Bioprocessing, Bioengineering and Process Chemistry in the Biopharmaceutical Industry

The explosion and exploitation of biotechnology and bioengineering toward the end of the twentieth century led to the invention of valuable biologic drugs for the benefit of patients around the world. Opportunities continue to expand in dramatic and unforeseen ways through the present time. Advances in individual fields as well as multidisciplinary integration across science, technology, and bioengineering drive this innovation, emphasizing the importance of reducing timelines and cost for drug development and clinical studies, all under a regulatory and ethical framework that is vital for delivering safe and effective therapies to patients.

Medical disorders with associated genetic components and the understanding of single mutations in disorders like sickle-cell disease, cystic fibrosis, and progeria have led to possible solutions by synthetic biologists for such diseases. The latest advancements in synthetic biology and emerging new technologies manifested in uncovering the expression of monoclonal antibodies, antibody drug conjugates (ADCs), gene therapy and immunotherapies, cell therapies (e.g., CAR-T [Chimeric Antigen Receptor-engineered T-cells]), gene editing (e.g., CRISPR [Clustered Regularly Interspaced Short Palindromic Repeats]), and vaccines among other applications. Efficient processes for generating monoclonal antibodies, gene therapy, and cell therapy products require significant advances in upstream and downstream processes, analytical methods, as well as automation leading to process efficiencies and green processes.

The opening chapter discusses the differences and the relationship between small molecules and biologic drugs and topics representing the latest advances of bioprocessing, bioengineering, and process chemistry in biologics and biotechnology applications of interest to global readers from both industry and academia.

In its simplest definition, synthetic biology is the creation of new biological entities for useful purposes. By manipulating an organism’s genome, synthetic biologists can produce novel proteins for a wide range of applications, from the biosynthesis of industrial chemicals to the discovery and optimization of biotherapeutics. The field of synthetic biology has experienced a renaissance in recent years as technological advances have lowered the barrier to entry and increased the potential for innovation. Principal among these advances has been the development of highly precise and large-scale DNA synthesis platforms.

The synthesis of genetic material is a non-trivial, yet integral component of synthetic biology. After all, behind every novel protein is a novel DNA sequence. Synthetic oligonucleotides are also irreplaceable components in CRISPR (clustered regularly interspersed palindromic repeats) gene-editing, DNA sequencing, and the myriad tools that enable researchers to manipulate and interrogate genomes.

Historically, oligonucleotide synthesis was a slow, error-prone process that severely limited its usefulness beyond niche studies. However, the advent of phosphoramidite chemistry and solid-phase synthesis marked an inflection point after which the scale, efficiency, and precision of DNA synthesis markedly increased. And with this increase came an ever-growing list of applications for synthetic biology.

The field of synthetic biology is on a trajectory to play a pivotal role in addressing many of the world’s most challenging and complex problems. Projects are underway to develop and apply engineered organisms in bioremediation, helping to clean polluted ecosystems. Crops, engineered to resist harsh weather conditions, have long been sought as a means to reduce starvation in drought-stricken environments. And with the ability to rapidly design and build DNA libraries, drug developers will be better equipped to discover and optimize novel therapeutic modalities.

Perhaps the most salient example of synthetic biology’s ability to advance therapeutic development is the rapid expansion of cell therapies to include chimeric antigen receptor (CAR)-T and CAR-NK (natural killer) cells. These highly engineered cells sit at the vanguard of clinical oncology and, as the tools of synthetic biology continue to evolve, will likely play an ever-larger role in patient care.

In the following sections, we provide a brief overview of synthetic biology and how this nascent field is catalyzing rapid development of novel biotherapeutics.

DNA synthesis is a naturally occurring process that happens during DNA replication. In the mid-twentieth century, a chemical process was developed to synthesize single-stranded oligonucleotides. By the early 1980s, multiple commercial companies were selling synthetic DNA. Gene synthesis (i.e., DNA assembly) methods were developed in parallel to generate long double-stranded DNA (dsDNA) fragments from many short, synthetic oligonucleotides. These classical methods have been improved upon and miniaturized over the past few decades, culminating in modern gene synthesis platforms that can simultaneously synthesize ~10,000 genes where legacy gene synthesizers could synthesize only one. In this chapter, we review the key advances that enabled modern-scale gene synthesis and how they set the stage for an expanded role for synthetic gene fragments in antibody biopharmaceutical development. We conclude that, in this world of nearly unlimited gene synthesis capacity, the test and learn phases of the design-build-test-learn cycle have supplanted the build phase as the new bottlenecks in biopharmaceutical development.

This chapter on recent advances in upstream process development will discuss the limitations of fed-batch processes, the recent resurgence of interest in traditional “classical steady-state” perfusion in which viable cell density is held nearly constant, and the development of non-conventional “dynamic” perfusion processes in which viable cell density is allowed to peak and decline. Many variants and hybrid processes that combine elements of both perfusion and fed-batch, or even link bioreactors together for a unique, isolated control of the growth phase and the more quiescent production phase will be explored. Other process intensification methodologies such as N-1 perfusion for high-density production reactor inoculation will also be examined.

A section on the pragmatic control of mammalian cell metabolism will explain recent advances designed to precisely control lactic acid formation and limit ammonium ion accumulation. Genetic engineering approaches such as enzyme knock outs and catabolic pathway reconstitution to limit the formation of previously unknown growth-inhibitory by-products of metabolism will also be delineated.

Antibodies as a therapeutic treatment have been the focus of numerous companies for many years, resulting in over 100 currently on the market. This has led to the creation of a rich understanding of how to develop and manufacture these molecules. The result has been the creation of platforms consisting of high productivity cell lines and optimized culture conditions that can generate titers as high as 10 g/L. These platforms have also seen the introduction of streamlined downstream processes which typically consist of two to three chromatography steps.

In recent years, the bispecific and trispecific (or multispecific) antibodies have become a large area of development for most companies, as the ability to bind two different antigens at a time opens new and unique therapeutic areas. Although these are antibody-like, the complexity created due to the structurally diverse molecular formats and engineering adjustments presents a challenge to the current antibody development and manufacturing paradigm. New steps such as reactions to bring these molecules together as well as new impurities and stability issues have meant that these platforms have needed to be adjusted to enable production of suitable quantities of high-quality product.

Expression and production of multispecific antibodies have brought new challenges and considerations to cell line generation. Depending upon molecular format, a strategy needs to be implemented encompassing choice of cell host organism (microbial vs. mammalian), and in the case of mammalian expression, number of cell lines generated (single vs. dual cell lines), as well as random versus targeted integration of transgenes. These initial choices can have far reaching implications, often necessitating expanded cell line screening efforts when compared to traditional monoclonals.

Once a suitable cell line is created, the production of multispecific molecules uses culture conditions similar to typical antibodies. Some adjustments are required however as the engineering of these molecules may result in a higher occurrence of clip species formation or presence of different molecule fragments or impurities. Attention has been paid to ways to reduce these impurities while increasing titers through culture growth conditions and setpoints.

After production there are several considerations that must be focused on to achieve a final high-quality product. These complex molecules can be less stable than traditional antibodies and they tend to contain new and unique impurities that must be removed. Due to the engineering of the molecules, there can be issues with stability that may prevent traditional operations from being conducted such as low pH viral inactivation. This results in the need to design or find additional means to achieve sufficient and robust viral safety. Also, most of the molecules have some level of homodimers present, and due to the similarity of these impurities to the heterodimer, the separation and removal can be difficult. This often results in either the addition of new steps or the use of less traditional steps being employed.

Biopharmaceuticals (also known as biologics) play an increasing role in the treatment of a wide range of diseases, particularly cancer, autoimmune diseases, and infectious diseases. In 2017 and 2018, 11 of the 15 best-selling drugs worldwide were biopharmaceuticals, generally produced in cultured mammalian cells. The vast majority of biopharmaceuticals are glycoproteins, in which the attached glycan moieties play important and often critical roles in controlling activity, clearance, and immunogenicity. In addition to glycoproteins, carbohydrates, particularly glycosaminoglycans (GAGs) such as heparin, the most widely used anticoagulant drug in the world, are critically important biopharmaceutical products.

Many blockbuster biopharmaceuticals such as adalimumab (Humira), trastuzumab (Herceptin), and bevacizumab (Avastin) have recently come off patent, providing an opportunity for production of biosimilar versions by companies other than the innovator. In addition, there is increasing interest in producing glycosaminoglycans from cultured mammalian cells, rather than the current purification from animal tissues with the attendant risks of contamination by adventitious agents and adulteration due to isolation under non-cGMP conditions. To successfully produce biosimilars and bioengineered GAGs, control of glycan composition and structures is critical. This control is challenging as glycan synthesis is a nontemplated process, which is controlled by a complex collection of factors including the glycoprotein being synthesized or the core protein to which the GAG is attached, production host, enzyme activities, and bioprocess conditions.

In this chapter, we review some basics of glycoprotein and GAG biosynthesis with a particular focus on our current understanding of how glycan structures are controlled in vivo. We then review studies in which glycan structures for recombinant proteins and glycosaminoglycans have been optimized by cell line metabolic engineering and bioprocess manipulations. In particular, CRISPR has permitted exquisite editing of host cells, allowing tailored production of glycan structures, facilitating the production of biosimilars and bioengineered GAGs as well as setting the stage for “biobetters” in which improved functionality is obtained by glycoengineering. However, complicated new products including bispecific antibodies, intricate Fc-fusion proteins and molecules not yet envisioned may necessitate new hosts and further advances in glycoengineering.

While several approaches have been considered for in vivo delivery of therapeutic genes, vectors based on a small defective parvovirus, adeno-associated virus (AAV), have proven to be safer in achieving durable therapeutic transgene expression. Nevertheless, the use of vectors based on a virus that infects humans carries the risk of pre-existing host immunity from previous exposure to the virus. Even in the absence of prior infection, administration of large doses of a viral vector can activate host immune responses that can reduce, or prevent, therapeutic gene expression. Hence, careful monitoring of host immune responses before and after vector administration is required when delivering AAV-based gene therapies. Here we review anti-AAV immune responses and the approaches currently being explored to mitigate host immunity and achieve successful therapeutic gene expression.

Over the past 30 years, immunotherapy has grown from bench experiments to a viable clinical option in the battle against cancer with nearly 1000 currently registered clinical trials Ivica NA, Young CM. Tracking the CAR-T Revolution: Analysis of Clinical Trials of CAR-T and TCR-T Therapies for the Treatment of Cancer (1997-2020). Healthcare (Basel) 19;9(8):1062.

In chimeric antigen receptor T-cell (CAR-T) immunotherapy, a patient’s own immune cells are engineered to express a CAR which recognizes cancer cells, expanded ex vivo and then introduced back into the patient, where the engineered cells will mount an immune response against the targeted cancer cells. Immunotherapy manufacturing requires careful consideration of the collection of immune cells from the patient, introduction of receptor into the cells, method of cell expansion, formulation for cryopreservation, and the unique supply chain that must connect clinics, hospitals, and manufacturers to supply individual patients. In this chapter, we provide a brief overview of the current clinical landscape, discuss current practices and key challenges at each manufacturing step using clinical examples where available, and highlight emerging technologies that may accelerate the progress of CAR-T therapies.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology is one of the most significant scientific breakthroughs of this century, revolutionizing therapeutics. A component of bacterial immune systems, CRISPR has been repurposed to precisely edit DNA sequences. The accuracy, efficiency, and simplicity of CRISPR technology have provided exciting new avenues to modify DNA and RNA for the treatment of human diseases. This includes both gene therapy, in which genes are edited in vivo in patients, and gene-edited cell therapy, in which cells are edited ex vivo and transfused into patients. This chapter is a comprehensive guide to CRISPR gene editing and its applications in biotherapeutics. It begins with an introduction to CRISPR-Cas9 technology, examining its discovery and adaptation, mode of action, advantages over previous tools, derivatives of the original CRISPR-Cas9 technology, and gene editing methods. This is followed by a review of the applications of CRISPR technology in biotherapeutics, including both gene therapies and gene-edited cell therapies for the treatment of three disease categories: genetic disorders, infectious diseases, and cancer. Relevant preclinical research and/or clinical trials are discussed for each disease category. The final section delves into the future of CRISPR therapeutics, including nascent applications, the industrialization and projected growth of the industry, safety and ethical considerations, and its transformative potential for human health.

Fusion proteins, consisting of the joined peptide chains of two or more proteins that are naturally not connected, are popular and highly successful recombinant protein drugs with a wide range of different functionalities and therapeutic applications. This chapter summarizes the main features of this protein class and explains the general design principles. Typical building blocks comprise the constant domain of immunoglobulins, linkers with specific properties, and a second protein module that carries some extra functionality such as an enzyme for instance. In some cases, the inherent ability to form higher order complexes is utilized to generate oligomers with novel characteristics. As the artificially combined protein subunits not necessarily share the same physicochemical characteristics, manufacturing becomes challenging. Often even the orientation of the fusion partners has an effect on activity and yield. Therefore, some efforts were undertaken to optimize the constructs through protein engineering. These unnatural proteins are unknown to the human body and immune system, although their components are derived from existing proteins. This can lead to immunogenic reactions particularly through novel epitopes at the junction positions between linkers and fusion partners. The different therapeutic concepts rely on some specific features such as half-life extension by either enlarging the diameter of the proteins to protect them from rapid kidney clearance or through recycling by Fc gamma receptor (FcRn). Both approaches have successfully been used to generate so-called biobetters with enhanced functions. The other important category of fusion proteins contains targeting abilities often conferred trough antibody derivatives. Some proteins from that subtype are immunocytokines that deliver cytokines to specific cell types or organs to exert the corresponding pharmaceutical effect. Overall fusion proteins represent a highly successful but heterogeneous class of recombinant therapeutic.

Antibody drug conjugates (ADCs) are a rapidly growing class of targeted cancer drugs in which a highly toxic small molecule (payload) is conjugated to a tumor-selective antibody. Over the past two decades, a total of 11 ADCs have been approved by the FDA in the United States; including: gemtuzumab ozogamicin (Mylotarg™), brentuximab vedotin (Adcetris™), ado-trastuzumab emtansine (Kadcyla™), inotuzumab ozogamicin (Besponsa™), polatuzumab vedotin (Polivy™), enfortumab vedotin (Padcev™), trastuzumab deruxtecan (Enhertu™), sacituzumab govitecan (Trodelvy™), belantamab mafodotin (Blenrep™), loncastuximab tesirine-lpyl (Zynlonta™), and tisotumab vedotin-tftv (Tivdak™). The path to commercial success for these ADCs has been challenging however, and new ADC approvals were rare prior to 2017 when only three ADCs had been approved by the FDA. Then in 2019 three more ADCs were approved, followed by two approvals in 2020 and two more in 2021. Dozens of ADCs are now in late-stage clinical trials and new approvals are expected to remain consistent for the near future. Following closely behind are over a hundred new ADCs in early clinical or preclinical development. This chapter will summarize the history of currently approved ADCs with emphasis on the challenges that were overcome during development and new technology that likely contributed to their success. The safety and efficacy of each ADC will be discussed from a critical but honest perspective based on personal experience. My intention in writing this chapter is to encourage readers to educate themselves about the real benefits and risks of ADC therapeutics so that informed decisions can be made by cancer patients in collaboration with their doctors.

Mylotarg (gemtuzumab ozogamicin) was the first cytotoxic antibody-drug conjugate approved by the FDA. After its approval, it was withdrawn from the US market in 2010 although it continued to be marketed in Japan. As new clinical data were amassed, an effort to have it reapproved by the FDA and approved in other major markets was initiated. The efforts to bring the Chemistry, Manufacturing, and Controls portion of the regulatory filings up to contemporary standards while still maintaining the original product profile provide an interesting journey through the advances in chemistry, analysis, and regulatory science since the late twentieth century.

Biotherapeutics are rapidly emerging as a successful class of pharmaceuticals, even though significant challenges to their discovery and development remain. In this book chapter, we establish a conceptual framework for potential computational interventions at every stage of biologic drug discovery and early development. We call this framework biopharmaceutical informatics. This chapter provides a comprehensive overview of our strategic vision. This vision calls for closer collaboration between drug discovery and development functions of biopharmaceutical industry by integrating the considerations of developability during early stages of drug discovery. Computational tools already available to enable biopharmaceutical informatics are reviewed in this work. While our focus is on monoclonal antibody-based biologics, the concepts discussed in this work are also applicable to novel formats such as multispecific biologics.

Biomanufacturing drug substance processes include cell culture (thaw, seed train, inoculum, and protein production), harvest, and purification steps. Due to the living nature, the bioprocess inherently has higher variability when compared to chemical reactions. Ensuring consistent process performance and continuous process improvement are ongoing challenges for bioprocess.

Thorough characterization of process and product attributes using statistical design of experiments (DoE) during the process design (PD) stage is a proven means to establish the process and product knowledge. Recent accelerated development timelines are putting pressure and limitations on the extent of characterization during the PD stage, and, therefore, increasing the importance of a lifecycle approach to accumulate process and product knowledge during the post-approval stage of process validation (i.e., continued process verification).

Following telecoms, advertising, and insurance, the biopharma industry has started to embrace methods like Bayesian statistics and advanced data analytics (ADA) to gain additional process and product understanding, improved process control, and process performance. Using ADA may elucidate previously undetected relationships between process inputs and outputs, which hold promise as an additional tool to augment traditional DoE as a means to gain actionable insights, process and product knowledge.

An internal multidisciplinary team (e.g., ADA) comprising data engineers, data scientists, bioprocess experts, and a translator /project manager is formed. IT infrastructure to support ADA projects is established, the standard approaches to run the case studies are developed, and a library of models for bioprocess is built. An upstream process case study to improve upstream productivity and process robustness is discussed. The team is able to extract, cleanse, ingest, and process 800 GB of data from 11 disparate data sources into a cloud environment to evaluate more than 100 hypotheses. Implementing the insights leads to improved process performance. Through the case study, the strategy to sustain the internal capability is developed. The importance of building ADA capabilities to enable more efficient and reliable bioprocesses is discussed.

As the technologies within the biopharmaceutical industry evolve so does the global chemistry, manufacturing, and control (CMC) regulatory environment with ever-present complexity of gaining regulatory approvals around the world for new products for patients. From the earliest pharmaceutical laws in the 1900s to the inception of the International Council for Harmonization (ICH) to the transfer of regulatory oversight of well-characterized biologics from CBER (Center for Biologics Evaluation and Research) to CDER (Center for Drug Evaluation and Research), there has been a continuum of regulatory growth and oversight to ensure compliance and safety of all products globally. Every country and/or region has a separate health authority (HA) that oversees commercial licensure and clinical studies for all biopharmaceuticals. History, regional laws, regional cultures, and resources are many of the factors that influence the regulatory environment around the world, and all HAs must be engaged appropriately for successful global rollouts of biopharmaceuticals.

This chapter describes monoclonal antibody (mAb) continuous bioprocess (CBP) from design, implementation to manufacturing based on the scientific understanding of mAb physicochemical properties, proven bioprocessing principles, available technologies, chemistry, manufacturing, and control (CMC) considerations, current industrial practices, regulatory guidelines, challenges, potential solutions, and future perspectives. The discussion addresses the conventional, intensified, integral, and/or fully automated end-to-end mAb CBP manufacturing process from cell line development (CLD); cell culture process development (e.g., upstream process); protein purification process development (e.g., downstream process); analytical method development, qualification, and validation for process performance; and product quality monitoring and control perspectives. The increasing interest in the application of CBP in biopharmaceutical manufacturing is associated with increased mAb market demand, demonstrated process consistency and product quality, and potential cost of goods (COGs) reduction. Some unprecedented challenges of CBP application are discussed. Some innovative technologies are assessed with practical solutions proposed. A case study is presented and discussed regarding a flowthrough mode of cation exchange chromatography for potential CBP implementation. The goal of this chapter is to propose a design and establish a fully automated CBP platform for an end-to-end mAb production from cell culture to drug substance (DS) formulation. Finally, the CBP technology is proposed for other bioprocess and manufacturing such as adeno-associated virus (AAV) vector for gene therapy.

Technology transfer of the manufacturing process and associated analytical methods is a critical process in the lifecycle of biotech products. It enables the production of clinical material to test a hypothesis developed in the drug discovery environment, expansion of clinical supplies, and establishment of a robust platform for commercial production. It can also provide flexibility in the product lifecycle by adding additional manufacturing nodes to supplement supply or provide business continuity.

In the early history of the development and manufacturing of biotech products, there was reticence to locate commercial manufacturing facilities too far from the locations where the processes were developed since the technology was viewed as complex and not easy to transfer successfully. Over time, as technology matured, and in some cases driven by a need to acquire capacity not available within the innovator, companies began to transfer processes around internal networks and to newly constructed purpose-built sites, as well as to business partners and contract manufacturing organizations (CMOs) that sprung up to meet the expanding needs of successful pipelines.

Technology transfer is a complex multi-disciplinary activity involving process and analytical development, engineering, operations, quality, and increasing automation and IT, as facilities are designed with high levels of automation and control. Factors influencing success include the level of process understanding and the understanding of the relationship between the process, equipment, and automation. Effective project management is a critical enabler that ensures successful orchestration of multiple functions and effective decision-making in a very complex and dynamic environment.

The typical pathway to a successful transfer will be through a facility fit and gap assessment between the donor and acceptor facility, design and execution of any facility and equipment modifications, raw material procurement, process trial lots, and ultimately process validation lot manufacture. All of these activities will also be dependent on the transfer and qualification of appropriate test methods.

Technology transfer, while not explicitly regulated by health authorities, is described in International Council for Harmonisation (ICH) Q10 as “the transfer of product and process knowledge between development and manufacturing and within manufacturing sites to achieve product realization. This knowledge forms the basis for the manufacturing process, control strategy, process validation approach and ongoing continual improvement.” Additionally, it is widely appreciated that the outcome of validation and comparability studies is critically reviewed as part of the regulatory approval of the process in the new location. To this end, care must be taken to ensure data integrity and continued compliance with U.S. 21 code of federal regulations (CFR) Part 11 and Eudralex Volume 4, Annex 11 across all of the steps from the donor to acceptor site. Clear definition of protocols and success criteria is critical to ensure effective transfer and decision-making, especially when projects are under time pressure.

The relationships between the donor and acceptor teams are sometimes overlooked. Generation of close, collaborative relationships with shared goals are major enablers of success. These cultural and behavioral enablers can be built into the program, and on-site presence and visits to facilities can enhance these relationships.

In the past 30 or so years, the transfer of robust processes like those used to produce monoclonal antibodies has become extremely effective and can be achieved with relatively modest investment of expert resources and can be completed in a matter of months. Mature biopharma companies have made significant advances in the development of platform processes and, with the associated maturity of expertise and capability and knowledge management within these companies, can perform effective technology transfer in a matter of months in a clinical setting and less than one year in a larger-scale commercial setting. Leveraging modeling software and other digital tools has also enabled speed and robustness. Despite these advances, it should be acknowledged that the timeline and investment may be much longer and costly for tech transfers to new facilities (e.g., biotech to external contract manufacturing organizations [CMOs] or between external CMOs) (i.e., where an effective technology transfer platform process is not in place). Interestingly, in the emerging and advanced therapies environment, the situation is more like the early days of recombinant protein production where process knowledge is still under development and the expertise is often localized within a small number of companies. Nevertheless, the same principles of technology transfer can be applied and over time will result in a broader proliferation of skills and the same flexibility that applies now for monoclonal antibody production.

The biopharmaceutical industry is driven to innovate by the discovery of new therapeutics and modalities, as well as shifts in regulatory guidelines, market forces, and intellectual property law. Most recently, gene therapy, drug conjugates, chimeric antigen receptor (CAR)-T therapies, and a variety of fusion proteins have demanded effective manufacturing antibody methods that do not conform to established platforms. In addition, patent expiration on historic blockbuster drugs has driven a gold rush for biosimilars, which will also require more efficient and inexpensive production methods in a newly competitive market. At the same time, the need for process mobility, flexibility, and miniaturization has increased, with the goal of serving new patient populations under ambitious business growth models. Thus, it is useful to examine the new technologies that are being developed to address these needs. In this chapter, we will review several emerging trends and technologies, with some attention to the philosophical and economic drivers for their development.