New Results

Conversion of mammalian cell culture media waste to microbial fermentation feed efficiently supports production of recombinant protein by Escherichia coli

Ciara D Lynch, David J O’Connell
doi: https://doi.org/10.1101/2021.07.01.450323

Abstract

Deriving new value from waste streams through secondary processes is a central aim of the circular bioeconomy. In this study we investigate whether chemically defined spent media (CDSM) waste from cell culture bioprocess can be recycled and used as a feed in secondary microbial fermentation to produce new recombinant protein products. Our results show that CDSM supplemented with 2% glycerol supported a specific growth rate of E. coli cultures equivalent to that achieved using a nutritionally rich microbiological media (LB). The titre of recombinant protein produced following induction in a 4-hour expression screen was approximately equivalent in the CDSM fed cultures to that of baseline, and this was maintained in a 16-hr preparative fermentation. To understand the protein production achieved in CDSM fed culture we performed a quantitative analysis of proteome changes in the E. coli using mass spectrometry. This analysis revealed significant upregulation of protein synthesis machinery enzymes and significant downregulation of carbohydrate metabolism enzymes. We conclude that spent cell culture media, which represents 100s of millions of litres of waste generated by the bioprocessing industry annually, may be valorized as a feed resource for the production of recombinant proteins in secondary microbial fermentations.

Introduction

Diversion of waste streams generated by bio-industries to secondary processes to produce valuable products through microbial and chemical engineering has become a central pillar of the circular bioeconomy [1]. One example of a waste stream from bioindustry that has yet to be diverted to the creation of new valuable products is the cell culture media used in the bioprocessing of protein drug molecules. The commercial production of monoclonal antibodies using Chinese hamster ovary (CHO) cell bioprocess in 2019 alone resulted in approximately 30 metric tonnes of protein product [2]. The chemically defined cell culture media used to feed the mammalian cells is itself a sophisticated chemical formulation that also represents a significant waste product in downstream processing. Based on a product titre of 10 g/L of IgG in batch and fed batch systems, with the total yield of protein product produced in 2019 as a reference, we estimate that up to 300 million litres of cell culture waste is generated annually [2, 3]. Systematic approaches to valorize this waste as part of a commitment to a circular bioeconomy have not been investigated. We hypothesise that spent media from CHO cell culture has the potential to support E. coli fermentation and the production of recombinant protein titres when recycled from bioprocessing systems.

The re-use of spent media has been investigated in certain process systems, mostly through feeding spent media from the original culture as a supplement to fresh media in the same system [4-8]. These have reported some improvement in protein expression and growth rates. For example, IgG expression titre in mouse hybridoma cell culture was increased by as much as 50% when the cells were grown in culture media with 33% spent media supplementation [6]. Conversely, feeding the cells with 100% spent media led to a significant decrease in growth and protein production by the mammalian cells. This decrease was attributed to the build-up of auto-inhibitory metabolites and lack of nutrients.

Genetic engineering of a wide array of bacteria has been a central pillar of the development of the circular bioeconomy, enabling new product development from diverse organic waste streams including food oils, agrochemical waste and used bioplastics [9-14]. We aimed to test our hypothesis using Escherichia coli, a robust and versatile bacteriological expression host that is widely used commercially to produce recombinant proteins. The production of recombinant human insulin in E. coli for example, is a major milestone in human drug production [15, 16]. E. coli has been shown to utilize breakdown metabolites such as lactate present in spent culture medias [17] and can grow successfully in spent media from the fermentation of other cell types [8].

Comparative analysis of culture growth rates of an E. coli BL21 strain harbouring a recombinant mCherry-EF2 expression construct in rich microbiological media (LB) and in chemically defined spent media (CDSM) that had previously been used to culture CHO cells, either with or without supplementation, confirmed CDSM as a viable feed for bacterial fermentation. Analysis of recombinant protein production by the cultures confirmed equivalent recombinant protein titres post-purification when compared to LB.

Results

Growth rate analysis of E. coli BL21 mCherry/EF2 in minimal media, baseline rich media and chemically defined spent media

Growth rates of E. coli cultures grown in M9 minimal media with carbon source supplementation were measured to determine the optimal supplementation conditions that could then be applied to the CDSM fed cultures. Glycerol was selected as a suitable carbon supplementation as it is also a waste product from other processes. Gradients of glycerol additions were tested at concentrations of 1%-3% (figure 1A). 2% glycerol supplementation was shown to produce a specific growth rate of 0.673 generations per hour, as compared to the growth rates of 1% and 3% at 0.578 and 0.647. Volumes (v/v) over 3% greatly reduced the growth rate, while 2% glycerol addition to the reference LB media did not affect the growth rate seen in LB alone (data not shown).

Figure 1.
Figure 1. E. coli culture growth rates in bacteriological and cell culture media.

(A.) Glycerol supplementation in M9 minimal media; ■ = baseline media, ● = minimal media, ◆ = minimal media + 1% glycerol, □= minimal media + 2% glycerol, ▲ = minimal media + 3% glycerol. (B). Unsupplemented and supplemented chemically defined spent media (CDSM). ■ = baseline media, ● = M9 media, ◇ = chemically defined spent media (CDSM) unsupplemented, □ = CDSM with 2% glycerol. All time points were completed in triplicate with standard deviation as error bars.

The chemically defined spent medium used was CHOgro® spent media harvested from CHO (Chinese Hamster Ovary) cell culture. The specific growth rate of the E. coli in the CDSM was 0.704 gen/hour, approximately 70% of the rate achieved in the baseline LB media. Supplementing CDSM with 2% glycerol led to a further increase in growth rates to ∼94% of that of LB (figure 1B). This supplementation was chosen for all medias tested for protein expression analysis.

Protein expression analysis in chemically defined spent media fermentation

Recombinant mCherry-EF2 expression was tested in cultures grown in CDSM supplemented with 2% glycerol and the baseline LB with 2% glycerol. A four-hour expression screen post-induction with IPTG showed that protein production in CDSM was equivalent to baseline LB, with an average yield of 100.58 mg/L compared to LB’s 92.57 mg/L (figure 2A).

Figure 2
Figure 2 Protein expression and purification analysis.

(A.) Table of growth characteristics, biomass, and protein yields. LB media; Lysogeny Broth baseline rich media. M9 media; minimal salts media. CDSM I; chemically defined spent media CHOgro®. CDSM II; chemically defined spent media Expi-CHO®. R2 coefficient is the percentage of variability in the growth curve dataset that is accounted for by linear correlation between the OD600 (nm) and the time (hr). For the equation for the specific growth rate, see Methods. * n = 2 for all standard deviation calculations. (B.) SDS-PAGE analysis of purified protein versus input supernatant sample. Lane 1 = protein marker, lane 3 = post-boil supernatant sample from CDSM, lane 5 = purified monomeric fraction from size exclusion chromatography of CDSM.

We next tested a 16-hour expression post-induction with IPTG using CDSM, and included a second chemically defined spent medium, Expi-CHO® (termed CDSM II), to examine the robustness of our finding with widely used commercial medias. Protein yield from the CDSM I fed culture closely matched the yield of that from LB with a protein yield of 159.82 mg/L compared to 168.92 mg/L (figure 2A). The alternate spent media type tested, CDSM II, managed a successful yield of 127.57 mg/L. It should be noted that the CDSM II condition also lost some yield during expression, seen by the presence of the recombinant protein in the extracellular media also. SDS-PAGE analysis confirmed that the monomeric peak taken from SEC corresponding to the mCherry-EF2 protein at 32 kDa.

Mass spectrometry analysis of E. coli cultures at the whole proteome level

We performed a proteomic analysis of the protein expression patterns in the bacterial cultures themselves using LC-MS/MS. The analysis of proteomic data files was carried out using MaxQuant. Using the protein expression pattern for E. coli grown in LB media as a baseline reference, a student’s t-test with a false discovery rate of <0.05 identified 655 differentially expressed proteins in E. coli cultures grown in unsupplemented CDSM. In 2% glycerol supplemented CDSM conditions there were 167 proteins differentially expressed by comparison (Figure 3).

Figure 3.
Figure 3. Number of statistically significant differentially expressed proteins compared to the baseline condition in LB media.

Orange bars represent the number of downregulated proteins while blue represents all upregulated proteins compared to baseline LB media after a student’s t-test with an FDR of < 0.05.

In CDSM I supplemented with 2% glycerol, 1,222 proteins were found to be dysregulated and of these, 167 proteins were statistically significant. From these 167 proteins, 87 proteins were identified as upregulated and 80 proteins as downregulated. A program that measures the quantitative differences in expression by difference in LFQ intensity was written using Python script and using a cut-off significance value of >0.5 or <-0.5 in intensity identified the most significantly changed expression levels, shown here for the first time as a pool table plot (figure 4). Proteins upregulated in the CDSM I + 2% glycerol condition were principally in the amino-acid and purine biosynthesis pathways, while proteins that were most significantly downregulated were in the carbohydrate metabolism pathway (figure 4).

Figure 4.
Figure 4. Pool-table plot of the proteomic analysis of the significantly dysregulated proteins from the sample condition of CHOgro® with 2% glycerol supplementation.

This graph was constructed using the Matplotlib library in Python. Significant upregulation of expression is shown to the right of the central threshold divider (−0.5 to 0.5), with significant downregulation to the left. Significance in this case was defined as having a mean difference in LFQ intensity compared to the baseline media of more than 0.5 in either direction after a student’s t-test and with an FDR of <0.05. The top three pathway proteins and the top one unannotated pathway protein are labelled directly below their representative point, for both up- and down-regulated.

Amino acid biosynthesis and purine metabolism functional pathways were up-regulated in all cultures grown in CDSM compared to the baseline. Carbohydrate metabolism by contrast was mainly downregulated. Within amino acid biosynthesis are some of the most statistically significantly upregulated enzymes, such as diaminopimelate decarboxylase and Aspartate-ammonia ligase, two enzymes that are upregulated with a mean difference of LFQ intensity of > 2, along with 10 other significantly upregulated enzymes in this pathway. Purine metabolism also features highly upregulated enzymes such as glutamine dependent amidophosphoribosyltransferase, with a difference in intensity of > 3 along with 12 other significantly upregulated enzymes.

Discussion

Can spent media from CHO cell culture be reused to feed E. coli fermentation?

The creation of chemically defined culture media has led to increasing recombinant protein titres and protein quality [18, 19]. These successful developments have resulted in this cell culture media becoming a significant waste stream with approximately 300 million litres sent for disposal annually [2].

Our data indicates that chemically defined spent media (CDSM) is a potentially valuable resource for producing new recombinant proteins when compared with microbiological media prepared with casein digests and yeast extract, such as the LB media studied here. In this study we investigated the expression of a recombinant fusion protein ligand (EF2) derived from mCherry and the calcium binding protein Calbindin D9k. This recombinant protein has been developed as a ligand for a highly specific and high affinity purification system [20-23]. The growth rate of the expression culture in the CDSM + 2% glycerol reached was similar to the LB at ∼94% while the 4-hour protein yield was approximately equal, a striking and unanticipated finding (figure 2A). This finding was further verified in the 16-hour fermentation, a model of a preparative scale expression, whereby the CDSM cultures successfully supported a protein expression over this longer period with approximately the same yield (159.82 mg/L) as those cultures grown in the nutritionally rich LB media (168.92 mg/L) (figure 2A). This finding prompted us to investigate the proteome of the cultures to identify proteins responsible for metabolic changes that may contribute to this phenotype.

Proteomic analysis

Out of the 629 upregulated proteins identified in the optimised condition of CDSM + 2% Glycerol, the most statistically significantly upregulated proteins are enzymes involved in the amino acid biosynthesis pathway. For example, both Diaminopimelate decarboxylase and Aspartate-ammonia ligase are two highly upregulated proteins with LFQ differences of > 2 (Figure 4, table 1), involved in lysine biosynthesis and asparagine biosynthesis pathways that are dependent on glutamine uptake [24]. Glutamine (4 mM) is a supplement added to the chemically defined media prior to CHO cell culture. Amino acid accumulation such as increased L-asparagine is a known feature of the E. coli stress response, to make available the building blocks needed for synthesis of stress response proteins [25]. This upregulation may indicate the activation of a stress response in the cultures that is not activated in rich microbiological media but that contributes to recombinant protein expression. Other highly upregulated proteins (>2 fold) include glycerol metabolism enzymes such as Phosphogluconate dehydratase, purine metabolism enzymes such as glutamine dependent Amidophosphoribosyltransferase, and iron uptake proteins such as Enterobactin non-ribosomal peptide synthetase EntF, among others [26-28]. Interestingly the increased expression of the glycerol metabolism enzyme Phosphogluconate dehydratase and glutamine dependent Amidophosphoribosyltransferase correlates well with the supplementation with both glycerol and glutamine and confirms the sensitivity of this MS analysis.

View this table:
Table 1. Most significantly dysregulated E. coli proteins and the associated annotated functional pathway.

Proteins highlighted by accession number in Figure 4 are described here with one example of an upregulated and downregulated protein lacking an annotated functional pathway.

Among the 595 downregulated proteins in the CDSM + glycerol condition are enzymes involved in the TCA cycle such as the probable malate:quinone oxidoreductase, some specific stress response proteins such as protein YcfR (an acid stress response protein), and nucleotide synthesis/salvage proteins, such as cytidine deaminase. Many of these proteins depend on the presence of glucose in order to be active, leading to a possible reason for their downregulation in the CDSM + glycerol condition [29, 30]. Other downregulated proteins which are statistically significant include D-amino acid dehydrogenase, involved in amino-acid degradation, and generally present in high levels in LB rich broth due to extracellular D-amino acids which are not present in the CDSM [31, 32].

One of the main metabolic pathways that was upregulated in the CDSM media compared to LB was the amino-acid biosynthesis pathway. The baseline condition of LB contains tryptone, a source of nitrogen-containing peptides for E. coli growth, whereas the spent CHOgro® media must rely on the proteins released by the CHO cell culture conditions [33]. CHO cell growth in chemically defined media produces metabolites such as ammonium and lactate that can inhibit further growth [34]. These factors however can be used by E. coli to grow, utilising ammonium as its preferred nitrogen source and can break down lactate as a possible carbon source if needed [35]. The 102 proteins that are significantly dysregulated and are yet to be assigned a pathway on Uniprot are involved in a wide array of metabolic processes in the E. coli cell, such as the acid stress response protein seen to be the most downregulated in table 1. Further study into these individual proteins will help to understand the proteomic adaptions undertaken by the CDSM fed E. coli.

Proteomic analysis of the nutritional content of CDSM

CHO cells generally produce approximately 1,400 host cell proteins (HCPs) which are detectable in the clarified spent culture media, with ∼80% of the top 1000 of these HCPs in common across multiple cell lines [36, 37]. Mass spectrometry-based analysis has been widely employed to characterise HCP’s across a number of studies [38-40].

Mass spectrometry analysis of the CDSM alone identified879 host cell proteins from CHO in the spent media after culturing (see Table S1). These proteins were identified from all cellular compartments suggesting they are debris from CHO cell lysis in addition to any active secretion by the growing cells. These host cell proteins represent a source of amino acid building blocks for the increased recombinant protein production capabilities of the E. coli cultures.

Conclusion

We have shown in this study that mammalian cell culture waste, the chemically defined synthetic media used to grow Chinese Hamster Ovary cells, is conditioned such that it provides a nutritious feed for the growth of E. coli cultures in secondary fermentation. The growth rate of the culture in this waste medium is similar to that of rich microbiological culture media and upon supplementation with another waste by-product, glycerol, the growth rate is enhanced. Importantly, the expression of a recombinant protein from an expression plasmid construct is seen to be equivalent in protein titre between 4 to 16 hours showing that this waste has a real value in a biotechnological context. This approach may be further developed based on a deeper understanding of the protein expression patterns analysed here, that show significant upregulation and downregulation of metabolic enzymes and pathways. This approach can begin a route for the capture of a bioprocessing waste stream for the circular bioeconomy.

Experimental Procedures

Mammalian Cell Culture

Chinese Hamster Ovary (CHO) cells were incubated in 20 ml of serum-free CHOgro® Expression media supplemented with 4 mM L-Glutamine in T75 adherent cell line flasks, at 37°C with 5% CO2 and 95% O2. Cells were split at 70-80% confluency every 3-4 days. Spent media was harvested at each split and was clarified of cells and cellular debris by centrifugation at 300 x g for 4 minutes prior to storing at 4°C for up to 14 days.

Growth and Expression Cultures of E. coli

E. coli BL21 Gold was transformed with an mCherry-EF2 fusion protein expression construct with a T7 promoter expression system. A 10 ml starter culture of BL21 transformed E. coli was grown by incubating a single, isolated colony in Lysogeny Broth (LB) media, 2% glucose, and 100 µg/ml ampicillin. The culture was grown in a shaking incubator at 250 rpm, 37°C for 16 hours. Growth curves were taken from 50 ml cultures grown in triplicate in 250ml flasks by measuring OD600 every half an hour for growth curve plotting until the OD600 reached 0.6. The four-hour expression was carried out in duplicate 50 ml cultures with 2% glycerol and a 1 in 20 dilution of starter culture in the expression media in a shaking incubator at 250 rpm, 37°C.Expression of the fusion protein was induced by addition of 1 mM IPTG after OD600 reached 0.6. Cultures were then incubated on a shaking incubator at 250 rpm, 30°C for 4 hours or for 16 hours for the overnight expression experiment. Cultures were then spun at 4°C, 4000 x g for 20 minutes to harvest the pellets which were weighed prior to protein purification.

Calculations of Specific Growth Rate

Embedded Image Specific growth rate (k) was calculated by the above equation, where k is generations per hour and t is time in hours. Plots of growth curves of OD600 (nm) versus time (hrs) were generated to gather this data in Excel. Xt is one OD600 value at a later position, and X0 is another OD600 value taken from an earlier position using a trendline.

Recombinant Protein Purification

Cell pellets were re-suspended using a buffer containing 10 mM Tris and 2 mM CaCl2 pH 7.4 before lysing by sonication. The lysate was then boiled at 85 degrees Celsius and spun at 15,000 rcf for 30 minutes. Supernatant was harvested and referred to as the post-boil (PB) sample. 0.5 ml of this PB was run on a Superdex 200 10/300 size exclusion chromatography column, using Hepes Buffer Saline as a running buffer. Protein concentration was measured with a DeNovix DS-11 Spectrophotometer, using the UV-Vis application. Measurements were taken at 585 nm for mCherry yield, with an extinction coefficient calculated at 44,854.2 M-1cm-1 and a protein molecular weight of 30,667.4 g/mol.

Proteomic Analysis

4 ml of E. coli culture was harvested after reaching OD600 of 0.6, but before IPTG induction. These cultures were re-suspended in 8M urea. 5mM DTT was then added, and samples were incubated at room temperature for 10 minutes. After incubation, 10mM iodoacetamide was added, and samples were incubated in the dark for 10 minutes at room temperature. Samples were brought to neutral pH by addition of 150 µl of 200 mM NH4HCO3. Samples were then digested with trypsin at 37°C and 300 rpm on a thermomixer overnight. Samples were spun at 4000 x g for 2 mins to prevent ZipTip® from getting blocked in the next steps. Digested peptides were filtered through ZipTip® columns and eluted using 70% acetonitrile in acidic water (0.1% formic acid). The elution was dried off by speed-vac at 45 °C. The dried peptides were then re-suspended in 30 µl Buffer A (5% acetonitrile and 0.1% TFA). Analysis of proteomic data files was carried out using MaxQuant [41] to assemble peptides using the E. coli BL21 proteome from Uniprot (UP000002032). The software Perseus [42] was used to analyse the resulting dataset for student’s t-tests and heatmap generation. A Python programme was generated to give the remaining graphs, using the t-test results generated from Perseus and annotation information from Uniprot for E. coli strain BL21 (available on GitHub at https://github.com/Ciara-Lynch/Mass_Spec_Analysis.git).

Conflict of interest

The authors declare no financial or commercial conflict of interest.

Author Contributions

CL performed the experiments, CL and DJO’C analysed the data, CL and DJOC wrote the paper. DJO’C designed the study.

Supplementary Information

View this table:
Table S1: Table of CHO host cell proteins detected by LC-MS/MS in a spent CHOgro® media sample.

Completed in triplicate with mean LFQ values displayed in third column above.

Acknowledgements

The authors wish to thank Science Foundation Ireland and the EPSRC for joint funding of Ms Ciara Lynch through the Centre for Doctoral Training – Atoms to Products. The A2P CDT is supported by the Science Foundation Ireland (SFI) and the Engineering and Physical Sciences Research Council (EPSRC) under Grant No. 18/EPSRC-CDT/3582. The work was also supported by the Science Foundation Ireland funded BiOrbic bioeconomy research centre under grant no. 16/RC/3889. Thank you to Eugene Dillon and Siobhan Kelly for guidance on mass spectrometry usage.

Footnotes

  • all sections of the paper have been rewritten and the figures have been updated

Abbreviations

CHO
Chinese hamster ovary
LB
lysogeny broth
CDSM
chemically defined spent media

References

  1. [1].↵
    Stegmann P, Londo M, Juninger M (2020) The circular bioeconomy: Its elements and role in European bioeconomy clusters. Resources, Conservation & Recycling: X 6:100029
    OpenUrl
  2. [2].↵
    Ecker and Patricia Seymour, D. (2020, October 26). Mammalian Biomanufacturing Industry Supply & Demand Trends. BioProcess Online. https://www.bioprocessonline.com/doc/mammalian-biomanufacturing-industry-supply-demand-trends-0001.
  3. [3].↵
    Kunert, R., & Reinhart, D. (2016). Advances in recombinant antibody manufacturing. Applied Microbiology and Biotechnology, 100(8), 3451–3461.
    OpenUrlCrossRef
  4. [4].↵
    Riese U, Lütkemeyer D, Heidemann R, Büntemeyer H, Lehmann J. Re-use of spent cell culture medium in pilot scale and rapid preparative purification with membrane chromatography. J Biotechnol. 1994;34: 247–257.
    OpenUrlPubMed
  5. [5].
    Wu J, Ruan Q, Lam HYP. Evaluation of spent medium recycle and nutrient feeding strategies for recombinant protein production in the insect cell– baculovirus process. J Biotechnol. 1998;66: 109–116.
    OpenUrl
  6. [6].↵
    Ogawa A, Takada N, Terada S. Effective antibody production by reusing culture medium previously used in antibody purification. Biosci Biotechnol Biochem. 2009;73: 719–721.
    OpenUrlPubMed
  7. [7].
    Beas-Catena A, Sánchez-Mirón A, Garía-Camacho F, Contreras-Gómez A, Molina-Grima E. The effect of spent medium recycle on cell proliferation, metabolism and baculovirus production by the lepidopteran Se301 cell line infected at very low MOI. J Microbiol Biotechnol. 2013;23: 1747–1756.
    OpenUrl
  8. [8].↵
    Yano T, Mori H, Kobayashi T, Shimizu S. Reusability of Broth Supernatant as Medium : Journal of fermentation technology. 1980;58(3):259–66.
    OpenUrl
  9. [9].↵
    Guzik MW, Kenny ST, Duane GF, Casey E, Woods T, Babu RP, et al. Conversion of post consumer polyethylene to the biodegradable polymer polyhydroxyalkanoate. Appl Microbiol Biotechnol. 2014;98: 4223–4232.
    OpenUrlCrossRef
  10. [10].
    Cerrone F, Davis R, Kenny ST, Woods T, O’Donovan A, Gupta VK, et al. Use of a mannitol rich ensiled grass press juice (EGPJ) as a sole carbon source for polyhydroxyalkanoates (PHAs) production through high cell density cultivation. Bioresour Technol. 2015;191: 45–52.
    OpenUrl
  11. [11].
    Narancic T, Verstichel S, Reddy Chaganti S, Morales-Gamez L, Kenny ST, De Wilde B, et al. Biodegradable Plastic Blends Create New Possibilities for End-of-Life Management of Plastics but They Are Not a Panacea for Plastic Pollution. Environ Sci Technol. 2018;52: 10441–10452.
    OpenUrl
  12. [12].
    Gervasi T, Pellizzeri V, Calabrese G, Di Bella G, Cicero N, Dugo G. Production of single cell protein (SCP) from food and agricultural waste by using Saccharomyces cerevisiae. Nat Prod Res. 2018;32: 648–653.
    OpenUrl
  13. [13].
    Ruiz C, Kenny ST, Narancic T, Babu R, Connor KO. Conversion of waste cooking oil into medium chain polyhydroxyalkanoates in a high cell density fermentation. J Biotechnol. 2019;306: 9–15.
    OpenUrl
  14. [14].↵
    Rechtin TM, Hurst M, Potts T, Hestekin J, Beitle R, McLaughlin J, et al. Production of recombinant protein in Escherichia coli cultured in extract from waste product alga, Ulva lactuca. Biotechnol Prog. 2014;30: 784–789.
    OpenUrl
  15. [15].↵
    Kroeff EP, Owens RA, Campbell EL, Johnson RD, Marks HI. Production scale purification of biosynthetic human insulin by reversed-phase high-performance liquid chromatography. J Chromatogr. 1989 Jan 6;461:45–61. doi: 10.1016/s0021-9673(00)94274-2. PMID: 2651467.
    OpenUrlCrossRefPubMed
  16. [16].↵
    1. Georgiou G,
    2. DeBernardez-Clark E.
    Storrs SB, Przybycien TM: Commercial-scale refolding of recombinant methionyl bovine somatotropin. In Protein Refolding. edited by Georgiou G, DeBernardez-Clark E. Washington, DC: American Chemical Society; 1991:197–205.
  17. [17].↵
    Caglar MU, Houser JR, Barnhart CS, Boutz DR, Carroll SM, Dasgupta A, et al. The E. coli molecular phenotype under different growth conditions. Sci Rep. 2017;7: 45303.
    OpenUrlCrossRef
  18. [18].↵
    Huang YM, Hu W, Rustandi E, Chang K, Yusuf-Makagiansar H, Ryll T. Maximizing productivity of CHO cell-based fed-batch culture using chemically defined media conditions and typical manufacturing equipment. Biotechnol Prog. 2010 Sep-Oct;26(5):1400–10. doi: 10.1002/btpr.436. PMID: 20945494.
    OpenUrlCrossRefPubMed
  19. [19].↵
    Xu J, Rehmann MS, Xu X, Huang C, Tian J, Qian NX, Li ZJ. Improving titre while maintaining quality of final formulated drug substance via optimization of CHO cell culture conditions in low-iron chemically defined media. MAbs. 2018 Apr;10(3):488–499. doi: 10.1080/19420862.2018.1433978. Epub 2018 Feb 20. PMID: 29388872; PMCID: PMC5916559.
    OpenUrlCrossRefPubMed
  20. [20].↵
    Berggård T, Julenius K, Ogard A, Drakenberg T, Linse S. Fragment complementation studies of protein stabilization by hydrophobic core residues. Biochemistry. 2001 Feb 6;40(5):1257–64. doi: 10.1021/bi0014812.
    OpenUrlCrossRefPubMed
  21. [21].
    Dell’Orco D, Xue WF, Thulin E, Linse S. Electrostatic contributions to the kinetics and hermodynamics of protein assembly. Biophys J. 2005 Mar;88(3):1991–2002. doi: 10.1529/biophysj.104.049189.
    OpenUrlCrossRefPubMed
  22. [22].
    Mhurchú Nn, Zoubak L, McGauran G, Linse S, Yeliseev A, O’Connell DJ. Simplifying G Protein-Coupled Receptor Isolation with a Calcium-Dependent Fragment Complementation Affinity System. Biochemistry. 2018 Jul 31;57(30):4383–4390. doi: 10.1021/acs.biochem.8b00469.
    OpenUrlCrossRef
  23. [23].↵
    McGauran G, Linse S, O’Connell DJ. Single Step Purification of Glycogen Synthase Kinase Isoforms from Small Scale Transient Expression in HEK293 Cells with a Calcium-Dependent Fragment Complementation System. Methods Mol Biol. 2020;2095:385–396. doi: 10.1007/978-1-0716-0191-4_22.
    OpenUrlCrossRef
  24. [24].↵
    Lomelino CL, Andring JT, McKenna R, Kilberg MS. Asparagine synthetase: Function, structure, and role in disease. J Biol Chem. 2017 Dec 8;292(49):19952–19958. doi: 10.1074/jbc.R117.819060. Epub 2017 Oct 30. PMID: 29084849; PMCID: PMC5723983.
    OpenUrlAbstract/FREE Full Text
  25. [25].↵
    Jozefczuk, S., Klie, S., Catchpole, G., Szymanski, J., Cuadros-Inostroza, A., Steinhauser, D., Selbig, J., & Willmitzer, L. (2010). Metabolomic and transcriptomic stress response of Escherichia coli. Molecular Systems Biology, 6, 364.
    OpenUrlAbstract/FREE Full Text
  26. [26].↵
    Gardner PR, Fridovich I. Superoxide sensitivity of the Escherichia coli 6-phosphogluconate dehydratase. J Biol Chem. 1991 Jan 25;266(3):1478-83. PMID: 1846355.
    OpenUrlAbstract/FREE Full Text
  27. [27].
    Gots JS, Benson CE, Jochimsen B, Koduri KR. Microbial models and regulatory elements in the control of purine metabolism. Ciba Found Symp. 1977;(48):23–41. doi: 10.1002/9780470720301.ch3. PMID: 204462.
    OpenUrlCrossRefPubMed
  28. [28].↵
    Messenger LJ, Zalkin H. Glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Purification and properties. J Biol Chem. 1979 May 10;254(9):3382-92. PMID: 372191.
    OpenUrlAbstract/FREE Full Text
  29. [29].↵
    Flores N, Xiao J, Berry A, Bolivar F, Valle F. Pathway engineering for the production of aromatic compounds in Escherichia coli. Nat Biotechnol. 1996 May;14(5):620–3. doi: 10.1038/nbt0596-620. PMID: 9630954.
    OpenUrlCrossRefPubMed
  30. [30].↵
    Van der Rest ME, Frank C, Molenaar D. Functions of the membrane-associated and cytoplasmic malate dehydrogenases in the citric acid cycle of Escherichia coli. J Bacteriol. 2000 Dec;182(24):6892–9. doi: 10.1128/jb.182.24.6892-6899.2000. PMID: 11092847; PMCID: PMC94812.
    OpenUrlAbstract/FREE Full Text
  31. [31].↵
    Baev MV, Baev D, Radek AJ, Campbell JW. Growth of Escherichia coli MG1655 on LB medium: monitoring utilization of amino acids, peptides, and nucleotides with transcriptional microarrays. Appl Microbiol Biotechnol. 2006 Jul;71(3):317–22. doi: 10.1007/s00253-005-0310-5.
    OpenUrlCrossRefPubMedWeb of Science
  32. [32].↵
    Franklin FC, Venables WA. Biochemical, genetic, and regulatory studies of alanine catabolism in Escherichia coli K12. Mol Gen Genet. 1976 Dec 8;149(2):229–37. doi: 10.1007/BF00332894. PMID: 13292.
    OpenUrlCrossRefPubMed
  33. [33].↵
    Sezonov, Guennadi, Danièle Joseleau-Petit, and Richard D’Ari. 2007. “Escherichia Coli Physiology in Luria-Bertani Broth.” Journal of Bacteriology 189 (23): 8746–49.
    OpenUrlAbstract/FREE Full Text
  34. [34].↵
    Pereira, Sara, Helene Faustrup Kildegaard, and Mikael Rørdam Andersen. 2018. “Impact of CHO Metabolism on Cell Growth and Protein Production: An Overview of Toxic and Inhibiting Metabolites and Nutrients.” Biotechnology Journal 13 (3): e1700499.
    OpenUrl
  35. [35].↵
    Merrick, M. J., and R. A. Edwards. 1995. “Nitrogen Control in Bacteria.” Microbiological Reviews 59 (4): 604–22.
    OpenUrlAbstract/FREE Full Text
  36. [36].↵
    Yuk, I. H. et al. (2015) ‘More similar than different: Host cell protein production using three null CHO cell lines’, Biotechnology and bioengineering, 112(10), pp. 2068–2083.
    OpenUrl
  37. [37].↵
    Valente, K. N. et al. (2018) ‘Applications of proteomic methods for CHO host cell protein characterization in biopharmaceutical manufacturing’, Current opinion in biotechnology, 53, pp. 144–150.
    OpenUrl
  38. [38].↵
    Tscheliessnig, A. L. et al. (2013) ‘Host cell protein analysis in therapeutic protein bioprocessing - methods and applications’, Biotechnology journal, 8(6), pp. 655–670.
    OpenUrl
  39. [39].
    Hogwood, C. E. M., Bracewell, D. G. and Smales, C. M. (2014) ‘Measurement and control of host cell proteins (HCPs) in CHO cell bioprocesses’, Current opinion in biotechnology, 30, pp. 153–160.
    OpenUrl
  40. [40].↵
    Levy NE, Valente KN, Lee KH, Lenhoff AM. Host cell protein impurities in chromatographic polishing steps for monoclonal antibody purification. Biotechnol Bioeng. 2016 Jun;113(6):1260–72. doi: 10.1002/bit.25882.
    OpenUrlCrossRef
  41. [41].↵
    Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized ppb-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008;26: 1367–1372.
    OpenUrlCrossRefPubMedWeb of Science
  42. [42].↵
    Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, et al. The Perseus computational platform for comprehensive analysis of proteomics data. Nat Methods. 2016;13: 731–740.
    OpenUrlCrossRefPubMed
Back to top
Posted December 22, 2021.
Download PDF
Email
Conversion of mammalian cell culture media waste to microbial fermentation feed efficiently supports production of recombinant protein by Escherichia coli
Ciara D Lynch, David J O’Connell
bioRxiv 2021.07.01.450323; doi: https://doi.org/10.1101/2021.07.01.450323
Digg logo Reddit logo Twitter logo Facebook logo Google logo LinkedIn logo Mendeley logo
Citation Tools

Subject Area