). In addition to differentiation, MSCs have the capacity for self-renewal, so their therapeutic potential is a topic of high interest for tissue engineering and regenerative medicine. In the lab, cultures of MSCs are fed cell media that has been specifically designed to maximize the MSC growth potential, and the media commonly includes serum. However, due to the variability in serum compositions and the risk associated with animal component-containing media, many research groups have adopted serum-free culturing practices to grow MSCs. This practice allows groups to have more consistency between experiments while meeting regulatory requirements for clinical studies. In the absence of serum, proprietary supplements are added to the MSC cultures that provide growth factors for both expansion and differentiation.

THE EXPERIMENT DISCUSSION Two different commercial serum-free MSC media There was no detected difference in the amino acid without glutamine were tested. The MSC media was levels of Media 1 when the supplement was added, tested both with and without the supplied supplements indicating that the supplement did not contain to evaluate the change in the amino acid profile additional amino acids in the formulation. However, between the two formulations. All media samples in Media 2, there was a significant increase in the were prepared following the manufacturers’ protocols concentration of nine amino acids - Gly, His, Ile, Phe, and diluted 10x before analysis on the RebelTM with no Pro, Ser, Thr, Trp, and Val. Of these amino acids, Gly additional sample preparation. (Figure 1) showed the lowest increase after the supplement was added (1.5x increase). On the other end, Ile (10.4x higher) and Pro (22.4x higher) both had the most substantial increases in concentration after the supplement was added. The other six amino acids increased their levels anywhere between 3x - 8.5x with the supplement added compared to the base media alone. To ensure consistency when culturing MSCs, researchers should understand what is being added to their media. The Rebel is a simple tool to analyze MSC media as it is freshly prepared and during the culturing process to probe the nutrient uptakes of their cells. Figure 1: Media amino acid profile from chemically-defined MSC media with and without the supplied supplements. Error bars are from the standard deviation of n = 5 replicates.

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Comparison of the essential amino acid contents of Sf9 and Sf21 insect cell media.

BACKGROUND Insect cell lines like Sf9 and Sf21 were isolated initially from Spodoptera frugiperda. They are commonly used for recombinant protein expression, vaccine development, and viral vector production. These cell lines are very adaptable to both culturing in adherent and suspension cultures, and in traditional media with serum, serum- free media, and protein-free media. The selection of the appropriate media is based on a variety of conditions, but one of the most important is the amino acid content. Many amino acids are not synthesized by insect cells, and some are crucial to be added to cell media for optimal growth and/or protein production. Knowledge of the insect cell metabolism coupled with an understanding of the amino acid content of the cell media allows for a more informed process development analytics strategy to correlate changes in cell growth/viability and productivity to the nutrient contents of the media.

THE EXPERIMENT DISCUSSION Three different cell medias intended for Sf9 or Sf21 There were varying levels of all the essential amino insect cell line growth were used in this study. The acids, regardless of the type of media formulation. The insect cell media tested included a traditional media, traditional media had substantially higher levels of His a chemically-defined media, and a serum-free compared to the chemically-defined and serum-free media. All media samples were handled following the media by 7.4x and 11.3x, respectively. However, the manufacturers’ instructions. The samples were diluted traditional media had the lowest levels of five amino 100x before analysis on the Rebel with no additional acids – Ile, Leu, Met, Phe, and Val. The chemically- sample preparation. For simplicity, only the essential defined media had the highest levels of Ile, Lys, Met, amino acids are shown for a comparison of the three Phe, Trp, and Val. Also, it was tied with the serum- media. (Figure 1) free media for the highest levels of Leu. Other than Leu, the serum-free media had the second or third most amount of any other essential amino acid shown here. This brief analysis demonstrates how it is vital to consider measuring the composition of amino acids in insect cell media. Researchers should consider how the amino acid composition, in conjunction with the undefined components in traditional media (e.g., serum) and serum-free media (e.g., peptones, hydrolysates, yeastolates), may all affect the growth and productivity of their processes.

Figure 1: Essential amino acid profile from traditional, chemically- defined, and serum-free insect media. Error bars are from the standard deviation of n = 5 replicates.

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Supplementing RPMI media with different amounts of fetal bovine serum.

BACKGROUND Roswell Park Memorial Institute (RPMI) developed RPMI-1640 media from modification to McCoy’s 5A media. RPMI media is one of the most used chemically-defined and protein-free cell media since it works across a variety of mammalian cell lines (e.g., stem cells, T and B cells, hybridomas, HEK293, THP-1 leukemia cells, etc.) grown in suspensions and as monolayer cultures. RPMI media is usually supplemented with fetal bovine serum (FBS) at levels of 1 to 10% to supplement growth factors necessary to sustain specific cell line processes. However, FBS supplemented media presents some issues when there is a desire to perform routine quantitative analysis of the media. The high serum protein contents in the mixed media may interfere with standard chromatography columns for amino acid analysis. Removal of the proteins with precipitation may be required to prepare the samples before analysis with traditional chromatographic platforms. This additional sample prep may affect amino acid recoveries resulting in biased results with conventional analytics approaches for media analysis.

THE EXPERIMENT A commercially available RPMI media (without glutamine) and cell culture grade FBS (USA origin) were mixed at the listed levels below. All media samples were handled following the manufacturer’s instructions. Final solutions were diluted 10x before analysis on the Rebel with no additional sample preparation. (Figure 1)

Figure 1: Media component concentrations from RPMI and RPMI with FBS supplementation. Error bars are from the standard deviation of n = 6 replicates.

DISCUSSION The analysis was able to quantitate 24 different media components, including 19 amino acids, at a single dilution level. There were virtually no differences in the media components’ concentrations between the base RPMI media and the RPMI samples with up to 10% FBS added. The exceptions to this observation were both Ala and Gln. Both Ala and Gln were not in the original formulation of RPMI. They were not detected in the base media and 1% FBS-supplemented media. However, in 5% and 10% FBS, both Ala and Gln were identified, which were consistent with the separate observation of detecting both these amino acids in pure serum tested at a 10x dilution (not shown). Corresponding to the formulation, Arg had the highest levels of all amino acids averaging 2.108 mM. In comparison, Trp had the lowest concentration with an average of 0.011 mM. With the Rebel, one can quickly and confidently screen serum-supplemented media formulations to ensure batch-to-batch media consistency before introducing the cell media to cultures.

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Supplementing IMDM media with different amounts of fetal bovine serum.

BACKGROUND Iscove’s Modified Dulbecco’s Medium (IMDM) is an amino acid, vitamin, and inorganic salt enriched version of Dulbecco’s Modified Eagle Medium (DMEM). IMDM is commonly used for high-density and high-proliferating cell cultures of fibroblast-like cell lines (e.g., COS-7), hematopoietic stem cells, macrophages, and T lymphocytes (e.g., Jurkat cells). Since IMDM is a chemically-defined media, it lacks growth factors, proteins, and lipids. To support cell growth, IMDM is often supplemented with fetal bovine serum (FBS) at various levels (e.g., 1 to 10%) depending on the cell line. When using chemically-defined cell media like IMDM supplemented with FBS, media analysis can be burdensome. Interferences arise between the high levels of the proteins in the added FBS with standard chromatography columns. Additional sample preparation may be required to remove and precipitate the proteins from the sample media. These added steps may affect recoveries of the trace media components (e.g., amino acids and vitamins), resulting in issues with conventional analytics approaches for media analysis.

THE EXPERIMENT A commercially available IMDM media without glutamine and cell culture grade FBS (USA origin) were mixed at the listed levels below. All media samples were handled following the manufacturer’s instructions. Final solutions were diluted 20x before analysis on the Rebel with no additional sample preparation. (Figure 1)

Figure 1: Media component concentrations from IMDM and IMDM with FBS supplementation. Error bars are from the standard deviation of n = 6 replicates.

DISCUSSION At a single dilution of 20x with Rebel diluent, the analysis was able to quantitate 24 different media components, including 18 amino acids and three B vitamins. There were virtually no differences in the media components’ concentrations between the base IMDM media alone and the IMDM samples with up to 10% FBS added. Amongst the amino acids, Trp had the lowest levels averaging 0.032 mM, and lysine had the highest average concentration of 1.165 mM across the four formulations tested. Vitamin B1 (thiamine) was the lowest concentration of all the media components detected with an average concentration of 0.004 mM. Gamma-aminobutyric acid (GABA) and beta-alanine (βAla), which are not universally present in all chemically-defined media formulations, were detected across all samples at average concentrations of 0.028 mM and 0.030 mM, respectively. With the Rebel, one can quickly and confidently screen serum-supplemented media formulations. This test ensures batch-to-batch supplemented media consistency before introducing the media to cell cultures.

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Don’t sweat your cell media additives when you run it on the Rebel.

BACKGROUND Surfactants, antifoams, salts solutions, detergents, and buffers are commonly used in bioprocesses and fermentations to address a variety of needs. For instance, antifoams are widely used in bioreactors and shake flasks to counteract the unwanted foaming that may happen when gassing is performed to maintain a desired dissolved oxygen level. If foams are left to develop, they can cause sterilization concerns if the foam escapes the reactor vessel. Antifoam supplementation can eliminate this issue, but researchers must select an appropriate type so as not to impact cell growth or process yield negatively. Additionally, the use of antifoams and other additives may interfere with the chemical analysis of samples taken during the process. Additional sample cleanup and preparation may be required before analysis to ensure that some sensors and instrumentation will not foul or that the results are not biased with chemical interferences.

THE EXPERIMENT Individual samples of fresh chemically-defined CHO Across the seven cell media additives, the essential media (without Gln) were prepared with seven different amino acid Met and non-essential amino acid Gly had additives. These seven samples, along with an untreated the most significant average concentration deviations (no additives) sample were diluted 100X with the Rebel from the control (no additives) media formulation of diluent, placed into a vial and run immediately on the only 5% and 6%, respectively. These small measurement Rebel with no additional sample preparation. (Figure 1) deviations from the original media demonstrate that with these types of additives, the Rebel does not need DISCUSSION any kind of extraction or sample clean up steps before For the 19 amino acids present in the original analysis. The lack of additional preparation maintains a chemically-defined CHO media, there were very minor fast turnaround time and keeps the analysis simple for effects on the reported values measured by the Rebel. engineers.

Figure 1: Amino acid concentrations from chemically-defined CHO media in the presence of various common additives.

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The Rebel handles non-ionic surfactants in cell media samples with ease.

BACKGROUND Non-ionic surfactants like Tween 20 and Tween 80 can be additives to cell media for a variety of purposes. If Tweens are used at low concentrations, they are considered mild surfactants since they do not interfere with protein-protein interactions. Tweens primarily work to disassociate protein-lipid and lipid-lipid interactions, so they are often used to lyse cells to improve protein extraction and isolate nuclei. Tween may improve protein solubility and secretion, in addition to preventing aggregation and stabilizing high-order structure. In some bioprocesses, Tweens may affect cell viability, growth rates, and biofilm formation. Additionally, Tween containing samples might encounter interference with traditional chromatographic approaches and require extraction or purification procedures for removal before analysis.

THE EXPERIMENT Individual samples of fresh chemically-defined cell just a single 100X dilution level, the Rebel was able to media (without Gln) were prepared with 1 % of either quantitate 19 amino acids and other media components Tween 20 or Tween 80. These two samples, along (e.g., choline, nicotinamide, and β-alanine) in all three with the regular media sample, were diluted 100X with samples tested in this study. There was no significant the Rebel diluent, placed into a vial, and run for three difference in the reported concentrations for any of the replicates each on the Rebel with no additional sample analytes in all the samples and replicates that were run. preparation. (Figure 1) Therefore, there was no evidence of interference with the addition of either 1% Tween 20 or Tween 80 in the DISCUSSION chemically-defined cell media. If non-ionic surfactants Gentle polysorbate surfactants like Tween have a variety like Tween 20 or 80 are commonly added to your of applications in bioprocessing. To ensure success processes or fermentations, and you do not want to when sampling from solutions containing Tween 20 or delay your routine cell media analysis measurements, 80, analysis workflows should be screened to ensure no run them at-line on the Rebel. interference or bias is included in the measurements. At

Figure 1: Media component concentrations from chemically- defined CHO media in the presence of the non-ionic surfactant Tween 20 and Tween 80. Error bars are from the standard deviation of n = 3 replicates.

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How comparable are different vendors’ LB media?

BACKGROUND Lysogeny broth (LB) is one of the most popular media used for growing bacteria, especially recombinant strains of E. coli. Formulations of LB media contain a mix of salts, tryptone, and yeast extract to provide the essential nutrients like trace minerals, vitamins, and amino acids. For E. coli cultures, LB media is usually used to support cloning, and the production of plasmid DNA, DNA, and recombinant proteins. Common additives to LB media include antibiotics for selective cultivations and agar for plate cultures. Due to its broad usage, LB media can be purchased in liquid or dry powder mix from many vendors. Still, each vendor’s source of the individual components of the media may differ. This variance could change the concentrations of individual components in the media, which may affect the culture’s growth rates.

THE EXPERIMENT All commercially available LB-Miller liquid media was tested fresh, and handled following the manufacturer’s instructions. All samples were diluted 250x before analysis on the REBEL with no additional sample preparation. (Figure 1)

Figure 1: Amino acid concentrations from LB media diluted by 250x before analysis. Error bars are from the standard deviation of n = 5 replicates.

DISCUSSION All but three amino acids had very comparable concentrations across all four vendors. The three amino acids that displayed large deviations were alanine, lysine, and threonine. For alanine, vendor 3 reported a 68% higher concentration than the average of vendors 1, 2, and 4. There was a 190% higher value for lysine detected in vendor 1 compared to the average concentration in vendors 2, 3, and 4. Additionally, vendor 1 had the highest concentration of threonine, which was 360% higher than the average of vendors 2, 3, and 4. Due to the high variation in some amino acids, it would be wise to routinely check fresh media formulations of LB media. Using the REBEL to quickly screen different vendor’s LB media may support reproducible cultures and media preparations for bacterial cultures.

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High variations of analyte levels are detected in Terrific broth (TB) media.

BACKGROUND Terrific broth (TB) is an enriched bacterial media commonly used for high-density cultures of recombinant E. coli for protein expression or plasmid purification. Compared to other conventional bacteria media like Lysogeny broth (LB), it has higher concentrations of peptone, yeast extract, and glycerol as a carbon source. Another critical difference between TB and LB is mono and dibasic phosphates in the media, which improve the buffering capacity. TB media is easily acquired premixed in liquid or dry formulations from commercial sources. Although all sources use the same known formulation to mix the media, the exact concentrations of the individual media components may differ due to the different sources of the raw materials. These differences may affect the culture’s growth or productivity.

THE EXPERIMENT All commercially available liquid TB media was tested fresh and handled following the manufacturer’s instructions. All samples were diluted 250x before analysis on the REBEL with no additional sample preparation. (Figure 1)

Figure 1: Media component concentrations from Terrific broth diluted by 250x before analysis. Error bars are from the standard deviation of n = 5 replicates.

DISCUSSION There were several media components with similar concentrations across the five different vendors tested. For instance, the individual concentrations of Arg, Asn, Asp, Glu, His, Ile, Leu, Met, Phe, Trp, Tyr, and Val across all five vendors were all within ±20% of the average of each individual component. For the two B-vitamins detected in this study, B6-OH (pyridoxine) and NAM (nicotinamide), vendors 1, 2, and 3 had no detectable levels of B6-OH, and vendors 1 and 2 had no detected levels of NAM at the dilution level used in this experiment. The most considerable deviations from the average detected levels were observed in the amino acids Ala, Gly, Lys, and Thr. Vendor 2’s level of Ala and Gly was 36% higher and 31% lower, respectively, than the average value of the five vendors’ media. Vendor 3 had a 79% higher concentration of Lys compared to the average of the other vendors. Finally, vendors 2 and 3 had a 30% lower and 89% higher level, respectively, of Thr compared to the averages. These extreme differences may influence growth or productivities, so a quick media screen before the start of culture is advised. Identifying concentration changes across subsequent runs can help build process knowledge to prevent unexpected deviations.

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Investigating the high nutrient variations in Super Optimal Broth.

BACKGROUND Super Optimal broth (SOB) is a nutrient-rich microbiological media used for bacterial growth, typically with recombinant E. coli. Compared to lysogeny broth (LB) media, SOB media results in higher plasmid transformation efficiencies. SOB media has more peptone compared to LB media and includes magnesium sulfate and potassium chloride. SOB media differs from SOC (SOB media with catabolite repression) media due to its lack of glucose. Like other undefined media prepared from mixtures of digested proteins and yeast extracts, there can be variability in formulations due to processing changes and raw material sources. Therefore, cultures’ growth rates and productivities may differ when different vendor’s media are used in otherwise identical experiments.

THE EXPERIMENT All commercially available SOB liquid media was tested fresh and handled following the manufacturer’s instructions. All samples were diluted 500x before analysis on the REBEL with no additional sample preparation. (Figure 1)

Figure 1: Media component concentrations from Super Optimal broth media diluted by 500x before analysis. Error bars are from the standard deviation of n = 5 replicates.

DISCUSSION There was minimal similarity in the concentrations of most components across all five vendors’ SOB media. In fact, there was not a single component that had variations within 30% of the average value in all five vendors. Pyridoxine (B6-OH) was only detected in vendor 5’s media. Asn was not detected in vendor 3, Met was not detected in both vendors 1 and 5, and Trp was not detected in the media of vendors 1 and 3. The most considerable deviations from the average individual components levels were Pro in vendors 1 and 3. It was detected 74% lower and 117% higher, respectively, than the average concentration. Also, vendor 4 had high detected levels of Arg, His, and Phe that were 78%, 77%, and 70% higher than the averages, respectively. The extremely high variability in the individual media components’ concentrations suggests little similarity across different commercial sources of SOB media. Teams should test media before use, and especially when switching suppliers, to maintain uniformity across experiments.

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Probing formulation differences in Super Optimal Broth with catabolite repression media.

BACKGROUND Super Optimal Broth with catabolite repression (SOC) media is identical to traditional Super Optimal Broth (SOB) media, but it includes 20 mM glucose. SOC media use maximizes the transformation efficiency of competent cells (e.g., E. coli). The added glucose makes SOC media an optimal choice to ensure the metabolic health of bacteria after transformation. Typically, freshly transformed E. coli are incubated for an hour in SOC before plating on selective media. Despite its implementation, there can be variations in the individual component concentrations between different media’s commercial sources. This can be due to varying sources of raw materials or preparation procedures during manufacturing. These changes may affect the culture’s performance after transformation, especially if processes have been established based on another vendor’s media.

THE EXPERIMENT All commercially available SOC liquid media was tested fresh and handled following the manufacturer’s instructions. All samples were diluted 250x before analysis on the REBEL with no additional sample preparation. (Figure 1)

Figure 1: Media component concentrations from SOC media diluted by 250x before analysis. Error bars are from the standard deviation of n = 5 replicates.

DISCUSSION When compared to each media component’s averages across the four vendors of SOC media, vendor 3 had the most considerable variations. Eleven different components had concentrations that were more than 40% higher than the average levels. The most significant deviations were with Asp, Gly, and Pro, which were all detected higher than the averages by 64%, 86%, and 71%, respectively. Trp showed the highest difference in vendor 1’s media being detected 42% lower than the average. Vendor 1 also had no detected amount of Met. Gly and Tyr were both detected at 52% lower and 50% lower, respectively, in vendor 2’s SOC media. The only significantly different media component in vendor 4 was Gly, as it was detected 42% lower than the average. As shown here, uniformity among vendors of SOC media should not be assumed. Testing media before the start of culture ensures that the levels of nutrients in a media are at a desirable level.

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Investigating the similarities between different vendors’ 2X YT liquid media.

BACKGROUND 2X YT media is a traditional media that gets its name from having twice the yeast extract as the traditional lysogeny broth (LB) media. It is commonly used to culture recombinant strains of E. coli for DNA, plasmid, or protein production as the tryptone and yeast extract provide sufficient sources of carbon, nitrogen, vitamins, minerals, and amino acids. 2X YT media is also used for the non-selective propagation of M13 bacteriophages since the media provides an appropriate nutrient and growth factor profile to allow the phages to reproduce without stressing or weakening the host cells. Like other microbiological media, 2X YT can be purchased in liquid or dried forms from many commercial sources. Therefore, different levels of the individual nutrients may be observed due to changes in the source and processing methods of the tryptone and yeast extract used in the formulation.

THE EXPERIMENT All commercially available 2X YT liquid media was tested fresh and handled following the manufacturer’s instructions. All samples were diluted 250x before analysis on the REBEL with no additional sample preparation. (Figure 1)

Figure 1: Media component concentrations from 2X YT media diluted by 250x before analysis. Error bars are from the standard deviation of n = 5 replicates.

DISCUSSION Among the four different vendors tested in this study, Asn, Asp, His, Lys, and Phe all had values within ±10% of each other. Vendor 1 showed the highest variability from the average concentrations of all the vendors for three amino acids. These amino acids were Ala, Met, and Trp, and they had values higher than the averages by 58%, 45%, and 43%, respectively. At the dilution preparation used in this study, the only undetected amino acid was Met in vendor 3. Researchers can utilize a quick media screen like this to survey their media supplier options to assess vendors’ comparability. This test can be standardized when new media is received, during storage, with a supplier or lot change, or a change in the media form (e.g., liquid or dry). Identifying variations in the expected nutrient profile before its use may prevent undesirable culture outcomes like poor growth and low productivity.

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Comparing standard microbial media from a single supplier.

BACKGROUND Lysogeny broth (LB) has several common derivatives, including Terrific broth (TB), Super Optimal broth (SOB), Super Optimal Broth with catabolite repression (SOC), and 2X YT medias. All of these media can be used in microbial cultures. E. coli is the most common recombinant organism that is grown in all five of these media for cloning, the production of plasmid DNA and proteins, propagation of bacteriophages, and transformation. Although the exact formulations of these media are similar, there are different levels of peptone and yeast extract between them. Additionally, some may contain a carbon source (e.g., glucose in SOC or glycerol in TB) or have different salt and mineral contents that may affect the media’s buffering capacity. Unfortunately, suppliers do not provide analytical reports of the media’s nutrient profile ahead of time so users must assume conformance without validation.

THE EXPERIMENT All commercially available liquid media was tested fresh and handled following the manufacturer’s instructions. LB, TB, SOC, and 2X YT media were diluted 250x, and SOB media was diluted 500x before analysis on the REBEL. There was no additional sample preparation. All concentrations were normalized to the values from LB media for comparison. (Figure 1)

Figure 1: Amino acid concentrations from five standard microbial media from a single vendor. All LB blocks are black because all other values are normalized to it. The white box means it was not detected.

DISCUSSION The LB media had the lowest concentration of 15 of the 18 amino acids detected in this study. Glu in 2X YT was detected 22% lower than in LB, and Gly in 2X YT was not detected. Also, Pro was 6% lower in SOC media compared to LB. SOB and TB media had the highest relative values compared to LB. Some of the highest relative differences were observed in Ala found in TB, which was 415% higher than in LB. Both Gly and Ser were 487% and 600% higher, respectively, in SOB compared to LB. The five different media tested in this study all contain yeast extract and tryptone levels that supply the essential building blocks for fast growth. Yeast extracts have significant differences in composition between batches and formulations, making it far removed from a consistent chemically-defined media ingredient. Additionally, tryptone can vary in composition as it may be enzymatically processed differently batch-to-batch. Rapid media tests can determine the amino acid level changes between media types to inform researchers on composition before selecting the type of media.

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Comparing component levels of basal cell media with 10% fetal bovine serum.

BACKGROUND Dulbecco’s Modified Eagle Medium (DMEM), Iscove’s Modified Dulbecco’s Medium (IMDM), and Roswell Park Memorial Institute media (RPMI) are three of the most common cell media formulations used in cell culture processes. These chemically-defined media are protein-free and have specific formulations of amino acids, vitamins, minerals, and other components added to suit a cell line’s nutrient needs. For certain types of cell cultures and processes (e.g., stem cells, T and B cells, fibroblasts, hybridomas, HEK293, leukemia cells, plant cells, insect cells, etc.), these basal media are usually supplemented with fetal bovine serum (FBS) at 10% v/v. The FBS addition supplements the proteins, lipids, and growth factors necessary to sustain cell viability and growth, and to make the cultures adaptable to changes in pH, temperature, osmolality, or other culture dynamics. However, FBS supplemented media is challenging to assay with traditional chromatographic approaches. The serum proteins can interfere with the chromatography. Sample preparation with protein precipitation may interfere with media component recoveries resulting in biased results of fresh or spent media analysis.

THE EXPERIMENT A commercially available DMEM, IMDM, and RPMI media without glutamine and cell culture grade FBS (USA origin) were tested at the levels indicated below. All media samples were handled following the manufacturer’s instructions. Final solutions were diluted 10x before analysis on the Rebel with no additional sample preparation. (Figure 1)

Figure 1: Media component concentrations from basal media without and with 10% FBS supplementation. Error bars are from the standard deviation of n = 5 replicates.

DISCUSSION When comparing standard basal media compositions with and without FBS supplements, there were almost no differences in the media component levels for each type of media. For DMEM media, the formulations without FBS were very similar, within experiment error, for all nutrients added. However, low levels of Ala, Glu, and Pro were all detected with 10% serum supplementation. These three amino acids are not present in the DMEM formulation, so FBS supplementation indicates the addition of those components at very low levels. There were no significant changes observed in the IMDM media with and without serum. RPMI media does not include Ala in the formulation, but Ala was detected in the 10% FBS supplemented sample. Like the DMEM results, this indicates that the small amount of Ala added from the FBS was large enough to be detected in the serum-supplemented media sample. Using the Rebel, one can routinely monitor these slight changes of media components as the serum is added to the basal media formulations. This practice can support reproducible culture processes and media preparations for a variety of cell lines.

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Are you getting the cell culture media you expected?

BACKGROUND Cell culture media provides the nutrients for the growth and incubation of cells. These media blends often include a primary carbon source (e.g. glucose), nitrogen source (e.g. glutamine), buffers and other components like amino acids, vitamins, salts, trace minerals and other additives. One of the most commonly used cell media types is DMEM/F-12. This media is a blend of two established chemically defined cell medias called DMEM (Dulbecco’s Modified Eagle’s Medium) and Ham’s F-12. DMEM/F-12 contains high concentrations of amino acids, its formulation is well known, and it can be purchased from numerous vendors.

THE EXPERIMENT DISCUSSION Reproducibility is at the basis of all scientific discovery, As this quick snapshot shows, consistency between so we wanted to glimpse into the consistency of vendors should not be assumed. In some cases, DMEM/F-12 media across several providers. We certain media components were as high as 2.5x the purchased the same liquid DMEM/F-12 formulation expected values whereas other components were not from five vendors and kept them refrigerated prior detected at all. It is vital for researchers to work with to analysis with the RebelTM. Each media was diluted established cell media providers to ensure that their cell 10X with Rebel diluent, placed into a vial and run media choice is correct for their application, and that immediately. The concentrations reported from handling of the media once received is appropriate. the Rebel were averaged across five replicates To help ensure reproducibility, researchers should for each sample, and these average values were consider frequent analysis of their cell media with normalized to the known vendor supplied component the Rebel whenever new batches are received, concentrations in the cell media (Figure 1). during storage and just before being introduced into bioreactors to ensure that variations in the process are not from fluctuations in the media.

Figure 1: Comparison of DMEM/F-12 cell culture media run on the Rebel. Colors represent the normalized deviation from the known formulation of DMEM/F-12: green, red and black represent higher, lower and the same concentrations,respectively, compared to the reported formulation. Components not detected are shown with white boxes.

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Simply monitor vitamin levels during cell media analysis with the Rebel.

BACKGROUND The use of chemically defined cell culture media has increased, resulting in a growing focus on components that may pose stability challenges in solution. Vitamins have various shelf lives and may be prone to degradation from changes in temperature, oxygen or light exposure. Discussed here are four vitamins which are commonly supplemented to meet the metabolic needs of cells. This study focuses on thiamine (vitamin B1), nicotinamide (vitamin B3), pyridoxine (vitamin B6) and choline, which are all present in many established cell culture formulations and chemically defined CHO medias.

THE EXPERIMENT DISCUSSION We tested identical formulations of DMEM/F-12 media The Rebel allows for an easy and fast assay of vitamin (without glutamine) from three commercial providers. composition in the same run as amino acids, biogenic All media was stored unopened and refrigerated and amines and dipeptides. As shown here, there can be run on the same day. Each sample was diluted 10X dramatic fluctuations in the measured concentrations with RebelTM diluent, placed into an amber vial and depending on the vitamin and the source. Compared to run immediately on the Rebel. The concentrations the known formulation, thiamine was not detected in reported were averaged across five replicates for each vendor 1 and was 55% and 53% higher in concentration sample (Figure 1). in vendor 2 and 3, respectively. Nicotinamide levels were very consistent between vendors with deviations from the expected formulation of only -5%, 4% and -11% in vendors 1, 2 and 3, respectively. Pyridoxine showed fluctuations in its measured levels across the samples, with deviations of 24%, 3% and -29% in vendors 1, 2 and 3, respectively. Choline results showed higher concentrations in all samples with values of 67%, 14% and 20% higher than the formulation from vendors 1, 2 and 3, respectively. As this quick snapshot shows, one should not assume uniformity when switching between cell culture media providers. Investigating your media prior to feeding your cells is an easy step Figure 1: Comparison of four vitamin levels in three different towards ensuring that variability in your experiment is commercially available DMEM/F-12 media blends. Error bars are the not due to unexpected fluctuations from the media standard deviations from n = 5 replicate measurements. formulation.

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Tease out variations in dry cell culture media with the Rebel.

BACKGROUND When ordering large amounts of cell culture media, a researcher often decides between premixed liquid and dry formulations of the same media. Dry formulations afford the advantages of easier storage, shipment and costs. However, dry formulations must be mixed ahead of time which may introduce variations of the nutrient composition based on how well a researcher follows the directions and the conditions of the media while in storage. Running a simple media assay prior to feeding your cells can ensure that the composition of the media is what is expected.

THE EXPERIMENT Medium 199 is a classic cell culture media developed all four medias tested. On the other hand, Arg, Lys and in the 1950s. The values reported below are from the Trp all exhibited variations greater than ±20% of the analysis of formulations of Medium 199 normalized to formulations across all the media. For the liquid media, the specification sheet values (n = 5, Figure 1). Every the three greatest outliers compared to the formulation formulation had identical concentrations for the amino were Arg (44% higher), Lys (26% higher) and both Trp acids and varied only in salt or phenol red content. and Tyr (24% higher). For the first dry media formulation, The manufacturer’s directions were followed for the Arg (40% higher), Trp (39% higher) and Val (24% higher) mixing of the three dry medias. Samples were tested were the three amino acids with the highest deviation with the Rebel at 10X dilutions. from the formulation. Arg (50% higher), Ser (39% lower) and Trp (38% higher) in the second dry media were the DISCUSSION most variable amino acids. Finally, the greatest variation The rapid quantitation of the amino acids in Medium in dry media three was found in Arg (33% higher), Lys 199 highlight that there are often fluctuations in the (20% higher) and Trp (33% higher). As this example cell media used to feed cells. Both Leu and Phe were demonstrates, researchers should verify the composition the most consistent amino acids with deviation of their of their media prior to introducing it in their cell cultures. levels of less than ±10% of the formulation values across

Figure 1: Comparison of the amino acid levels between liquid and three dry formulations of Medium 199. The color scale is the normalized level of the average value for each amino acid compared to the formulation.

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Serum supplemented media analysis is routine with the Rebel.

BACKGROUND Fetal bovine serum (FBS) is a common cell media supplement due to its supply of growth factors necessary for certain culture processes. FBS is typically supplemented at less than 10% v/v and can be purchased from many commercial vendors. When using media supplemented with FBS, media analysis may be complicated due to the high levels of proteins in solutions. These proteins may interfere by loading preferentially to chromatography columns so specialized sample preparation protocols are required to remove (e.g. precipitate) the proteins. The resulting supernatant is then available for additional preparation and derivatization steps required prior to analysis of media components like amino acids and vitamins.

THE EXPERIMENT DISCUSSION The nutrient composition of a commercially available FBS supplemented media is common for many chemically-defined media (without Gln) wascell culture applications, and the Rebel is suited supplemented with FBS at various concentrations (0% for its analysis. Across all concentrations of FBS - 10%). Samples were diluted 100X with Rebel diluent, supplemented media, there were no major changes placed into a vial and run immediately by the Rebel. No in the reported levels of the components. Most additional protein precipitation step was performed components had concentrations between 0.5 and and all samples were run for five replicates. (Figure 1) 1.5 mM. Both Asn and Ser had the highest average concentrations of 4.66 ± 0.15 mM and 3.07 ± 0.14 mM, respectively. The lowest concentration components were choline, Gly, Lys, Met, and Trp with average concentrations of 0.42 ± 0.07 mM, 0.44 ± 0.02 mM, 0.30 ± 0.04 mM, 0.33 ± 0.02 mM and 0.38 ± 0.01 mM, respectively. For each media preparation, the average relative standard deviation across all components was 3%. As demonstrated here, at-line media analysis of FBS supplemented chemically-defined media by the Rebel provided precise results without the complexity of additional sample preparation steps like a dedicated protein precipitation. If your process requires FBS supplementation, and you have been struggling to Figure 1: Concentrations of media components in FBS supplemented implement media analysis due to the complexity of the chemically-defined media tested on the Rebel. media, the Rebel is the tool for the job.

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Faster, easier, clearer – accelerate process development with the Rebel.

BACKGROUND Cell culture analysis is routinely conducted in core laboratories with liquid chromatography instruments with absorbance detection (e.g. HPLC-UV). Since many common components in cell media do not have fluorophores, a chemical derivatization step is required to add a tag to the molecules for detection. This additional sample preparation step slows the analysis cycle from when a sample is taken to when it is measured resulting in significant time delays for scientists.

THE EXPERIMENT DISCUSSION Here we will compare the results from the same Not all media formulations are exact. Changes in the chemically defined formulation of a DMEM/F-12 environment (e.g. temperature, light, oxygen exposure) aliquot on an HPLC instrument and the RebelTM. and age may cause changes from the labeled values. For HPLC, the samples needed to be analyzed over In Figure 1, the values reported by the Rebel analyzer two runs. In the first, the media was run using an and the HPLC instrument are complimentary and established 3rd party derivatization strategy for the support that the actual composition of the media amino acid analysis. A separate run was conducted does not perfectly match what is shown on the for the identification of vitamins since a different formulation sheet. The Rebel leverages its simplicity to column and solvent system was required. In contrast, allow bioprocess engineers to run their own samples the media was simply diluted 10X with Rebel diluent, anytime which eliminates weeklong delays, sample placed into a vial and ran immediately with the Rebel. prep headaches, and data alignment nightmares. If A single run was all it took to identify both amino acids you struggle with the bottleneck of cell media analysis and vitamins within a fraction of the time. and would like more rigorous statistics to back your decisions, run them on the Rebel.

Figure 1: Comparison of HPLC (n=1) and the Rebel (n=5) quantitation of DMEM/F12 media compared to the formulation sheet values. Note: The HPLC method was not compatible for the detection of choline so no value was recorded.

[email protected] | 908Devices.com SPECIFICATIONS SPEC SHEET

Get cell media analysis at-line, anytime.

The Rebel puts the control in your hands.

Its small, self-contained footprint fits right on your bench allowing you to run samples at-line in less than 7 minutes. SPECIFICATIONS

SAMPLE INFO Get quantitative identification and analysis on a panel of >30 analytes including all the key amino acids, biogenic amines, water soluble vitamins and dipeptides.

Analysis time: Amino Acids Alanine Arginine Asparagine 7 minutes per well Aspartic acid Glutamic acid Glutamine Volume and format: Glycine Histidine Isoleucine 10 µL minimum volume (prior to dilution) Leucine Lysine Methionine 96-well plate or chromatography vials Phenylalanine Proline Serine Cooled sample storage is standard Threonine Tryptophan Tyrosine Valine Type: Fresh or spent cell media from Biogenic beta-Alanine GABA Hydroxyproline mammalian or microbial cultures Amines L-Citrulline Methyl-L-histidine Sarcosine and fermentations Vitamins Choline Nicotineamide Pyridoxal Pyridoxine Thiamine Dipeptides Alanine-glutamine Cystine

THE DEVICE YOUR DATA OPERATING CONSIDERATIONS Calibration: Output: Ambient temperature: Automated calibration and Values reported in “mM” 15 - 25°C QC with consumable kits concentration over 2.5 Ambient humidity: System Interfaces: orders of magnitude linear 20 - 80% RH (non-condensing) Ethernet, USB port (2) dynamic range Solvent waste collected in dedicated vessel Report format: with level sensing CSV and PDF Condensate (water) collected in dedicated vessel with level sensing Power requirements: COMPLIANCE SOFTWARE 100-240 VAC, 50-60 Hz Safety/Compliance: UL/CSA/ Designed for operation in Physical dimensions: IEC 61010-1 3rd Edition; cGLP/cGMP, with support for Height (22 in, 56 cm), width (13 in, 33 cm), CE marked; EU & China RoHS; 21 CFR Part 11-compliance depth (27 in, 69 cm), weight (83 lbs, 38 kg) EU REACH

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