You might believe that bioprocess development is simple, perhaps even lacking in innovation, a perception shared by many scientists in basic research. However, this view is misguided. 

While it's true that bioprocess development may not be "rocket science" and some advancements may not be groundbreaking, it's undeniably a complex scientific discipline. It's a form of process science that intricately integrates elements from diverse fields such as biology, microbiology, biochemistry, chemistry, chemical engineering, and even modeling. 

To excel in bioprocess development requires both creative thinking and logical thinking, making it anything but simple. This concept is discussed in greater detail with Wei Zhang in an episode of the Smart Biotech Scientist Podcast, hosted by David Brühlmann, founder of Brühlmann Consulting.

A Journey into Biotherapeutics: From Small Molecules to Antibodies

Wei Zhang's journey into biotechnology began with a childhood keenness for biopharmaceutical-related fields. This passion led him to pursue both his bachelor's and master's degrees in medicinal chemistry, with a primary focus on creating small-molecule drugs through organic chemical synthesis.

During a pivotal period of growth in biotechnology, Wei decided to pivot her acquired skills and knowledge towards biotech-related endeavors for her PhD studies and first job. She delved into biotransformation, a domain that still involved the production of small-molecule drugs but through utilizing biological tools, such as enzymes and biocatalysis.

It was during this time that Wei began to recognize the emergence and significance of antibodies and other biotherapeutics as the next generation of pharmaceuticals. This realization prompted her decisive move after her first job to join the biotech industry. 

Here, she continues to contribute to pharmaceutical advancements, but with a distinct focus on the production of antibodies and biotherapeutics. This comprehensive journey has provided Wei Zhang with a thorough understanding of the bioprocessing industry.

Core Challenges in Bioprocess Development and Biologics Manufacturing

The bioprocessing industry faces several key challenges in bioprocess development and biologics manufacturing.

The Hurdle of Process Scaling Up

The first significant challenge is scaling up the process. Transitioning a process from laboratory scale to pilot scale and eventually to GMP (Good Manufacturing Practice) production scale is a profoundly challenging task. It is essential to maintain both process performance and product quality consistency throughout the entire scaling-up process, which is far from easy.

Ensuring Product Stability

Another critical challenge is maintaining product stability throughout the manufacturing process and during the final product storage. Many biologic products are inherently unstable; they can aggregate or degrade under unfavorable conditions. Therefore, proper process development and formulation development are crucial to ensure the drug's quality and efficacy.

The Imperative of Interdisciplinary Learning

A constant challenge for biotech scientists is the necessity to learn from colleagues outside their core areas. Broadening perspectives and deepening knowledge and technical skill sets are vital. 

For example, as a downstream scientist working in purification, one must understand upstream culture and QC (Quality Control) analytics to perform downstream processing effectively. Looking beyond one's immediate sector is key to understanding others' work and challenges.

Chromatography and filtration are complementary techniques used in bioprocessing, but both of them serve as essential tools for purification in biotechnology field.

Purification Techniques: Mainstays and Evolving Complexities

In purification, traditional methods such as precipitation, extraction, and crystallization are employed, but chromatography and filtration remain the most common techniques in bioprocessing.

Chromatography and Filtration: Complementary Approaches

Based on their modes of separation mechanisms:

• Chromatography can be classified into affinity, ion exchange, and hydrophobic interaction. It separates components based on their differential affinity for the stationary and mobile phases. Chromatography is highly useful for purification due to its ability to provide high resolution and high selectivity. This makes it valuable for achieving high separation efficiency and purifying closely related molecules, such as product-related impurities.
• Filtration, on the other hand, can be classified into dead-end filtration and cross-flow filtration. It separates components based on size difference. Filtration is typically applied to remove particles, such as cells or cell debris, during cell harvest, or the preventive removal of potential contaminants, like microorganisms or viruses.

Regardless of the drug modality (recombinant proteins, viruses, or other forms), chromatography and filtration remain the mainstay for purification process development. They are complementary techniques, both of which are essential tools in the biotechnology field.

Challenges in Purification Process Development

Developing purification processes today faces specific challenges, primarily driven by the increasing complexity of drug modalities.

• Heterogeneity of Complex Molecules: Complex molecules often exhibit heterogeneity in size, charge, or structure. This requires highly selective and specific purification techniques to separate the target from product-related impurities, which is crucial for new emerging drug modalities, such as viruses, RNAs, and bispecific antibodies.
• Analytical Tools for Process Monitoring: Another challenge in complex target purification is the need for precise analytical tools to monitor the process. While traditional methods, such as HPLC or micro CE, are often sufficient for protein purification development, viral vectors and RNA molecules require consideration of additional Critical Quality Attributes (CQAs), including the field MT particle ratio for viral vectors or the double-stranded RNA content for mRNA. These parameters currently require time-consuming analytics, which can slow down the development of purification processes for these new drug modalities.

Advancements in Purification Methods

Despite the perception that advancements in bioprocessing have been slow over many decades, with chromatography and filtration remaining mainstays, significant developments have occurred.

Affinity Ligand Development

Recent advancements include a broader spectrum of affinity purification tools resulting from developments in affinity ligand technology. These affinity tools provide higher sensitivity and selectivity for separating target molecules. 

For example:

• For the purification of bispecific antibodies, multiple affinity resins are available, each designed to bind to specific domains of antibody structures.
• For viral vectors, affinity resins are available to purify different serotypes of AAV and lentivirus.
• In the case of mRNA, several oligo(dT) affinity products are now available, designed explicitly for mRNA purification.

These advancements in the bioprocessing field simplify and facilitate the purification of new drug modalities. They offer very efficient separation, especially during the capture step, where most impurities can be removed. 

This enables the shortening of subsequent polishing steps, thereby accelerating the timeline for process development of new drug modalities. This ability to reduce additional purification steps is a significant game-changer, leading to substantial cost savings, reduced labor, and decreased complexity, especially when scaling up.

The Need for Dedicated Tools

However, there is a current lack of new techniques or purification tools dedicated explicitly to emerging modalities. Purification tools for new modalities still rely on older techniques developed for protein purification. The advancement of various therapeutics is swift, but there is a lag in bioprocessing tool development to catch up. Scientists in the bioprocessing field need to put more effort into developing new tools for bioprocessing new drug modalities.

Unique Considerations for Modality-Specific Purification

Let's look at specific modalities more closely.

Purifying Viral Particles

Virus purification presents unique challenges compared to the purification of proteins or antibodies.

• Stability: Viruses, particularly enveloped viruses, can be sensitive to physical and chemical stressors, such as high salt concentrations or low pH levels. Therefore, gentle purification methods are often required to maintain the integrity or infectivity of the viral particles after purification.
• Removal of Host Cell Impurities: It's relatively more challenging to remove host cell impurities, such as whole-cell proteins and nucleic acids, during viral purification. This is because most viruses, nucleic acids, and most host cell proteins have a negatively charged surface. Thus, the purification process must be carefully designed to ensure the efficient removal of these impurities.
• Viral Clearance: Achieving virus removal for viral clearance in virus purification is challenging. Viral removal is typically achieved through size exclusion methods, such as nanofiltration. However, in viral purification, the size of the target virus can be similar to the model viruses used in viral clearance. In such scenarios, size cut-off-based methods, such as nanofiltration techniques, no longer work, necessitating the development of alternative techniques for virus removal.

Historically, viruses have been purified using precipitation, ultracentrifugation, or density gradient centrifugation. However, these techniques have limitations, particularly concerning scaling up. Nowadays, there's a greater reliance on more scalable methods, such as column-based, filtration-based, or enzymatic digestion-based methods. These approaches are practical for removing impurities and separating viruses from other host cell impurities.

Key Techniques and Innovations in RNA Purification

Techniques and innovations in RNA purification evolve with the advancement of RNA therapeutics. Before the COVID-19 pandemic, RNA-based therapeutics typically referred to short-stranded RNAs, such as ASOs, siRNAs, or microRNAs, which could be produced by chemical synthesis followed by simple purification. Today, RNA therapeutics primarily involve long-stranded mRNA (messenger RNAs), famously used for COVID-19 vaccines.

One key technological innovation in RNA purification is the accessibility of RNA affinity ligands. These ligands accelerate RNA purification from the post-IVT (in vitro transcription) process because they selectively bind to poly(A) tails on mRNA structures, resulting in high-purity and high-yield isolated RNA molecules.

However, challenges remain in the downstream purification of mRNA, such as how to remove double-stranded RNA and how to produce and purify circular RNA. Researchers and industry professionals are actively developing new tools and workflows for the purification of RNA, particularly circular RNA.

Advancements in Traditional Vaccine Downstream Processing

For traditional vaccines, such as inactivated virus vaccine purification or attenuated virus purification, there's a shift from methods that heavily rely on precipitation (like PEG precipitation) and ultracentrifugation (such as density gradient centrifugation or sucrose/cesium chloride centrifugation). These older methods are not scalable for volumes of 100 liters or higher.

Now, there's a reliance on more scalable methods, like column-based or filtration-based methods, to scale up the process. Column-based methods also offer higher separation efficiency, which contributes to improved drug safety and accuracy.

Strategies for Seamless Scale-Up in Downstream Process Development

For those working in R&D, developing a downstream process with an eye towards seamless scale-up requires key considerations.

Cultivating a Scale-Up Mindset

It's essential to cultivate a scale-up mindset during laboratory-scale process development. This means ensuring that all unit operations chosen for the process and all raw materials utilized have an equivalent GMP-grade version that can be used in eventual GMP production.

Collaboration Across Disciplines

Another crucial aspect is cooperation with scientists, engineers, and production technicians. Engaging in top-to-bottom and collaborative efforts with these groups is vital for smoothly developing and transitioning the process from lab scale to pilot scale and eventually to production scale.

Balancing Cost and Quality in Material Selection

When selecting types of resins or equipment, a critical pitfall to avoid is neglecting the balance between cost and quality. For process development aimed at final commercialization production, this balance is paramount. One should not be solely cost-driven or solely quality-driven. Careful analysis and evaluation at the lab scale are necessary to select cost-effective raw materials and processes without compromising product quality.

I think it's essential to cooperate with the scientists, with engineers, or even production technicians in the process development, need to talk to them and also coordinate with them in order to smoothly develop and transit the process from lab scale to pallet scale to eventually the production scale.

The Future Trajectory of Downstream Processing

Five to ten years into the future, several trends are expected to shape downstream processing.

Continuous Bioprocessing

Continuous chromatography and other continuous bioprocessing techniques are expected to gain wider acceptance and adoption. This is a current hot topic in downstream processing.

Mixed-Mode Chromatography

Mixed-mode chromatography will likely see increased application. While not widely used in monoclonal antibody purification, it's expected to play a much bigger role in the purification of new drug modalities. Mixed-mode chromatography combines multiple modes of interaction on a single resin, offering unique selectivity that enables more efficient purification processes and potentially further reduces the number of purification steps.

Dedicated Purification Tools for New Modalities

More advancements in purification tools will be dedicatedly developed for vaccines, gene therapies, or RNA-based therapeutics. Currently, tools for RNA purification still rely on older techniques designed for protein purification, highlighting a critical area for future development.

Integration of AI and Machine Learning

Artificial Intelligence (AI) and modeling are expected to have a significant impact on downstream processing. Currently, process development still relies on traditional tools or numerous experimental runs to identify optimal purification conditions. With the assistance of AI and modeling, the timelines for developing the purification process can be shortened. These technologies can provide highly accurate predictions of "sweet spots" in process conditions, optimizing purification for specific drugs or molecules. This will significantly accelerate timelines and enhance prediction for precise demands.

Practical Steps for Aspiring Downstream Scientists

For anyone about to embark on downstream development, the first steps involve broad learning and collaboration. It's essential to acquire knowledge and skills from diverse disciplines, including chemistry, chemical engineering, and biology. The most effective way to learn quickly is to consult with industry professionals, friends, and colleagues in the field to acquire this basic knowledge as soon as possible. This lays a strong foundation for becoming an experienced scientist in the field.

Final Remarks

Ultimately, it's essential to reiterate that bioprocess development is a form of process science. It's a relatively easy field to enter, but it demands years of effort and a wealth of knowledge to excel in truly. It's not easy to do well in this complex and evolving discipline.

About Wei Zhang

Wei Zhang is the DSP Group Head at the A*STAR Bioprocessing Technology Institute in Singapore. In this role, he leads a team of over 10 researchers dedicated to downstream process and product analytics development across a diverse range of modalities. 

These include monoclonal antibodies (mAb), bispecific antibodies (bsAb), mRNA, viruses, adeno-associated viruses (AAV), virus-like particles (VLP), vaccines, and enzymes.

Connect with Wei Zhang on LinkedIn.

David Brühlmann is a strategic advisor who helps C-level biotech leaders reduce development and manufacturing costs to make life-saving therapies accessible to more patients worldwide.

He is also a biotech technology innovation coach, technology transfer leader, and host of the Smart Biotech Scientist podcast—the go-to podcast for biotech scientists who want to master biopharma CMC development and biomanufacturing.  

Hear It From The Horse’s Mouth

Want to listen to the full interview? Go to Smart Biotech Scientist Podcast. 

Want to hear more? Do visit the podcast page and check out other episodes. 
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