Dr. Sunny Zhou, Professor, Chemistry & Chemical Biology at Northeastern University recently shared insights on his upcoming keynote presentation "Systematic and Deep Characterization of Host Cell Proteins (HCPs)", to be delivered at the 4th Annual Detection, Characterization and Control of Impurities in Biologics conference at the Bioprocessing Summit in Boston.

KEYNOTE PRESENTATION: Systematic and Deep Characterization of Host Cell Proteins (HCPs)
Sunny Zhou, PhD, Professor, Chemistry & Chemical Biology, Northeastern University

Host Cell Proteins (HCPs) in biotherapeutics have the potential to affect product quality and immunogenicity in patients. In this talk, we will discuss the regulatory requirements for HCPs, profiling parameters, and characterization tools and methodologies.

Q.  Please share a little bit about yourself and what inspired you to work in this field?
In the past 30 years, my research has been centered on protein analysis, chemistry, engineering, and enzymes. In addition to basic research, I have been actively collaborating with biopharma companies and regulatory agencies to characterize and develop protein pharmaceuticals. One program is “Hybrid Modality Engineering of Proteins”—a platform to introduce non-canonical chemical moieties and/or scaffolds into peptides and proteins to confer novel functions otherwise unavailable via recombinant technology.

Host cell proteins (HCPs) are particularly interesting to me because they encompass all my research areas. Moreover, I am very interested in the discovery and elucidation of unknowns, such as novel post-translational modifications (PTMs), and unknown substrates and functions of enzymes (i.e., functional proteomics). As you will see, unknowns abound for HCPs.

Q. What are HCPs and why are they important?
Host cell proteins (HCPs) are proteins produced by the host cells, and particularly those carried over to the final purified drug substances. It has been well documented that residual HCPs can cause various problems, such as proteolysis of drug substances and degradation of excipients; and in a few cases, detrimental clinical outcomes, such as immunogenicity.

Despite of their importance, HCPs remain poorly characterized and somewhat misunderstood. The practical implication of this lack of knowledge is that the process to remove them remains largely empirical and lacks guiding principles. Several factors contribute to the impediments.

First, the analytical challenges. The total HCP levels are typically under 10 ppm (parts per million). Moreover, an individual HCP is likely to be under 1 ppm. Of note, some HCPs, particularly enzymes, can have observable activities even at this ultra-low level. As such, currently there are only limited characterizations of HCPs. 

Second, they are process dependent. The final HCPs level, composition, and activity can be affected by every step, both upstream (e.g., cell lines and growth conditions) and downstream (e.g., purification and formulation). This has a huge implication because there will be a lot of variability, even in the same product at one company. The confidential nature of processes also makes it difficult to compare and generalize across the industry. 

Third, they are dynamic. It is well known that many enzymes are produced in their zymogen forms, which are inactive or less active. The conversion from zymogen to the active enzyme takes time and may also require external factors such as metal ions that may slowly leach from the containers, such as glass. 

Lastly, the conceptual framework. For instance, the existence of proteoforms of drug substances (e.g., charge variants) and biological proteins are well recognized. On the other hand, a given HCP is typically treated as a static and single molecular entity. The good news is that we, and others, have already started to introduce new approaches and concepts as highlighted below.

Q.  How is your lab tackling them?
First and foremost, my laboratory is addressing the challenge of ultra-low abundance. We, and others, have been developing various enrichment schemes. One approach that my laboratory is exploring is activity-based protein profiling, particularly enzymes that can be selectively and covalently tagged. However, this type of approach still has some limitations, which I will elaborate on in my talk. I will also discuss how to optimize all the steps involved, such as higher hit rates during mass spectrometric analysis. 
An exciting new development comes out from my collaboration with Mike Dolan, Sheldon Oppenheim, and colleagues at Takeda. We have developed a general affinity capture workflow.  In my talk, Mike and I will discuss our new site-specific way to immobilize anti-host cell (HCP) antibodies. Our workflow should work well for both targeted analysis (one or a few selected HCPs) or global unbiased analysis. 

Q. How did COVID affect your work?
As everyone else, our research has been slowed down. My master and undergraduate students had no access to the laboratory for bench work for a long time. However, they made up for this by writing a nice review on site-specific conjugation of native antibodies. Luckily, they all were able to find excellent positions in the biopharma companies or attend to the graduate programs they liked. On the other hand, while I was teaching, I could see the huge mental toll the pandemic had on my students and their families.

Q. What have you learned during the pandemic?
The biggest lesson I have learned is that things may go will most likely go wrong. Second, forewarned is forearmed. Since the very beginning, I had been closely monitoring the situation in China. On January 27th 2020, I gave a quiz on COVID (before the term was officially coined) to my class. After that, we had weekly discussion and update, and were the first ones who went to online teaching. I am happy to report that, because our early preparation, all my students and their families were safe and healthy. Back to HCPs, we should be proactive about them just as for COVID. 

Q. I am getting ready for my first hybrid event...what are you looking forward to as in person conferences return?
What I miss a lot from in-person meetings is having “by the way” moments. When talking in person, conversations naturally go off topic and we have stimulated thoughts, often leading to new ideas and deeper insights. Naturally, I really enjoy getting to know my fellow scientists as human beings, past their profession.

Q. Why did you choose to speak at The Bioprocessing Summit?
I have attended this conference and others organized by the Cambridge Healthtech Institute (CHI) several times over the past decade. I always find the organizers of this summit to be proactive and collaborative with both the speakers and the attendees. Perhaps not everybody is aware of this, but the organizer for this meeting is always actively communicating with the speakers and advisers before the meeting to solicit topics, and after the meetings, seeking feedback on how to improve and accommodate everybody and the industry.
Always well organized, the summit also has the advantage of being relatively small, so there is ample time and opportunities for productive and pleasant exchanges among the speakers and attendees. Moreover, the summit is a really great place to meet new people with common interests and different expertise. Last but not least, Boston is a great place to visit–especially in the summer.

Q. What can the audience expect from your talk(s)?
It is a privilege to have 60 minutes for my talk, so I will start with an overview of the field and new trends, such as various enrichment approaches. Furthermore, I will share some of my thoughts on the broader topic, and cover some concepts and issues that may have been overlooked, such as the limitations (e.g., false negatives or low hit rates) for conventional mass spectrometry work flow.  \

Q. Do you have any message for your peers or any closing remarks?
The study of host cell proteins is a very exciting field. It is important, complex, complicated, and highly variable, thus offering ample opportunities for creative approaches. Some new concepts, approaches, discoveries, and technology developments have emerged in the past years, and many more will be forthcoming. For example, new modalities, such as adeno-associated virus (AAV), will present their own unique issues. Finally, the investigation of HCPs is perfect for collaboration across the whole industry. Certainly, I hope my talk and this conference will catalyze the formation of a focus group or consortium. Please reach out to me if you are interested in joining the efforts. 

SPEAKER BIOGRAPHY:

Sunny Zhou, PhD, Professor, Chemistry & Chemical Biology, Northeastern University
Professor Zhou’s laboratory applies protein chemistry, analysis and engineering to biology and medicine. One program is “Hybrid Modality Engineering of Proteins”—a platform to introduce non-canonical chemical moieties and/or scaffolds into peptides and proteins to confer novel functions otherwise unavailable, such as photomedicine. The second is to devise chemo-enzymatic methodologies to characterize protein modifications, such as crosslinking, isoaspartic acid formation (asparagine deamidation) and methylations. In collaboration with biologists and clinicians alike, we also investigate their biological effects, and moreover, as critical attributes in protein pharmaceuticals. Over the past decade, Professor Zhou has been actively collaborating and consulting with biotech and pharm companies on biotherapeutics, enzymes and protein chemistry. These collaborations have led to the elucidation of product and process-related modifications (many of which were previously unknown). He also developed and now teaches a new advanced course entitled “Chemistry and Design of Protein Pharmaceuticals,” as well as workshops on antibody-drug conjugates (ADC), and training courses for scientists from industry, academia, and regulatory agencies in the US, China, and APEC economies.