Inside the Laboratory: The Zydney Group at Pennsylvania State University

"Inside the Laboratory" is a joint series with LCGC and Spectroscopy, profiling analytical scientists and their research groups at universities all over the world. This series spotlights the current chromatographic and spectroscopic research their groups are conducting, and the importance of their research in analytical chemistry and specific industries. In this edition of “Inside the Laboratory,” Andrew Zydney of Pennsylvania State University discusses his laboratory’s work with using high performance liquid chromatography (HPLC) to analyze biopharmaceutical products before and after membrane separation processes.

Membranes are essential for the downstream purification of biotherapeutics like monoclonal antibodies. They are used for clarification, virus removal, concentration, and sterile filtration, enabling the production of life-saving products (1). In 2016, the global market for membrane filtration in biopharmaceuticals was valued at $3.5 billion (1). Recent developments, such as economic pressures from low-cost manufacturers, the rise of biosimilars, and the shift toward continuous bioprocessing, have heightened interest in membrane technologies, positioning them as key innovations in addressing challenges in biotherapeutic production and enhancing efficiency in the bio-pharmaceutical sector (1).

Andrew Zydney of The Pennsylvania State University is conducting research in this space. He is the Bayard D. Kunkle Chair and Professor of Chemical Engineering at The Pennsylvania State University. He received his BS in Chemical Engineering at Yale University in 1980, and his Ph.D in Chemical Engineering at the Massachusetts Institute of Technology in 1985 (2).

The campus of Penn State University in summer sunny day, State College, Pennsylvania. | Image Credit: © lucky-photo – stock.adobe.com

Zydney’s research areas of interest include studying membrane separations systems for bioprocessing and medical devices. Some of the work his laboratory conducts involves applying membrane technology to purify high-value biological products, such as gene therapy agents and recombinant proteins (2).

In this edition of “Inside the Laboratory,” Zydney sat down with LCGC International to talk about his group’s current research endeavors.

Dr. Andrew Zydney of The Pennsylvania State University. | Photo Credit: Andrew Zydney

Can you describe your laboratory for our audience? How many researchers do you supervise and what is the focus of your work?

I currently have 13 PhD students, one post-doc, and eight undergraduates working in my laboratory. My research group focuses on the development and application of membrane technology for the purification of monoclonal antibodies, vaccines, and gene therapies.This includes projects examining the initial clarification of cell culture fluid using depth filtration, virus removal filtration, sterile filtration to ensure sterility of the biotherapeutic, and the use of ultrafiltration membranes for concentration and buffer exchange in the final formulation of biological products.

Can you talk about some of your laboratory group’s recent work using high-performance liquid chromatography (HPLC)?

Although our group is focused on membrane technology, HPLC is critical for the analysis of the purity and composition of the biopharmaceutical product both before and after the membrane separation processes that we are studying. For example, we use HPLC to evaluate the distribution of host cell proteins present in monoclonal antibody (mAb) feed streams, to quantify the amount of residual nucleotides in the transcribed messenger RNA (mRNA) generated by in vitro transcription, and to evaluate the purity of adeno-associated virus that are of interest as gene therapies.

How have membrane technologies revolutionized the downstream purification of biotherapeutics like monoclonal antibodies?

Membranes are a critical component of the downstream process for monoclonal antibody purification. Membranes are used in antibody processes to remove cells and cell debris, to remove viruses and bacteria that might have inadvertently contaminated the product, and to formulate the antibody at the appropriate concentration and in the desired buffer so that the drug can be safely stored and delivered to the patient. In addition to studying all of these processes, my laboratory is also exploring the use of membranes for antibody purification using high performance countercurrent membrane purification and by selectively precipitating the antibody and then washing away the impurities using membrane filtration.

Can you elaborate on how membranes contribute to critical steps such as virus removal, product concentration, and sterile filtration?

Because antibodies are produced in mammalian cells (most commonly Chinese hamster ovary cells), it is absolutely essential that the downstream process include multiple steps that are designed to remove or inactivate viruses that might be present in the mammalian cell culture. Virus filtration has become a standard method for virus removal since it provides a highly robust size-based technology for removing both enveloped and non-enveloped viruses. The smallest viruses are approximately 20 nm in size, whereas antibodies are 10–12 nm in size. This leads to a highly challenging separation, but membrane companies have developed a range of products that can provide high levels of virus removal (>99.9%) while recovering >95% of the antibody.

Membrane ultrafiltration is the method of choice for the final concentration and formulation of nearly all biopharmaceutical products. In this case, the membranes are designed to have pores that provide nearly complete retention of the product, with water, salts, and small buffer components removed in the filtrate during the ultrafiltration process.

Because most biopharmaceuticals are damaged at high temperatures, it is not possible to sterilize these products using high temperatures. Instead, they are all “sterile filtered” by passing the products through membranes that have pores that retain even the smallest bacteria while allowing the biopharmaceutical to pass through the membrane and directly into a vial for distribution to the patient.

With the global membrane filtration market in the biopharmaceutical industry valued at $3.5 billion in 2016, how has the market evolved in response to rising economic pressures and the growth of biosimilars?

In general, membrane technology is less expensive than chromatography and more easily scaled up to large-scale manufacturing. It is also easier to implement membrane systems as part of a continuous downstream process. Thus, the rising economic pressures and the development of biosimilars has created exciting opportunities for the development of new membrane processes that can provide lower cost purification in commercial biomanufacturing.

What role do low-cost international manufacturers and biosimilars play in driving innovation in membrane technologies?

Although biosimilars have played a role in spurring the development of lower cost downstream processes using membrane systems, the most significant driver of innovation in membrane technology is probably the development of new classes of biopharmaceutical products. This includes mRNA, lipid nanoparticle delivery systems, and adeno-associated virus (AAV) for gene therapies (among others). Many of these new biotherapeutics require a complete rethinking of the downstream process, including how to best employ membrane technologies for the purification of these products. In some cases, this will likely require the development of new membranes and membrane modules that are specifically targeted to the purification of these novel biopharmaceuticals.

What recent advancements in membrane technology hold the most promise for improving efficiency in biotherapeutic production?

One of the exciting areas of development in membrane technology is single pass tangential flow filtration (SPTFF). In contrast to traditional batch membrane processes, in which the product needs to be recirculated through the membrane module multiple times to achieve the desired degree of purification, SPTFF enables concentration of the product in a single pass through the membrane device. This technology is ideal for the development of intensified andcontinuous downstream processes, which has become a major focus throughout the biopharmaceutical industry. This approach can also have unique benefits for the concentration of shear sensitive products like RNA, DNA, and many viral vectors.

Are there specific innovations or research areas that you think will define the next generation of membrane separations in bioprocessing?

This is a difficult question to answer. I think the next generation of membrane separations in bioprocessing will be specifically designed to address key challenges in purifying new classes of biopharmaceuticals. For example, sterile filtration is relatively straightforward for a monoclonal antibody since an antibody is about 10 nm in size while the smallest bacteria are around 300 nm in size. The situation is entirely different when trying to perform sterile filtration on a lipid nanoparticle containing an mRNA vaccine or cancer therapeutic.In this case, the mRNA-lipid nanoparticle complex is around 150 nm in size, often with a fairly broad size distribution. There is a critical need for the development of new sterile filtration membranes that are specifically targeted to this application. Such membranes could significantly reduce the cost and increase the performance of this critical step in the downstream process.

Are there any emerging trends that you are observing in biopharma that you would like to highlight for our readers?

This is an incredibly exciting time in biopharma as the industry looks to exploit recent scientific advances to develop novel gene therapies. This includes the development of mRNA vaccines (similar to those that provide protection against Covid-19), the development of adeno-associated viral vectors for delivery of gene therapies, and the development of CRISPR-based therapies that can performing gene editing in the treatment of genetic disorders. There is a critical need to develop lower-cost and higher performance processes for the manufacture of these novel therapeutics so that they can become widely accessible to patients around the world.

References

1. Zydney Research Group, Research. PSU.edu. Available at: https://sites.psu.edu/zydney/research/ (accessed 2024-11-18).
2. Penn State College of Engineering, Andrew Zydney. PSU.edu. Available at: https://www.che.psu.edu/department/directory-detail-g.aspx?q=alz3/ (accessed 2024-11-18).