Process Analytical Technologies and Data Analytics for the Manufacture of Monoclonal Antibodies
2020; Elsevier BV; Volume: 38; Issue: 10 Linguagem: Inglês
10.1016/j.tibtech.2020.07.004
ISSN0167-9430
AutoresMurali Kannan Maruthamuthu, Scott R. Rudge, Arezoo M. Ardekani, Michael R. Ladisch, Mohit S. Verma,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoProcess analytical technology (PAT) has evolved from hardware-based analyses for defined biological, biomolecular, and biochemical analytes to a toolbox that encompasses data analytics and soft sensors to monitor and control monoclonal antibody (mAb) manufacture.Engineered cell lines used in batch processes and continuous manufacturing have helped improve qualities and production rates for mAbs.Data analytics has become increasingly important as sensors become smaller, more robust, and increasingly ubiquitous, with soft sensors enabling determination of a rolling baseline of process conditions and consequences during the production of biologics.In-line sensors utilized for downstream processes provide a template for how such sensors might be used as part of PAT in the real-time monitoring of the manufacture of biotherapeutic proteins in both upstream and downstream unit operations. Process analytical technology (PAT) for the manufacture of monoclonal antibodies (mAbs) is defined by an integrated set of advanced and automated methods that analyze the compositions and biophysical properties of cell culture fluids, cell-free product streams, and biotherapeutic molecules that are ultimately formulated into concentrated products. In-line or near-line probes and systems are remarkably well developed, although challenges remain in the determination of the absence of viral loads, detecting microbial or mycoplasma contamination, and applying data-driven deep learning to process monitoring and soft sensors. In this review, we address the current status of PAT for both batch and continuous processing steps and discuss its potential impact on facilitating the continuous manufacture of biotherapeutics. Process analytical technology (PAT) for the manufacture of monoclonal antibodies (mAbs) is defined by an integrated set of advanced and automated methods that analyze the compositions and biophysical properties of cell culture fluids, cell-free product streams, and biotherapeutic molecules that are ultimately formulated into concentrated products. In-line or near-line probes and systems are remarkably well developed, although challenges remain in the determination of the absence of viral loads, detecting microbial or mycoplasma contamination, and applying data-driven deep learning to process monitoring and soft sensors. In this review, we address the current status of PAT for both batch and continuous processing steps and discuss its potential impact on facilitating the continuous manufacture of biotherapeutics. mAbs have evolved from being scientific tools derived from murine hybridomas in 1975 to biotherapeutic molecules based on humanized antibodies (see Glossary). The first mAb for therapeutic use in humans was approved in 1986 and the first bispecific mAb (bsAb; catumaxomab) was approved in 2009 [1.Buss N.A. et al.Monoclonal antibody therapeutics: history and future.Curr. Opin. Pharmacol. 2012; 12: 615-622Crossref PubMed Scopus (316) Google Scholar]. Humanized antibodies include IgG1s, 2s, and 4s grafted onto the Fc and Fv regions, which comprise human sequences. Currently, there are ~570 antibody therapeutics at various clinical phases, with 62 in late-stage trials [2.Kaplon H. Reichert J.M. Antibodies to watch in 2019.MAbs. 2019; 11: 219-238Crossref PubMed Scopus (379) Google Scholar]. Global mAb sales have grown from US $18.5 billion in 2010 [1.Buss N.A. et al.Monoclonal antibody therapeutics: history and future.Curr. Opin. Pharmacol. 2012; 12: 615-622Crossref PubMed Scopus (316) Google Scholar] to US $98 billion in 2017 with 57 mAbs and 11 biosimilars in clinical use as of the end of 2017' [3.Grilo A.L. Mantalaris A. The increasingly human and profitable monoclonal antibody market.Trends Biotechnol. 2019; 37: 9-15Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar]. Of these, 93% are produced in USA and Europe and half are based on fully human genetic sequences [3.Grilo A.L. Mantalaris A. The increasingly human and profitable monoclonal antibody market.Trends Biotechnol. 2019; 37: 9-15Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar]. Over time, several classes of mAb have evolved [1.Buss N.A. et al.Monoclonal antibody therapeutics: history and future.Curr. Opin. Pharmacol. 2012; 12: 615-622Crossref PubMed Scopus (316) Google Scholar]. Early products (Erbitux, Remicade, and rituximab) were obtained by grafting antigen-specific variable domains of mouse antibodies onto constant domains of a human antibody. Humanized mAbs (e.g., Avastin, Mylotarg, and Herceptin), based on a murine hypervariable region grafted onto a human antibody framework, resulted in decreased immunogenic properties and reduced formation of antidrug antibodies. Ultimately, human mAbs emerged from research that utilized phage display technology and transgenic mouse strains that express human variable domains (i.e., Humira, Simponi, and Yervoy). Approximately 20 bsAbs for non-oncology indications have entered various stages of testing since 2000 [4.Udpa N. Million R.P. Monoclonal antibody biosimilars.Nat. Rev. Drug Discov. 2016; 15: 13-14Crossref PubMed Scopus (37) Google Scholar,5.Mullard A. Bispecific antibody pipeline moves beyond oncology.Nat. Rev. Drug Discov. 2017; 16: 667-668Crossref Scopus (23) Google Scholar]. Candidates have the potential to attack Pseudomonas aeruginosa, treat type 2 diabetes mellitus, or provide postexposure protection against Ebola viruses. Approximately 500 fully human antibodies have been identified in the blood of a survivor of coronavirus disease 2019 (COVID-19), and are being assessed for effectiveness against COVID-19 with the goal of rapidly developing a therapeutic antibodyi. Both Biogen and GlaxoSmithKline have separately partnered with Vir to produce mAbs found capable of binding to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)ii. Administration of biologics at concentrations of 150 mg/ml or higher in formulated matrices suitable for injection [6.Whitaker N. et al.A formulation development approach to identify and select stable ultra-high-concentration monoclonal antibody formulations with reduced viscosities.J. Pharm. Sci. 2017; 106: 3230-3241Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar] opens the possibility of subcutaneous home self-administration as the number of biotherapeutic mAbs increases. Dosages range from 50 to 1000 mg per patient per treatment. The patient's need for the therapeutic may last from months to years, and some mAbs or biosimilars will apply to a large population of patients. These combined requirements will necessitate total global production of metric ton quantities annually [4.Udpa N. Million R.P. Monoclonal antibody biosimilars.Nat. Rev. Drug Discov. 2016; 15: 13-14Crossref PubMed Scopus (37) Google Scholar,7.Kelly B. Industrialization of mAb production technology: the bioprocessing industry at a crossroads.mAbs. 2009; 1: 443-452Crossref PubMed Scopus (526) Google Scholar]. PAT will be an important component of achieving enhanced productivity. PAT is a framework for ensuring the quality of a pharmaceutical product by monitoring process streams and unit operations, thereby providing a real-time understanding of the manufacturing process. Determining the sources of variability in a process, how the variability is managed by the process, and whether the product quality may be predicted from process parameters is central to PAT. This knowledge is used to decide which material and process attributes need to be measured and controlled during manufacturing. Implementation of PAT tools (e.g., multivariate analytics, process analyzers, process controllers, and continuous improvement tools) for these critical attributes helps to ensure product quality. The FDA introduced the PAT framework in their Guidance to the Industry document in 2004iii,iv. Since its introduction, the PAT framework has been implemented for the development of small-molecule active pharmaceutical ingredients (APIs) to improve the understanding of process chemistry, as highlighted by the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ Consortium) [8.Chanda A. et al.Industry perspectives on process analytical technology and development.Org. Process R&D. 2015; 19: 63-83Crossref Scopus (125) Google Scholar]. Implementation of PAT for the development and manufacture of mAbs is now gaining momentum with pilot-scale demonstrations of multiattribute monitoring and potential for process control [9.Rolinger L. et al.Multi-variate PAT for UF / DF of proteins – monitoring concentration, particle sizes, and buffer exchange.Anal. Bioanal. Chem. 2020; 412: 2123-2136Crossref PubMed Scopus (33) Google Scholar]. The need for reliable scalable manufacturing of mAbs (and other biologics) continues to increase (e.g., antibody-based therapies for the COVID-19 pandemic). In this review, we highlight the current status of mAb manufacturing, associated challenges, and how PAT and data analytics can help overcome these challenges to develop a new therapeutic product. Chinese hamster ovary (CHO) cells, introduced over 30 years ago and widely used in batch processes, have become a major cell line for the commercial production of mAbs in submerged culture using bioreactors with volumes of up to 15 000 l. CHO cells produce 60% of all mAbs produced, with myeloma cells producing the remainder [3.Grilo A.L. Mantalaris A. The increasingly human and profitable monoclonal antibody market.Trends Biotechnol. 2019; 37: 9-15Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar]. Improvements in cell lines used in batch culture and development of end-to-end continuous manufacturing of mAbs (Figure 1A,E ) has been proposed as a way to increase productivity, decrease equipment footprint, and control cost [10.Alper H.S. Wittmann C. Systems metabolic engineering approaches for rewiring cells.Biotechnol. J. 2019; 14: 1-2Crossref PubMed Scopus (1) Google Scholar, 11.Fisher A.C. et al.The current scientific and regulatory landscape in advancing integrated continuous biopharmaceutical manufacturing.Trends Biotechnol. 2018; 37: 253-267Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 12.Somasundaram B. et al.Progression of continuous downstream processing of monoclonal antibodies: current trends and challenges.Biotechnol. Bioeng. 2018; 115: 2893-2907Crossref PubMed Scopus (88) Google Scholar, 13.Pollock J. et al.Integrated continuous bioprocessing: economic, operational, and environmental feasibility for clinical and commercial antibody manufacture.Biotechnol. Prog. 2017; 33: 854-866Crossref PubMed Scopus (121) Google Scholar, 14.National Academies of Sciences, Engineering, and Medicine et al.Continuous Manufacturing for the Modernization of Pharmaceutical Production: Proceedings of a Workshop. National Academies Press, 2019Google Scholar]. PAT is key to tracking the health, productivity, and titer of large-scale CHO cell cultures over a period of months in a manufacturing environment and is welldeveloped for monitoring the recovery and purification of the mAb product regardless of whether batch or continuous production is used [15.Kornecki M. Strube J. Process analytical technology for advanced process control in biologics manufacturing with the aid of macroscopic kinetic modeling.Bioengineering. 2018; 5: 25Crossref Scopus (41) Google Scholar].Figure 1Unit Operations for the Manufacture of mAbs.Show full caption(A) Schematic of batch process sequence for monoclonal antibody (mAb) manufacture; (B) perfusion culture system: (i) reactor on left side of drawing; (ii) membrane-based separation system on right side of drawing; (for details of numbers, see [16.Petrossian, A. and DeGiovanni, A. Porton International. Perfusion airlift reactor. US4806484Google Scholar]); (C) downstream processing sequence for recovery and purification of mAb; (D) product capacities of mAb processes; (E) comparison with a continuous process; (F) tangential flow filtration (TFF) in a continuous process configuration. For both chromatographic separations (E,F), TFF batch processes operate continuously by cycling discrete unit operations between service and regeneration steps. Reproduced, with permission, from [16.Petrossian, A. and DeGiovanni, A. Porton International. Perfusion airlift reactor. US4806484Google Scholar] (A), [16.Petrossian, A. and DeGiovanni, A. Porton International. Perfusion airlift reactor. US4806484Google Scholar] (B), [17.Godawat R. et al.End-to-end integrated fully continuous production of recombinant monoclonal antibodies.J. Biotechnol. 2015; 213: 13-19Crossref PubMed Scopus (158) Google Scholar] (C), and [7.Kelly B. Industrialization of mAb production technology: the bioprocessing industry at a crossroads.mAbs. 2009; 1: 443-452Crossref PubMed Scopus (526) Google Scholar] (D). Abbreviations: CEX, cation exchange chromatography; DF, diafiltration; LC, liquid chromatography; UF, ultrafiltration.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Schematic of batch process sequence for monoclonal antibody (mAb) manufacture; (B) perfusion culture system: (i) reactor on left side of drawing; (ii) membrane-based separation system on right side of drawing; (for details of numbers, see [16.Petrossian, A. and DeGiovanni, A. Porton International. Perfusion airlift reactor. US4806484Google Scholar]); (C) downstream processing sequence for recovery and purification of mAb; (D) product capacities of mAb processes; (E) comparison with a continuous process; (F) tangential flow filtration (TFF) in a continuous process configuration. For both chromatographic separations (E,F), TFF batch processes operate continuously by cycling discrete unit operations between service and regeneration steps. Reproduced, with permission, from [16.Petrossian, A. and DeGiovanni, A. Porton International. Perfusion airlift reactor. US4806484Google Scholar] (A), [16.Petrossian, A. and DeGiovanni, A. Porton International. Perfusion airlift reactor. US4806484Google Scholar] (B), [17.Godawat R. et al.End-to-end integrated fully continuous production of recombinant monoclonal antibodies.J. Biotechnol. 2015; 213: 13-19Crossref PubMed Scopus (158) Google Scholar] (C), and [7.Kelly B. Industrialization of mAb production technology: the bioprocessing industry at a crossroads.mAbs. 2009; 1: 443-452Crossref PubMed Scopus (526) Google Scholar] (D). Abbreviations: CEX, cation exchange chromatography; DF, diafiltration; LC, liquid chromatography; UF, ultrafiltration. Once an expression system is selected, production is carried out in a sequence of cell culture, recovery, and purification (Figure 1A). Cell culture entails that batch processes that are carried out in specially designed glass or stainless-steel bioreactors or, alternately, in single-use systems (see Box 1 for the evolution of single-use bioreactors). Perfusion culture, first patented in 1989 [16.Petrossian, A. and DeGiovanni, A. Porton International. Perfusion airlift reactor. US4806484Google Scholar] (Figure 1B), is semicontinuous, with media being replenished at the same rate that the bioreactor fluid is separated from cells. The cells return to the bioreactor (Figure 1B) and the mAb product is subsequently harvested, recovered, and purified (Figure 1C), with detailsof the overall sequence given in Figure 1D [17.Godawat R. et al.End-to-end integrated fully continuous production of recombinant monoclonal antibodies.J. Biotechnol. 2015; 213: 13-19Crossref PubMed Scopus (158) Google Scholar]. As summarized in Box 1, Kelly correctly anticipated increases in production capacity and bioreactor volumes (Figure 1D [7.Kelly B. Industrialization of mAb production technology: the bioprocessing industry at a crossroads.mAbs. 2009; 1: 443-452Crossref PubMed Scopus (526) Google Scholar]).Box 1Single-use bioreactorsSingle-use bioreactors are functionally equivalent to stainless steel reactors except the wetted surfaces are made of disposable materials that contact media, cell culture fluids, and process streams. These materials are constructed of multilayer films comprising a structural layer, a barrier layer, and a fluid contact layer. The polymeric films have the requisite inertness and sealing properties [64.Barbarous M. Setie A. Properties of materials used in single-use flexible containers: requirements and analysis.Biopharm Int. 2006; 2006 (Suppl.)Google Scholar]. Single-use bioreactors consist of disposable filter capsules and presterilized containers resembling large plastic bags with volumes of up to 2000 l. Introduced to the industry ~35 years ago, single-use technology has evolved to include fittings, presterilized, prepackaged sensors, and plastic tubing connections comprising'welded' junctions, where tubing is spliced together and then bonded during set-up of a run. Sterility is achieved during the manufacture of single-use components by beta or gammairradiation. Single-use bioreactors are functionally equivalent to stainless steel reactors except the wetted surfaces are made of disposable materials that contact media, cell culture fluids, and process streams. These materials are constructed of multilayer films comprising a structural layer, a barrier layer, and a fluid contact layer. The polymeric films have the requisite inertness and sealing properties [64.Barbarous M. Setie A. Properties of materials used in single-use flexible containers: requirements and analysis.Biopharm Int. 2006; 2006 (Suppl.)Google Scholar]. Single-use bioreactors consist of disposable filter capsules and presterilized containers resembling large plastic bags with volumes of up to 2000 l. Introduced to the industry ~35 years ago, single-use technology has evolved to include fittings, presterilized, prepackaged sensors, and plastic tubing connections comprising'welded' junctions, where tubing is spliced together and then bonded during set-up of a run. Sterility is achieved during the manufacture of single-use components by beta or gammairradiation. The potential impact of the combined application of biology with continuous manufacturing technology is particularly relevant for treating infectious diseases, such as COVID-19. As succinctly stated by Walker [18.Walker J. Lack of blood samples stalls virus-drug work.Wall Street J. 2020; (18 March)Google Scholar]: 'An antibody drug could be developed far more quickly' (than a small molecule drug)… 'because the cure for the new coronavirus likely already exists in the blood of survivors.' Once antibodies are identified and an expression system developed, their combination with continuous manufacturing of mAbs is arguably a key step in scaling availability to accelerate release of the final product for broad clinical use. In the meantime, blood plasma of survivors could serve a limited role for treating patients with COVID-19. Economic impacts could be large, given the US$2 trillion cost of COVID-19 to the US economy as of 10 April 2020. PAT is a set of analytical techniques for which probes are in contact with the process flow and provide frequent and fully automated measurements in realtime for process control with minimal operator intervention [15.Kornecki M. Strube J. Process analytical technology for advanced process control in biologics manufacturing with the aid of macroscopic kinetic modeling.Bioengineering. 2018; 5: 25Crossref Scopus (41) Google Scholar]. These tools are required for maintaining the product quality and for helping to understand and identify critical process attributes. The FDA guidance document intends to encourage innovation in the development, manufacturing, and quality assurance of pharmaceuticalsiii,iv. Key elements of PAT fall into several basic categories, as illustrated for granulocyte colony-stimulating factor (GCSF) by Hebbi and colleagues [19.Hebbi V. et al.Process analytical technology implementation for protein refolding: GCSF as a case study.Biotechnol. Bioeng. 2019; 116: 1039-1052Crossref PubMed Scopus (18) Google Scholar]. PAT applies to: process sensors and process analyzers; process control tools (hardware and software); and, more broadly, utilizes multivariate design; data acquisition and analysis; and continuous improvement and knowledge management tools. The upstream and downstream components of a mAb manufacturing sequence, together with identification of PAT most likely to be associated with the specific unit operations,are depicted in Figure 2. Figure 2 summarizes key areas for continued advances in PAT, including efficient probes and sensors for data collection, comprehensive data libraries, algorithms for deep learning, and real-time data analysis for making real-time decisions about process operations. Combined with improvements in cell lines and unit operations throughout the entire sequence of manufacturing unit operations, PAT has the potential to have a catalytic role in accelerating the transition from batch to continuous manufacture of mAbs. Upstream processes for batch and single-use bioreactors have well-developed cell culture media and clonal cell lines [20.Jayapal K.P. et al.Recombinant protein therapeutics from CHO Cells - 20 years and counting.Chem. Eng. Prog. 2007; 103: 40-47Google Scholar,21.Grav L.M. et al.Application of CRISPR/Cas9 genome editing to improve recombinant protein production in CHO cells.Methods Mol. Biol. 2017; 1603: 101-118Crossref PubMed Scopus (24) Google Scholar]. Culture conditions that maintain appropriate cell density, stability, and integrity are well defined and readily monitored [22.Zydney A.L. Continuous downstream processing for high value biological products: a Review.Biotechnol. Bioeng. 2016; 113: 465-475Crossref PubMed Scopus (216) Google Scholar,23.Rudge S.R. Ladisch M.R. Industrial challenges of recombinant proteins.Adv. Biochem. Eng. Biotechnol. 2020; 171: 1-22PubMed Google Scholar]. Viable cell count, cell metabolites, changes in media components, and mAb accumulation may be measured near-line by liquid chromatography-mass spectrometry (LC-MS), MS-MS, Coulter counters, osmometers, and rheometers within a timeframe of 30 min to several hours. In addition, chemometrics may be applied for chemical analyses combined with data analytics to help guide the analysis of the manufacturing processes. Perfusion processes operate for up to 40 days and retain cells in the bioreactor while product is removed and media is replenished, resulting in high cell densities (108/ml) within a small footprint. A fed-batch reactor run for 10–21 days results in 10× lower cell counts(i.e., 107 cells/ml). A perfusion reactor may be operated for a longer period of time since waste products, proteins, and spent media are continually removed in several ways, including a membrane separation system (Figure 1Bii). Run time in a fed-batch reactor is limited by the bioreactor volume available for the addition of nutrients after the run has started, as well as the concentration of nutrients needed for optimal cell growth.Batch and perfusion reactors have evolved to encompass single-use components, thereby helping to enable contract manufacture where products change from one run to the next. Cell culture media required to reach high cell densities (such as Ham's F10 or Dulbecco's Modified Eagle Media) are free of animal blood serum supplements and are chemically defined [24.Gronemeyer P. et al.Trends in upstream and downstream process development for antibody manufacturing.Bioengineering (Basel). 2014; 1: 188-212Crossref PubMed Scopus (235) Google Scholar]. One example is an off-the-shelf chemically defined medium for CHO cells (EX-CELL® Advanced™ HD Perfusion Medium Cell Vento CHO 100, and others)vi. A comparison of initial amino acid compositions by Reinhart and colleagues [19.Hebbi V. et al.Process analytical technology implementation for protein refolding: GCSF as a case study.Biotechnol. Bioeng. 2019; 116: 1039-1052Crossref PubMed Scopus (18) Google Scholar] for commercially available media summarized the changes in amino acid compositions that occur during fed-batch culture. Their comprehensive discussion of culture media indicated components that could be monitored by PAT, and also captured the complexity of cell culture fluids, resulting in the introduction of background that may interfere with online analysis of target molecules and that must be accounted for if PAT is to be utilized. The whole-genome sequence for CHO cells, completed in 2011 [25.Xu X. et al.The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line.Nat. Biotechnol. 2011; 29: 735-741Crossref PubMed Scopus (611) Google Scholar], enabled more facile applications of gene-editing tools, including clustered regularly interspaced short palindromic repeats (CRISPR) coupled to the enzyme Cas9 [21.Grav L.M. et al.Application of CRISPR/Cas9 genome editing to improve recombinant protein production in CHO cells.Methods Mol. Biol. 2017; 1603: 101-118Crossref PubMed Scopus (24) Google Scholar]. Improved cell lines resulted from accurate annotations for the CHO genome, including introns, exons, transcription start sites, promoter regions, and noncoding RNAs [26.Kuo C.-C. et al.The emerging role of systems biology for engineering protein production in CHO cells.Curr. Opin. Biotechnol. 2018; 51: 64-69Crossref PubMed Scopus (56) Google Scholar]. Transgene integration improved productivity by achieving a high gene transcript for the target protein (e.g., mAb) [27.Chen C. et al.Upstream process intensification and continuous manufacturing.Curr. Opin. Chem. Eng. 2018; 22: 191-198Crossref Scopus (54) Google Scholar]. PAT sensors (Table 1) monitor media conditions (pH and dissolved oxygen) and measure cellular processes, media supplementation, and extrinsic factors that influence protein expression [28.Claβen J. et al.Spectroscopic sensors for in-line bioprocess monitoring in research and pharmaceutical industrial application.Anal. Bioanal. Chem. 2017; 409: 651-666Crossref PubMed Scopus (109) Google Scholar]. In addition to genome editing during cell line research and development, process control of the quantity and quality of expressed proteins achieved by directing the metabolism of the cellsin a scalable system depends on measurements from in-line sensors or near-line analytical methods [20.Jayapal K.P. et al.Recombinant protein therapeutics from CHO Cells - 20 years and counting.Chem. Eng. Prog. 2007; 103: 40-47Google Scholar]. Since cells are suspended in a medium into which air is introduced, sensors for three phases are needed: air, liquid, and solid (Table 1) [29.Bluma A. et al.Process analytical sensors and image-based techniques for single-use bioreactors.Eng. Life Sci. 2011; 11: 550-553Crossref Scopus (16) Google Scholar]. Temperature, shear force, conductivity, pH, pO2, pCO2, leachables, bioburden, secondary metabolites, and cell morphology, as well as cell viability, density, stability, and productivity [30.Li F. et al.Cell culture processes for monoclonal antibody production.MAbs. 2010; 2: 466-477Crossref PubMed Scopus (527) Google Scholar], protein aggregation and secondary structure [21.Grav L.M. et al.Application of CRISPR/Cas9 genome editing to improve recombinant protein production in CHO cells.Methods Mol. Biol. 2017; 1603: 101-118Crossref PubMed Scopus (24) Google Scholar], osmolality, and ammonia are measurable, and measured, by in-line sensors or near-line analysis of culture fluids. However, determination of viral load [31.Gillespie C. et al.Continuous In-line virus inactivation for next generation bioprocessing.Biotechnol. J. 2019; 141700718Crossref Scopus (43) Google Scholar] or mycoplasmamay require several days and, thus, is currently less amenable to online methods. We focus on in-line sensors in Table 1 because defining the state of a new process during development requires the use of as many attributes as possible to explain the sources of variability. When moving from research to production, the number of parameters to be monitored in-line may be reduced if a lower number of distinct parameters are shown to be sufficient to describe the state of the process. While some components in single-use reactors may be used only once and standard sensors for temperature, pH, and pO2 are now also disposable, many sensors are reused after cleaning and recalibration.Table 1Different Types of In-Line Sensors Used in Biotechnology Process TechnologyaAbbreviations: DSP, downstream Processing; USP,upstream processing., bAdapted, with permission, from [28,29].Type of measurementSensor techniqueSegment of installationMode of measurementPhysical parametersTemperatureThermometerUSP and DSPIn-linePressureThermocoupleUSP and DSPIn-lineRedox potentialMembrane pressure sensors, redox (Pt) electrodeUSP and DSPIn-lineChemical parameterspHpH electrodesUSP and DSPIn-lineDissolved gasAmperometry electrodes, CO2 electrodesMostly USPIn-lineGas phaseParamagnetic CO2 sensorsMostly USPIn-lineDissolved compoundsSpectroscopic sensors (glucose, lactate)USP and DSPIn-line (mostly in research)Biological parametersBiomassSpectroscopic sensorsUSP and DSPImpedance sensorsMostly USPCell morphologyFluorescence sensorsUSPIn-line (mostly in research)Impedance sensorsUSPViabilityIn situ microscopyUSPDNA/RNA/contentSpectroscopic sensorsUSP and/or DSPa Abbreviations: DSP, downstream Processing; USP,upstream processing.b Adapted, with permission, from [28.Claβen J. et al.Spectroscopic sensors for in-line bioprocess monitoring in research and pharmaceutical industrial application.Anal. Bioanal. Chem. 2017; 409: 651-666Crossref PubMed Scopus (109) Google Scholar,29.Bluma A. et al.Process analytical sensors and image-based techniques for single-use bioreactors.Eng. Life Sci. 2011; 11: 550-553Crossref Scopus (16) Google
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