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Hardware and software are the focus here in Part 1. HTTPS://STOCK.ADOBE.COM

The past couple of decades have witnessed significant advances in upstream bioprocess technologies and approaches. Since its establishment, BPI has been a facilitator of discussion in both print and professional conferences, as well as in webcasts and news online. To mark the 20th anniversary of the publication, we surveyed articles published over the past two decades and found hundreds that highlight significant advances in both emerging and established themes in biopharmaceutical production:

• “hardware” and assets (e.g., analytical instrumentation, bioreactors, and facilities) — in Part 1
• “software” and knowledge (e.g., process controls, quality by design (QbD), and process analytical technologies (PAT)) — in Part 1
• “wetware” technology (e.g., expression systems, cell-line development, culture media, and emerging modalities such as cell and gene therapies) — in Part 2.

Advances in single-use technologies (SUTs) are so extensive that they merit dedicated coverage elsewhere in this special issue. Since the turn of the century, they have been influencing the directions taken by biopharmaceutical developers, solution suppliers, and services providers alike. The resulting technological progress has changed the expectations of regulators, and will continue to. All stakeholders share the common goal of improving outcomes for patients through providing them with accessible, affordable, safe, and effective biological therapies.

Note that the references listed as Further Reading are a small sampling of the hundreds we could have provided on these topics from the BioProcess International archives so far. The topics often overlap, so many articles also illustrate more than one type of advance. You can find them all in the issue-archives section of BPI’s website online at https://bioprocessintl.com.

Your local hardware store once was a little shop where you could pick up tools and supplies for basic carpentry and repair work. Now big-box stores sell everything from lumber to appliances and fixtures, as well as tools and accessories. Here we apply the same sort of all-encompassing view to biotechnology “hardware,” from the bioreactors at the heart of it all to the facilities that house bioprocess operations.

Bioreactor Systems: As is noted elsewhere in this special issue, the expansion of SUT in bioprocessing is one of the most significant developments in the biopharmaceutical industry in the past 20 years. Nowhere has that made a greater impact than in upstream production processes, from seed-train cultures through commercial production.

Early limitations on the disposable-bioreactor concept were addressed by suppliers over time. Stirred-tank mixing (rather than the revolutionary rocking technology that proved to be impractical at large scales) has enabled working volumes now up to 5,000–6,000 L. Adherent-cell cultures are accommodated by single-use supports and other innovative technologies. Ports for sampling and feeding are increasingly sophisticated, as are connector and disconnector designs. Those have led to enhancement of traditional fed-batch approaches for extended periods of cell viability, even as other technologies (e.g., specialized clarification methods) enabled more widespread use of perfusion-mode production.

Especially over the past decade, high-density cell cultures (>150 million cells/mL) became both more feasible and more commonly used as downstream limitations were addressed — taking production yields of target proteins well into double-digit g/L territory. At-line and on-line sampling — and even testing — have gone from a “pipe dream” to a reality in many instances, all to the benefit of process control. Hardware has made all the difference in these advancements, providing the means for process engineers to envision and then realize the types of innovations described in the sections below.

Perfusion: Ever since BPI’s first publication, the industry has trended slowly but steadily toward a preference for perfusion processes over batch/fed-batch cultures. This trend has been driven by specific product requirements — because perfusion systems can maintain cells at higher viabilities, which can be a critical factor for many labile protein products — as well as general advantages afforded by smaller footprint requirements and the ability to run long continuous campaigns. Robustness of perfusion has increased over time, especially in response to early perceptions regarding increased contamination risk. Meanwhile, process development tools and technology advances have accommodated this trend with, for example,

• improved process monitoring
• long-lasting, robust sensors
• perfusion-tailored SUTs
• small-scale high-throughput (HTP) systems for scaled-down perfusion screening and process development.

Scalability: Whether up, down, or out, scaling is a critical aspect of process developers’ work. The HTP afforded by small-scale systems greatly shortens overall timelines. This helps development teams select clones that will serve as the basis of future cell banks as well as optimize media formulations that enable cultures to express products that meet both critical quality attributes (CQAs) and necessary process economics (e.g., regarding cellular productivity and viable-cell growth rates). To be implemented successfully, results obtained from those small-scale studies must be reliably representative of the production/commercial scale. Scale dependence must be minimized or accommodated.

BPI authors have demonstrated progress in technologies and provided results (e.g., from early adopters) through several reports. This began with scale-down of traditional stainless-steel batch and fed-batch processes and has continued with similar work using perfusion bioreactors. Scalability assurance continued as the implementation of single-use bioreactors and probes gained momentum to the current state at which scalability can be maintained faithfully across modes of operation, vessel and ancillary component types, and materials of construction — as well as for all cell types (e.g., mammalian, insect, and microbial cells). Successful and rapid scale-up of a process that’s developed using small (even miniaturized) HTP systems depends on many factors in addition to robust scalability technologies. Key among those is a deep process understanding that can be leveraged through using standardized approaches and platforms that apply to different drug products in development (e.g., using a common cell source and process as the starting point for process development).

The importance of scalability is not limited to early process-development phases; it continues throughout each product’s life cycle. For example, using a faithful scale-down model can assist in implementation of process changes by enabling teams to conduct validation activities using qualified scale-down models without sacrificing production capacity. Likewise, scale-down models can be helpful for conducting investigations into deviations and excursions that can occur during production campaigns. A truly faithful small-scale model should recapitulate the issue being investigated and thus help investigators develop a proven correction without taking a production unit off line. Such developments have been beneficial in addressing production issues and making continuous improvements deep into a product life cycle without sacrificing production capacity or patient supplies.

Aseptic Processes and Contamination Control: The upstream stage of bioprocessing is an area of concern with regard to pathogen safety because growth media and production cells used at this stage are potential sources of biological contamination. That may include adventitious viruses or bacteria (a potential source of endotoxins). Moreover, growth conditions used upstream can propagate live pathogens if they are introduced inadvertently. So whereas strategies for contamination control are expected to be holistic — covering all aspects of a process — the upstream stage has been an area of focus for prevention (e.g., through sterilization of raw materials), testing, and improved detection. Downstream activities focus more on the means of killing, inactivating, and removing potential adventitious with validated reduction methods while continuing to control for potential introduction of pathogens (e.g., bacteria, viruses, and mold) from people, raw materials, and the environment.

SUTs also have improved the containment of culture processes for prevention of both cross-contamination from other process streams and adventitious agents from operators and the environment. Because biopharmaceutical final products cannot be sterilized, they must be made with a sterility mindset throughout the manufacturing process. Thus, contamination control always has been vitally important even at the upstream-production stage — not only to protect patients, but also to protect the living cells on which bioprocessing depends. Healthy cells are happy cells, so to speak, and happy cells perform best.

Before the era of SUT, the goal of aseptic processing was achieved primarily through rigorous and tightly validated cleaning protocols. Those remain important, especially to legacy facilities and long-established manufacturing processes, but modern strategies can focus more on true containment based on closed, modular cell-culture isolator systems. Biotech-hardware developers have developed — and continue to innovate on — a range of options for bioprocess engineers to experiment with. No single solution works for every product candidate, but the overall trend is a movement away from the primary emphasis on cleaning and toward making enclosure the priority.

Facilities: Enabled by the expanding applications of SUTs, biopharmaceutical manufacturing facilities have undergone a revolution over the past two decades as well. With equipment and instruments mostly uncoupled from hard-piped utilities and infrastructure, companies are free to modularize and mobilize unit operations such as media preparation, cell culture, and harvesting within a warehouse-like environment. Two decades ago, the typical bioprocess facility was dedicated to making a single blockbuster product; it featured miles of gleaming steel piping, specialized pressure vessels, and highly segregated cleanrooms; and it took years to design, build, and qualify for operation. Some such facilities remain in use — but they are no longer the norm.

Modern biomanufacturing is more likely to take place in the bright white environs of modular cleanrooms, with isolators and single-use systems that reduce the highest levels of cleanliness to the smallest footprint required. New biologics are less likely to serve blockbuster-level populations, so more products can be made at smaller scales for niche markets, making multiproduct facilities the new norm. Technology transfer is more straightforward than it once was — not only because the typical range of process scales has narrowed for most upcoming produts, but also because equipment suppliers have addressed the needs of users and developed “families” of directly scalable bioreactors.

20 Areas of Advancement in 20 Years
(1) Facility considerations: ballroom and modular manufacturing concepts, equipment installation, technology transfer, instrument qualification, and process validation(2) Closed, modular cell-culture isolator systems and related advances in aseptic processing(3) Single-cell cloning and selection systems(4) Bioreactor systems, with options proliferating for mode of operation (fed-batch/continuous, suspension/adherent), mixing technologies, capacity for high-density and extended-viability cultures, (at/on-line) sampling and monitoring technologies, and process controls(5) Perfusion cell-culture systems that operate across scales and productsSoftware
(6) Options for integration into cell-culture systems(7) Capabilities for quality by design (QbD), statistical design of experiments (DoE), high-throughput screening, and scale-down modeling

(8) Automation and process analytical technology (PAT) for improved design, analysis, and control of critical quality attributes (CQAs) through measurement of critical process parameters (CPPs)

(9) Pathogen safety and contamination control

(10) Culture-media components: serum-free and chemically defined formulations and supplements coupled with mammalian host-cell and cell-adaptation approaches

(11) Culture-media preparation: media batching, media-powder hydration, media cartridges, and tools for agglomeration prevention

(12) Cell banking and recovery for seeding robustness

(13) Mammalian cell-line development: improvements to transfection efficiency and metabolic engineering; development of expression hosts for increased product expression levels with consistent product quality attributes (PQAs)

(14) Cell-therapy culture systems (autologous and allogeneic) and viral production systems for gene therapies

(15) Genetic engineering game changers (targeted expression and genome editing)

(16) Bacterial protein expression: improvements to Escherichia coli and Pseudomonas fluorescens expression systems, inclusion-body solubilization, product secretion, and overall recovery

(17) Alternative expression systems: growing application of yeast cells, insect cells with the baculovirus expression-vector system (BEVS), plant cell-culture systems, cell-free expression, and whole-organism production (e.g., proteins expressed in transgenic-animal milk and secretions from whole plants)

Overall Advancements
(18) Scalability with faithfulness across scales (up and down) in process development and investigational studies

(19) Improvements in upstream product-quality control and impurities detection, such as for host-cell proteins (HCPs)

(20) The bottom line: reduced costs, increased efficiencies, shortened timelines, and consistent product quality

For the information-technology (IT) world, software is a familiar and well-defined term. But in a bioprocessing context, we use it herein to cover information in general: the collection, interpretation, analysis, and application of process data and the knowledge arising from putting such information to use. From computer modeling and multivariate analysis methods in process development to monitoring and control in manufacturing, software is vital to many other advancements described herein and throughout this special anniversary issue. As of now, the industry truly has entered the “bioprocess 4.0” era, but that era has only just begun.

Process Control: Robust, reproducible, and efficient upstream bioprocesses begin with identification of critical process parameters (CPPs) such as pH, dissolved oxygen (DO), temperature, viable-cell density, culture viability, specific growth rate, nutrient levels, and other measurable parameters. Advancing sensor technologies — both single- and multiuse — are key to monitoring CPPs, and the data they gather must be organized, interpreted, and analyzed to translate process monitoring into process control.

Over the past two decades, instrument suppliers such as Cytiva, Eppendorf, Pall, and Sartorius have developed sophisticated software — e.g., the Unicorn, DASware, mPath, and BioPAT systems, respectively — for handling those data and helping users optimize and manage their upstream processes. That allows companies to address CQAs of their drug products from the very start of drug-substance manufacturing. The modern upstream process engineer understands how CPPs affect such CQAs as proper glycosylation and higher-order protein structure — and can use that knowledge to tune cultures for the best possible outcomes.

Process Development: The primary impact of all that available information has been to provide much greater precision in process engineering than could have been possible in years past. Regulators saw this coming 20 years ago when they introduced the quality by design (QbD) initiative — a concept that initially was met with incredulity and skepticism but that has encouraged and progressed alongside the technological advancements we have seen since then. When the US Food and Drug Administration’s (FDA’s) process validation guidance was updated a decade ago, it changed the way that developers talked about qualification and validation of instruments, equipment, and processes. The risk-based methodologies that followed have been made possible by implementing HTP screening and statistical DoE methods once familiar only to drug discovery laboratories.

All of that progress has made upstream process development much more of a science than an art. Trial-and-error remains a key part of it; the experiments just move faster now than ever before. Scientists can vary multiple process variables together now rather than one at a time, with microbioreactors and other radically scaled-down systems making it possible to run dozens of tests in parallel. This helps an upstream group quantify the design-space limits of each process by identifying CPPs that need the most stringent specification limits.

Looking Ahead
Although the term had been used in niche scientific papers since the 1950s, science-fiction writers popularized the concept of “wetware” in cyberpunk novels such as Bruce Sterling’s Schismatrix, published in 1985. When applied to biotechnology, we use it similarly to the “wet chemistry” description of some laboratories. Living cells, tissues, and organisms are “wet” by nature in that they are highly water-based entities, and bioprocesses themselves take place in a fluid environment.

In BPI’s September issue, Part 2 will highlight advances in mammalian cell-line development, genetic engineering, single-cell cloning and selection, cell banking, culture media, and other expression systems including microbials and transgenics.

Further Reading, Part 1
You can find all of these and more in the issue-archive section of BPI’s website at https://www.bioprocessintl.com.

Tholudur A, et al. Using Design of Experiments To Assess Escherichia coli Fermentation Robustness. October 2005

Gryseels T. Considering Cell Culture Automation in Upstream Bioprocess Development. December 2008

Collins B, Bornsztejn V. A Modular Approach to Facility Validation. November 2009

Minow B, Rogge P, Thompson K. Implementing a Fully Disposable MAb Manufacturing Facility. June 2012

Bauer I, et al. Novel Single-Use Sensors for Online Measurement of Glucose. September 2012

Klykov SP, Kurakov VV. A New Kinetic Structured Model for Cell Cultivation in a Chemostat. October 2012

Ratcliff A, Preisig C. Advances in Sensor Technology Improve Biopharmaceutical Development. April 2013

Weber A, et al. Development and Qualification of a Scalable, Disposable Bioreactor for GMP-Compliant Cell Culture: Suppliers Put Quality By Design Concepts into Practice. April 2013

Levine HL, et al. Single-Use Technology and Modular Construction. April 2013 (supplement)

Hemmerich J, Kensy F. Automation of Microbioreactors. September 2013
Sinclair A, Titchener-Hooker N. Inactivated Poliovirus Vaccine Made in Modular Facilities with Single-Use Technology. October 2013 (supplement)

Glauche F, et al. Design of Experiments for Fed-Batch Process Development in Shaken Cultures. January 2014

Shimoni Y, et al. Qualification of Scale-Down Bioreactors: Validation of Process Changes in Commercial Production of Animal-Cell-Derived Products, Part 1 — Concept. May 2014

Shimoni Y, et al. Qualification of Scale-Down Bioreactors: Validation of Process Changes in Commercial Production of Animal-Cell-Derived Products, Part 2 — Application. June 2014

DeWilde D, et al. Superior Scalability of Single-Use Bioreactors. September 2014 (supplement)

Weber A, et al. Development and Qualification of a Scalable, Disposable Bioreactor for GMP-Compliant Cell Culture. September 2014 (supplement)

Hutchinson N. Understanding and Controlling Sources of Process Variation: Risks to Achieving Product Critical Quality Attributes. October 2014

Wright B, et al. A Novel Seed-Train Process: Using High-Density Cell Banking, a Disposable Bioreactor, and Perfusion Technologies. March 2015

Chi B, et al. Multiproduct Facility Design and Control for Biologics: Challenges and Considerations. April 2015

Simón M. Bioreactor Design for Adherent Cell Culture — The Bolt-On Bioreactor Project, Part 3: Containment, Sterility. May 2015

DiCesare C, et al. Development, Qualification, and Application of a Bioreactor Scale-Down Process: Modeling Large-Scale Microcarrier Perfusion Cell Culture. January 2016

Carvell J, et al. Monitoring Live Biomass in Disposable Bioreactors. March 2016

Gahtan S, Fablet P, Hilbold N. Insulin in Demand: Engineering a Facility to Serve Local and International Markets. March 2016

O’Kennedy R. Multivariate Analysis of Biological Additives for Growth Media and Feeds. March 2016

Nienow AW, Isailovic B, Barrett TA. Design and Performance of Single-Use, Stirred-Tank Bioreactors. November 2016

Jorjorian P, Kenyon D. How to Set Up a Perfusion Process for Higher Productivity and Quality. April 2017

Waniger S, Wozniak E, Biesecker K. Difficult-to-Express Proteins: Resolving Bioprocessing Challenges with a Scalable Perfusion Bioreactor. May 2017

Shimoni Y, et al. Reducing Variability in Cell-Specific Productivity in Perfusion Culture: A Case Study. January–February 2018

Maischberger T, Krainer F. Improving Bioreactor Performance: Measuring Dissolved Oxygen to Determine kLa. October 2018

Simón M, Henriksen-Lacey M, Aiastui A. Efficient Production of Adherent Cells – The Bolt-On Bioreactor Project, Next Phase. June 2019 (eBook)

Solbach D. Production of Transient Lentiviral Vectors in HEK 293T Cells: Cultivation on Fibra-Cel Disks in a Single-Use, Packed-Bed, Stirred-Tank Bioreactor. October 2019

Pathange LP, Shimoni Y, Srinivasan V. Product Quality Attribute Shifts in Perfusion Systems, Part 1: Identifying Shifts When They Occur. September 2020

Shimoni Y, Prasad Pathange LP, Srinivasan V. Product Quality Attribute Shifts in Perfusion Systems, Part 2: Elucidating Cellular Mechanisms. November–December 2020

Kis Z. Enhancing Vaccine Platforms: Computational Models Accelerate Development, Manufacturing, and Distribution. January–February 2021 (supplement)

Yamanaka H, Murato Y, Cizdziel PE. Bioreactor Automation Driven by Real-Time Sensing: Enhancing Productivity Through Accurate, Efficient Glucose Control. January–February 2021 (supplement)

Yuval Shimoni is associate director and product quality leader at BioMarin Pharmaceutical, 105 Digital Drive, Novato, CA 94949; [email protected]. Cheryl Scott is cofounder and senior technical editor of BioProcess International, part of Informa Connect Life Sciences, 1-212-600-3429, [email protected].

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