On-demand manufacturing of clinical-quality biopharmaceuticals

Abstract

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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Figure 1: Schematic of the InSCyT system for on-demand biomanufacturing and demonstration of consistent operation across three distinct InSCyT systems.
Figure 2: Production of hGH on the InSCyT system.
Figure 3: Accelerated process development using the InSCyT system and production of IFNα-2b.
Figure 4: Production of G-CSF on three identical InSCyT systems.

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Acknowledgements

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.

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Contributions

L.E.C., A.E.L. and K.R.L. designed experiments, analyzed data and wrote the manuscript. J.C.L., K.R.L., R.D.B., A.S., A.E.L., N.J.M. and L.E.C. designed and built the InSCyT system. A.E.L. and R.D.B. developed controls for the InSCyT system. L.E.C., W.D. and A.B. performed experiments on the InSCyT system and performed quality assessments. D.W., Y.A.W., Y.L., S.-L.W. and W.S.H. assessed quality by mass spectrometry and isoelectric focusing. S.M.T., N.V., C.G. and S.M.C. developed the purification processess and assessed quality by RPLC, size exclusion chromatography and CD. J.R.B., N.C.D. and K.R.L. developed and performed product-specific ELISAs. K.R.L., K.A.S., N.J.M. and J.R.B. engineered the strains used in production. J.J.C., N.A.C., D.L., C.A.M., C.B.M., N.J.M. and K.R.L. contributed to development of upstream processes. A.C. and L.B. developed the purification process for hGH. J.C.L., S.M.C., R.D.B. and W.S.H. designed the experimental strategy, supervised analysis and wrote the manuscript. All authors reviewed the manuscript.

Corresponding author

Correspondence to J Christopher Love.

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Integrated supplementary information

Supplementary Figure 1 Detailed piping and instrumentation diagram (P&ID) for the InSCyT system.

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.

Supplementary Figure 4 Product quality data for InSCyT-produced hGH as analyzed by LC–MS.

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.

Supplementary Figure 8 Production of IFNα-2b on the InSCyT system.

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.

Supplementary Figure 9 Production of G-CSF using multiple InSCyT systems.

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).

Supplementary Figure 10 Product quality data for InSCyT-produced G-CSF as analyzed by LC–MS.

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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