By Weichang Zhou, PhD
Since the first monoclonal antibody (OKT3) was approved in 1986, the FDA has granted approval to more than 100 antibody-based therapeutic products, including monoclonal antibodies (mAbs), fusion proteins, antibody-drug conjugates (ADCs), and bispecific antibodies (bsAbs).
In particular, over 70 therapeutic antibody-based products, including many blockbuster drugs such as tumor necrosis factor-α (TNF-α), PD-1, and PD-L1 antibodies, have been approved since 2014. The global sales of the biologics were expected to exceed $300 billion in 2021.1,2 Biologics in recent years have begun to dominate all drug sales. For instance, in the first nine months in 2021, 13 of the top 20 medicines were biologics, including two mRNA-based COVID-19 vaccines and 10 antibody-based products.
Biologics are highly complex molecules produced by living cells and purified through a multistep manufacturing process. The key characteristics of these molecules, known as critical quality attributes (CQAs), can vary based on post-translational modifications that occur in the cellular environment or during the subsequent manufacturing process. Hence, the process defines the product, and it is in many cases said that the “product is the process.”3
As we look back on the last few decades, the capabilities and the efforts of our industry have been tremendous in developing drug substance and product processes, along with analytical methods for manufacturing these life-saving biologics to meet their demands despite many technical and regulatory challenges.
The four main challenges (described below) to ensure product quality and robust supply in a cost-effective way will continue to drive bioprocessing innovations going forward as we seek to develop novel biologics and manufacture them with consistent quality even faster, especially to combat pandemic outbreaks.
Accelerating timelines from ideas to clinical approvals and beyond
Since COVID-19 emerged, our industry has been demonstrating its ability to act collaboratively and quickly. It has expedited the development and manufacturing of multiple vaccines (including two mRNA-based vaccines) and biologics (including several mAbs).
To accelerate development timelines—from DNA sequence to IND status and, ultimately, approval—for multiple neutralizing mAbs against COVID-19, our industry has applied integrated and transformative methodologies without compromising product quality and safety. The timeline from DNA to IND was shortened to 3–6 months, and neutralizing mAbs were developed from DNA to EUA within 14 months. Thousands of kilograms were manufactured within months for therapeutic applications across the globe.4
This unprecedented speed recently achieved for the pandemic-related therapeutics may start to revolutionize biologics development and be a major feature in the future of bioprocessing. In addition to continuing to apply innovative technologies to shorten the development timelines, future bioprocessing development will focus on further reducing manufacturing costs. New and smaller “facilities of the future” that utilize integrated continuous processes, single-use systems and sophisticated digitalization, automation, and artificial intelligence tools will offer greater flexibility, higher productivity, more efficient use of space, and reduced cost of goods.
Bioprocessing improvements prompted by the COVID-19 response may facilitate the development of other more complex biologics, including ADCs and bispecific or multispecific antibodies. More direct applications of the knowledge and experience gained from the rapid development of COVID-19 vaccines and biologics include the development of new vaccines and therapeutic biologics for combating life-threating diseases besides COVID-19. No longer will the development of new vaccines and biologics take 10 or so years.
Since the advent of therapeutic biologics, strategies for production cell line selection have been continuously improved for greater productivity and efficiency. These recent advances enable us to reshape the chemistry, manufacturing, and control (CMC) strategy so that we can supply clinical materials in as little as three months.
Codon optimization before DNA synthesis is a common practice to get high protein expression levels. Its algorithm, derived from dedicated codon and codon-pair usage of a specific host cell line, may be more effective than algorithms derived from a generalized species-based codon usage database to increase protein expression in a given host cell line. In addition, custom codon optimization effort is useful throughout development as the strategy is effective for both transient and stable cell line expression systems.
As CMC development timelines have been accelerated over the years, the use of pool-derived materials for toxicology studies has been winning increased acceptance by global regulatory agencies. It is crucial, therefore, to select a final clone with product CQAs similar to the material used for to the toxicology studies. Importantly, next-generation sequencing (NGS)-based cDNA is applied instead of peptide mapping by liquid chromatography-mass spectrometry (LC-MS) to quickly screen for clones without any sequence variants to those pool material cells used in early development. Concurrent cell line stability passage can be done during clone screening to further reduce timelines.
Truncation or cleavage of recombinant proteins is often observed when developing these complex molecules. This poses challenges in downstream purification and results in lower yield. Batch refeeding is usually applied in the early stages of cell line selection to screen for clones with less clipping, once again demonstrating how advancements in understanding the product and process early in development can help save significant time, costs, and effort from occurring in the later development stages.
Greater adoption of continuous bioprocessing and single-use systems
Continuous bioprocessing will increasingly be applied to the manufacture of various biologics modalities, including mAbs and bsAbs, fusion proteins, and recombinant proteins. The establishment of continuous processing represents an ongoing shift in the industry to address the growing demand for biologics of high quality, high volume, and affordability.
For upstream processing, perfusion culture has been widely adopted for clinical and commercial production. Perfusion culture has significant advantages over fed-batch culture regarding yield, quality, flexibility, and cost-effectiveness. A well-developed perfusion culture system—such as the WuXi Biologics Ultra-High Productivity Platform, or WuXiUP™—could enable 5–10-fold improvement on the cell density and productivity of almost any type of biologic, compared to a fed-batch culture. Additionally, utilizing continuous product harvest reduces the residence time of the product within the bioreactor, leading to improved product quality.
Integrated continuous downstream processing at lab or pilot scales has been achieved by advances in single-use systems, flow-through chromatography, and single-pass tangential flow filtration. Yet, industry-wide investment at large scale is lagging behind.
Improvement in downstream productivity within larger-scale manufacturing settings is needed to keep up with the dramatic improvements made in expression titers from upstream processes using automated steady-state perfusion culture coupled with continuous cell-free harvest, and an elongated harvesting period.5
The International Conference on Harmonization (ICH) recently issued a draft of its Q13 guideline for the continuous manufacturing of drug substances and drug products.6 The guideline, for which comments are currently being reviewed, is widely anticipated to invigorate interest and efforts in the development and production of biologics using continuous processing approaches, which would heavily rely on either partially integrated or fully connected continuous downstream unit operations.
A foreseeable bottleneck for both continuous downstream options is the lack of cost-effective off-the-shelf or custom-fit process analytical technology (PAT) tools and automation systems for process monitoring and real-time control. Through the collaborative efforts of instrument and automated control solution providers, academics, engineers, and the biopharmaceutical industry, this bottleneck may be mitigated in the near future for much wider application of fully continuous downstream processing.
As cell culture titers continue to increase with advanced cell line development strategies and optimized cell culture media, the demand for bioreactors with a smaller footprint will increase. This, in combination with continuous processing, will further promote the adoption of single-use systems.
A completely disposable biomanufacturing process may be achieved by expanding the use of single-use technologies in other unit operations, such as introducing disposable tangential flow depth filtration systems and single-use ion exchange membrane chromatography devices. Disposable technologies also enable new facility designs such as modular manufacturing units that can provide flexible capacity, reduce time-to-market, and ease changeover between products while also minimizing cross-contamination.
There is a strong trend toward intensified continuous systems that promise to enhance productivity achieved by these approaches. Fully continuous processes may be achieved through process integration and harmonization of different unit operations to minimize holding times and maximize productivity. Through continuous bioprocessing, large quantities of products may be manufactured with smaller equipment footprints and reduced overall capital investment.
Other improvements include implementation of auto-feeding, auto-sampling, and minimal tubing assembly in large-scale manufacturing processes that can enable greater process control, resulting in more robust performance and product quality. PAT tools, such as Raman spectroscopy, as well as other inline testing methods will be implemented into large-scale manufacturing to enable real-time testing and control of important process parameters and performance.
Other new analytical technologies have impacted biomanufacturing, such as the peptide-based multi-attribute method (MAM) with enhanced sensitivity and product selectivity compared to capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) methods. Additionally, emerging technologies, such as surface plasmon resonance (SPR) and biolayer interferometry (BLI), could enhance the workflow in product release.
The utilization of PAT tools offers two main advantages: fast decision making and advanced process control. PAT platforms have been developed for on-line monitoring of product aggregation and fragmentation in continuous processing,7 as well as for control of viable cell density,8 which helps to develop a better process with accelerated CMC timeline.
Applications of digitalization and artificial intelligence
The manufacturing “facilities of the future” will be more digitalized. Artificial intelligence (AI) applications and innovative algorithms will provide better process control and ensure process and product consistency from batch to batch.
The use of AI to enable, expedite, and innovate the R&D of biologics has been intensifying. For example, AI prediction models can be used to further optimize the manufacturing process and increase efficiency. With the advent of protein structure prediction AI models, such as AlphaFold2, and the use of AI in automation via the Internet of Things (IOT), opportunities for the use of AI in drug discovery, bioprocessing, and manufacturing have begun to take definitive shape.
In the early stages of drug discovery, AI-aided research is advancing from protein structure prediction to the determination of drug developability attributes such as protein aggregation, binding affinity, solubility, stability, and in vivo and in situ efficacy, among other key criteria for determining drug candidacy and drug design optimization.
With an optimized drug candidate, AI models can provide in silico bioprocess development predictions and guidance to further optimize development of biologics on criteria such as production yield and product quality. By combining AI’s experimental simulation and guidance with experimental procedure automation through IOT-connecting lab equipment and operating machineries, experiments can be performed with minimal human involvement, avoiding human error and expediting the overall drug development process. AI combined with IOT (AIOT) can further improve the process consistency and accelerate development.
Bioprocessing trends for next-generation antibody-based modalities
Bioprocessing for new product modalities such as bsAbs and ADCs can be challenging due to the complexities of these molecules. With rapid growth of ADC pipelines worldwide, there is strong demand to access fully integrated ADC platforms that can support process development and manufacturing from DNA to IND. These fully integrated ADC platforms include not only the development and manufacturing of the bioconjugate product but also mAb, linker, and conjugation technologies. Besides reducing DNA-to-IND timelines, these platforms can reduce risks. These platforms also reflect a greater focus on conjugation process robustness and the control of drug and antibody ratios (DARs).
Conjugation technologies encompass various linker mechanisms as well as different payloads, conjugation chemistries, conjugation sites, and DARs. All this diversity adds variability to manufacturing, complicating processes for purification and the characterization of ADC structure and potency. In the last few years, ADC developers have made significant strides to improve the developability, manufacturability, and functionality of these complex drugs, but challenges still remain to move these modalities into a more streamlined development process. To cope with these difficulties, biomanufacturers need more sophisticated analytics.
In the manufacture of bsAbs, associated byproducts resulted from processes such as chain mispairing, unbalanced chain expression, and incomplete assembly pose considerable challenges to downstream purification. To help manufacturers cope with variable byproduct species and quantities, researchers have proposed a toolbox-based approach for bsAb purification.9
Besides downstream approaches tailored for bsAbs, perfusion cell culture can significantly improve bsAb quality (for example, in terms of increased percentage of monomer, as determined by Caliper or capillary isoelectric focusing assays). Therefore, perfusion cell culture can be a better choice than conventional fed-batch culture.10 The downstream and upstream strategies discussed here for the development of bsAbs may be applied to the development of multispecific antibodies as well.
Although issues in biologics development still remain, especially for new and complex drug modalities such as ADCs and bsAbs, the industry has risen to meet these challenges with innovative AI approaches, new analytical tools, and high-throughput matrix-driven experimental designs. Utilizing more comprehensive, integrated, and single-source drug development and manufacturing platforms, along with the knowledge gained from developing multiple biologics in the fight against the pandemic, has provided biologics developers the opportunity to produce biotherapeutics and vaccines faster and more cost-effectively than thought possible just a couple of years ago.
Weichang Zhou, PhD, is executive vice president and chief technology officer of WuXi Biologics.
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