Conventional manufacturing of protein biopharmaceuticals in centralized, large-scale, single-product facilities is not well-suited to the agile production of drugs for small patient populations or individuals. Previous solutions for small-scale manufacturing are limited in both process reproducibility and product quality, owing to their complicated means of protein expression and purification1,2,3,4. We describe an automated, benchtop, multiproduct manufacturing system, called Integrated Scalable Cyto-Technology (InSCyT), for the end-to-end production of hundreds to thousands of doses of clinical-quality protein biologics in about 3 d. Unlike previous systems, InSCyT includes fully integrated modules for sustained production, efficient purification without the use of affinity tags, and formulation to a final dosage form of recombinant biopharmaceuticals. We demonstrate that InSCyT can accelerate process development from sequence to purified drug in 12 weeks. We used integrated design to produce human growth hormone, interferon α-2b and granulocyte colony-stimulating factor with highly similar processes on this system and show that their purity and potency are comparable to those of marketed reference products.
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Adiga, R. et al. Point-of-care production of therapeutic proteins of good-manufacturing-practice quality. Nat. Biomed. Eng. https://doi.org/10.1038/s41551-018-0259-1 (2018).
Boles, K.S. et al. Digital-to-biological converter for on-demand production of biologics. Nat. Biotechnol. 35, 672–675 (2017).
Pardee, K. et al. Portable, on-demand biomolecular manufacturing. Cell 167, 248–259.e12 (2016).
Perez-Pinera, P. et al. Synthetic biology and microbioreactor platforms for programmable production of biologics at the point-of-care. Nat. Commun. 7, 12211 (2016).
Dolsten, M. & Søgaard, M. Precision medicine: an approach to R&D for delivering superior medicines to patients. Clin. Transl. Med. 1, 7 (2012).
Anonymous. Patient-centered drug manufacture. Nat. Biotechnol. 35, 485 (2017).
Schellekens, H., Aldosari, M., Talsma, H. & Mastrobattista, E. Making individualized drugs a reality. Nat. Biotechnol. 35, 507–513 (2017).
Love, K.R. et al. Comparative genomics and transcriptomics of Pichia pastoris. BMC Genomics 17, 550 (2016).
Ahmad, M., Hirz, M., Pichler, H. & Schwab, H. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl. Microbiol. Biotechnol. 98, 5301–5317 (2014).
Hamilton, S.R. Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 313, 1441–1443 (2006).
Konstantinov, K.B. & Cooney, C.L. White paper on continuous bioprocessing. May 20–21 2014 Continuous Manufacturing Symposium. J. Pharm. Sci. 104, 813–820 (2015).
Genentech Inc. Nutropin prescribing information. https://www.gene.com/download/pdf/nutropin_aq_prescribing.pdf (2016).
Center for Biologics Evaluation and Research & Center for Drug Evaluation and Research. Guidance for industry: for the submission of chemistry, manufacturing, and controls information for a therapeutic recombinant DNA-derived product or a monoclonal antibody product for in vivo use. https://www.fda.gov/downloads/biologicsbloodvaccines/guidancecomplianceregulatoryinformation/guidances/general/ucm173477.pdf (1996).
The European Agency for the Evaluation of Medicinal Products. CPMP position statement on DNA and host cell proteins (HCP) impurities, routine testing versus validation studies. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003322.pdf (1997).
Jawa, V. et al. Evaluating immunogenicity risk due to host cell protein impurities in antibody-based biotherapeutics. AAPS J. 18, 1439–1452 (2016).
World Health Organization. Guidelines on the quality, safety, and efficacy of biotherapeutic protein products prepared by recombinant DNA technology. http://www.who.int/biologicals/biotherapeutics/rDNA_DB_final_19_Nov_2013.pdf (2013).
Canova-Davis, E. et al. Properties of a cleaved two-chain form of recombinant human growth hormone. Int. J. Pept. Protein Res. 35, 17–24 (1990).
Merck & Co. Inc. Intron A product information 1–39 https://www.merck.com/product/usa/pi_circulars/i/intron_a/intron_a_pi.pdf (1986).
Matthews, C.B. et al. Reexamining opportunities for therapeutic protein production in eukaryotic microorganisms. Biotechnol. Bioeng. 114, 2432–2444 (2017).
Timmick, S.M. et al. An impurity characterization based approach for the rapid development of integrated downstream purification processes. Biotechnol. Bioeng. 115, 2048–2060 (2018).
Reinl, S.J. & Pogue, G.P. C-terminally truncated interferon. US Patent US20090025106A1 (2011).
Hermeling, S. et al. Structural characterization and immunogenicity in wild-type and immune tolerant mice of degraded recombinant human interferon alpha2b. Pharm. Res. 22, 1997–2006 (2005).
Gibson, S.J. et al. N-terminal or signal peptide sequence engineering prevents truncation of human monoclonal antibody light chains. Biotechnol. Bioeng. 114, 1970–1977 (2017).
Amgen Inc. Neupogen prescribing information. https://www.accessdata.fda.gov/drugsatfda_docs/label/2013/103353s5157lbl.pdf (2015).
Krishnan, S. et al. Aggregation of granulocyte colony stimulating factor under physiological conditions: characterization and thermodynamic inhibition. Biochemistry 41, 6422–6431 (2002).
Lu, H.S. et al. Chemical modification and site-directed mutagenesis of methionine residues in recombinant human granulocyte colony-stimulating factor: effect on stability and biological activity. Arch. Biochem. Biophys. 362, 1–11 (1999).
Bönig, H. et al. Glycosylated vs non-glycosylated granulocyte colony-stimulating factor (G-CSF)—results of a prospective randomised monocentre study. Bone Marrow Transplant. 28, 259–264 (2001).
Sörgel, F. et al. Comparability of biosimilar filgrastim with originator filgrastim: protein characterization, pharmacodynamics, and pharmacokinetics. BioDrugs 29, 123–131 (2015).
Adamo, A. et al. On-demand continuous-flow production of pharmaceuticals in a compact, reconfigurable system. Science 352, 61–67 (2016).
US Food and Drug Administration. Points to consider in the manufacture and testing of monoclonal antibody products for human use (US Government Publishing Office, 1997).
Matthews, C.B., Kuo, A., Love, K.R. & Love, J.C. Development of a general defined medium for Pichia pastoris. Biotechnol. Bioeng. 115, 103–113 (2018).
Hornbeck, P., Winston, S.E. & Fuller, S.A. Enzyme-Linked Immunosorbent Assays (ELISA). in Curr. Protoc. Mol. Biol. 11.2.1–11.2.22 (Wiley, 1991).
Wang, Y.A. et al. Integrated bottom-up and top-down liquid chromatography-mass spectrometry (LC-MS) for characterization of recombinant human growth hormone degradation products. Anal. Chem. https://doi.org/10.1021/acs.analchem.7b03026 (2017).
United States Department of Agriculture (USDA). Animal and Plant Health Inspection Service. 9 CFR Ch. 1, Subchapter A - Animal Welfare (US Government Publishing Office, 2013).
Office for Laboratory Animal Welfare (OLAW). Public Health Service policy on humane care and use of laboratory animals. Health Research Extension Act of 1985 (US Government Publishing Office, 1985).
National Research Council. Guide for the Care and Use of Laboratory Animals. (National Academies Press, 2011).
Biological Evaluation of Medical Devices - Part 2: Animal Welfare Requirements ISO 10993-2 (International Organization for Standardization, 2006).
Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. https://www.aaalac.org/ (2018).
This work was supported in part by the Defense Advanced Research Projects Agency (DARPA) and SPAWAR System Center Pacific (SSC Pacific) under contract no. N66001-13-C-4025. This work was also supported in part by the Koch Institute Support (core) grant P30-CA14051 from the National Cancer Institute and funding from the Department of Chemical Engineering, School of Engineering, Massachusetts Institute of Technology. J.R.B., N.C.D. and N.J.M. were supported by a NIGMS/MIT Biotechnology Training Program Fellowship under NIH contract no. 2T32GM008334-26. K.A.S. was supported by a Mazumdar Shaw International Fellowship. J.C.L. is a Camille Dreyfus Teacher-Scholar. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute, NIH, DARPA or SSC Pacific.
The authors have filed patents related to this work.
Integrated supplementary information
Supplementary Figure 2 Comparison of innovator and InSCyT manufacturing processes for hGH, IFNα-2b, and G-CSF.
Box color indicates the type of unit operation including fermentation (red), solids separation or resuspension (orange), chromatography (purple), pH adjustment (blue), formulation (black) and others (green). AEX, anion exchange; B+E, bind and elute; CEX, cation exchange; SEC, size exclusion chromatography; MMCEX, multi-modal cation exchange; FT, flow through; IEX, ion exchange; HCIC, hydrophobic charge induction chromatography.References39. Waites, M. J., Morgan, N. L., Rockey, J. S. & Higton, G. Industrial Microbiology: An Introduction. (2009).40. Leibowitz, P. & Weinstein, M. J. Extraction of Interferon from Bacteria. (1982).41. Nagabhushan, T. L. & Trotta, P. P. Interferons. Ullmann's Encyclopedia of Industrial Chemistry 375-391 (2012). doi:10.1002/14356007.a14_36542. Boone, T. C., Miller, A. L. & Andresen, J. W. Method for purifying granulocyte colony stimulating factor. (1998). doi:10.1016/j.(73)43. Souza, L. M. Production of Pluripotent Granulocyte Colony-Stimulating Factor. (1989).
Supplementary Figure 3 Additional product quality data for InSCyT-produced hGH from the first-generation process.
Analysis of purity by isoelectric focusing (IEF). M, IEF protein marker. Circular dichroism (CD) analysis of InSCyT hGH samples from day 6 on each system alongside a reference standard (A and B represent technical duplicates). MRW, mean residue ellipticity. Analysis of additional product-related variants. Each of the 12 data points represents a unique sample (four time points from each of three InSCyT systems). Black boxes represent the range of InSCyT hGH samples with an additional line at the mean.
Peptide mapping used to confirm identity (100% sequence coverage). Example LC–MS chromatograms used to quantify purity, including base peak chromatograms and extracted-ion chromatograms of two-chain and deamidated product variants. Data is representative of all 15 InSCyT hGH samples analyzed in this work.
Supplementary Figure 5 Typical product-related variant levels in reference standards for hGH, IFNα-2b and G-CSF.
Summary of product-related variant levels found in reference standards for hGH, IFNα-2b, and G-CSF compared with values found for marketed products in literature44-47. hGH and G-CSF reference standards are de-identified marketed products; IFNα-2b reference standard is a manufactured drug substance. References 44. Jiang, H., Wu, S., Karger, B. L. & Hancock, W. S. Mass Spectrometric Analysis of Innovator, Counterfeit, and Follow-On Recombinant Human Growth Hormone. Artif. Cells Blood Substitutes Immobil. Biotechnol. Cells Blood Substit Immobi 10–15 (2009). doi:10.1021/bp.7245. Riggin, R. M., Shaar, C. J., Dorulla, G. K., Lefeber, D. S. & Miner, D. J. High-performance size-exclusion chromatographic determination of the potency of biosynthetic human growth hormone products. J. Chromatogr. A 435, 307–318 (1988).46. Herman, A. C., Boone, T. C. & Lu, H. S. Characterization, Formulation, and Stability of Neupogen(R) (Filgrastim), a Recombinant Human Granulocyte-Colony Stimulating Factor. in Formulation, Characterization, and Stability of Protein Drugs (eds. Pearlman, R. & Wang, Y. J.) 303–328 (Plenum Press, 1991). doi:10.1080/10643389.2012.72882547. Rathore, A. S. & Bhambure, R. Establishing analytical comparability for ‘biosimilars’: Filgrastim as a case study. Anal. Bioanal. Chem. 406, 6569–6576 (2014).
Supplementary Figure 6 Extended fermentation and on-demand purification and formulation of hGH on the InSCyT system using the second-generation manufacturing process.
Dose size was 1.75 mg12. Center points and error bars represent the mean and range, respectively, of technical triplicates unless otherwise noted. (a) Timeline and yields for production of hGH using the InSCyT system. Unpurified (orange) and formulated (blue) hGH doses from each batch are shown. Grey circles represent individual data points. Product quality analyses for InSCyT-produced hGH alongside a de-identified marketed product. Quantification of host-cell protein and host-cell DNA contaminants in formulated InSCyT hGH (per Fig. 2b). Each data point represents a single batch. Paired data points indicate analyses from a single batch. Analysis of additional product-related variants. Each data point represents a single batch. Black boxes represent the range of InSCyT hGH samples with an additional line at the mean. Analysis of purity by isoelectric focusing (IEF). M, IEF protein marker. Analysis of tertiary structure by circular dichroism (CD). Example reversed-phase liquid chromatography (RPLC) and size exclusion chromatography (SEC) chromatograms used to quantify purity of hGH (representative of all three batches). (b) Fermentograms and UV profiles from chromatography operations on the InSCyT system during the extended production of hGH on demand. DO, dissolved oxygen; SLPM, standard liters per minute.
Supplementary Figure 7 Detailed comparison of product quality across experiments during the at-scale process development for IFNα-2b.
(a) Summary of product-related variants detected in formulated InSCyT IFNα-2b during at-scale process development and final process qualification. Center points and error bars for the qualification run represent the mean and range, respectively, of four different batches produced during the run. The timeline (bottom) indicates when each characterization experiment was executed, where day 0 corresponds to the day when at-scale process development first began. Horizontal colored bars represent the modules that were used in each experiment (USP – orange, DSP – purple, TFF – blue). Each bar represents a set of experimental conditions on that module. HMW, high molecular weight. (b) Quantity of oxidized product-related variants as influenced by various upstream parameters. N-terminal variant, aggregate, and host cell protein (HCP) content associated with different purification processes. Purification process A was used in IFNα-2b Process Development Experiment 4, and purification process B was used in IFNα-2b Process Development Experiment 3 and the Process Qualification Run.
Dose size was 12ug18. (a) Yields for production of IFNα-2b using the InSCyT system during the Process Qualification Run. Wet cell weight (WCW) (black) and cumulative unpurified (orange) and formulated (blue) doses of IFNα-2b are shown. Grey circles show individual data points. Complete process yield is approximately 11%. Additional product quality data from the process qualification run for IFNα-2b alongside a reference drug substance. Analysis of purity by isoelectric focusing (IEF). M, IEF protein marker. Example RPLC and SEC chromatograms used to quantify purity of IFNα-2b (representative of all four time points). Differences in chromatographic behavior of InSCyT IFNα-2b and the reference standard were confirmed by MALDI as a naturally-occurring C-terminal truncation. (b) Fermentograms and UV profiles from chromatography operations on the InSCyT system during the process qualification run for IFNα-2b. DO, dissolved oxygen; SLPM, standard liters per minute. (c) Product quality data for InSCyT-produced IFNα-2b analyzed by LCMS. Peptide mapping used to confirm identity (96.3% sequence coverage). Example LCMS chromatograms used to quantify purity, including base peak chromatograms and extracted-ion chromatograms of N-terminal variant.
Dose size was 300 μg24. Center points and error bars represent the mean and range, respectively, of technical triplicates unless otherwise noted. Fermentograms and UV profiles from chromatography operations on three InSCyT systems during G-CSF production. DO, dissolved oxygen; SLPM, standard liters per minute. Timeline and yields for production of G-CSF using three InSCyT systems. Wet cell weight (WCW) (black circles) and cumulative unpurified (orange) and formulated (blue) doses of G-CSF are shown. Grey circles show individual data points. Analysis of the tertiary structures of InSCyT G-CSF and a de-identified marketed product using circular dichroism (CD). Analysis of additional product-related variants. Black boxes represent the range of InSCyT G-CSF samples (Batches 1-6) with an additional line at the mean. SDS-PAGE (12% tris-glycine) analysis of samples from the USP during biomass accumulation (Perfusate Sample #0) and production (Perfusate Sample #1,2), and a final, formulated InSCyT sample (Formulated Sample #1,2) alongside a G-CSF marketed drug product (Std). M, molecular mass marker. Analysis of product purity by isoelectric focusing (IEF) for a formulated sample from each system. M, IEF protein marker. Example RPLC and SEC chromatograms used to quantify purity of G-CSF (representative of all six batches).
Peptide mapping used to confirm identity of G-CSF (100% sequence coverage). Example LC–MS chromatograms used to quantify purity of G-CSF, including base peak chromatograms and extracted-ion chromatograms of N-terminal and O-linked glycan product variants (T134 O-2mannose).
Supplementary Figure 11 Summary of results from repeat-dose study of InSCyT G-CSF in Sprague Dawley rats.
All hematology and clinical chemistry parameters demonstrating statistically significant differences at a 5% or 1% significance level are shown. All significance levels were determined using 1-way ANOVA. Mean values, standard deviation (SD), and number of animals (N) are shown for each relevant parameter for the vehicle control group, InSCyT G-CSF and Neupogen®.
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Crowell, L., Lu, A., Love, K. et al. On-demand manufacturing of clinical-quality biopharmaceuticals. Nat Biotechnol 36, 988–995 (2018). https://doi.org/10.1038/nbt.4262
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