October 1, 2016 (Vol. 36, No. 17)

Polymer-Based Protein Engineering Outperforms PEGylation, Says Biohybrid Solutions

Biohybrid Solutions is pioneering a new approach to bioconjugate production using polymer-based protein engineering. This innovation allows more control, expands the available polymer library, and minimizes the issues inherent with attaching polyethylene glycol (PEG) to proteins. Polymer-based protein engineering achieves the same outcomes as the more common molecular biology-dependent approach of classic protein engineering.

As Biohybrid Solutions co-founder and CEO Alan Russell, Ph.D., says, “Polymer-based protein engineering provides another option to scientists seeking to overcome the challenges of current PEGylation technology.”

PEGylation, which decorates the surface of protein with PEG, typically reduces a protein’s immunogenicity, extends a protein’s time in the body, and can improve its solubility. PEGylation, however, is not an easily controlled reaction. “You essentially place PEG and proteins together and hope for the best,” Dr. Russell explains. “There are few levers available to control and fine-tune the reaction.” Although PEGylation is notoriously difficult to commercialize, there are 10 FDA-approved protein-PEG conjugates currently marketed in the United States.

When standard development methods are used, the functionality of PEGylated products also is limited. “It’s very difficult to create a dense coating on the protein by reacting an existing polymer with the protein surface,” notes Dr. Russell. “Each reaction slows the next reaction, so most PEGylated drugs on the market have few attached PEG chains.”

Immunogenicity also is becoming an issue. According to Dr. Russell, some people “are developing an immune response against PEG.”

To back this assertion, Dr. Russell cites studies that have detected anti-PEG antibodies in blood. In a 1984 study, anti-PEG antibodies were found in about 0.2% of people tested. In a 2015 study, they were found in approximately 25% of the 250 people tested.

It is high time, Dr. Russell insists, for the field to advance.

This 3D structure for a chymotrypsin macro-initiator was derived from a 10 ns molecular dynamics simulation of the structure in water. The ball-and-stick figures depict how a lysine residue and the N-terminus were modified. The image was obtained by Sheiliza Carmali, Ph.D., who used the UCSF Chimera molecular graphics package.

Biohybrid Solutions’ Approach

 “Rather than grafting a polymer onto a protein, we employ a ‘grafted from’ approach using polymer-based protein engineering,” Dr. Russell reveals. “We choose a protein and modify it with the initiator for an atom transfer radical polymerization (ATRP), which is a controlled technique to grow polymers.”

The reaction, says Dr. Russell, is quite efficient and results in polymers growing from 80 to 100% of the available initiator binding sites. He adds that when this process is used, the density, length, and nature of the polymers that are grown can be controlled: “Because we can control the precise chemistry of the polymer, we can grow polymers that are either inert or responsive to their environment.”

If protein engineers want to mimic PEGylation, they typically replace PEG with an acrylic polymer, one that has a side chain that resembles PEG. This acrylic polymer is called POEGMA—poly(oligo(ethylene glycol) methyl ether methacrylate). “Unlike PEG, which is analogous to a piece of string, POEGMA resembles a comb,” points out Dr. Russell. Researchers are showing that carefully designed comb-like polymers have advantages over PEGylated proteins in terms of greater immunogenicity and a longer lifetime in the body.

When compared to PEGylated interferon-alfa-2a (known commercially as Pegasys), the POEGMA conjugate exhibited significantly better pharmacokinetics, tumor accumulation, and anticancer efficacy. According to a research from the Tsinghua University laboratory of Weiping Gao, Ph.D., the POEGMA conjugate “possessed a 7.2-fold higher antiproliferative bioactivity in vitro than Pegasys” and completely inhibited tumor growth. This research, which appeared July 2016 in Biomaterials, also indicated that in mice, 75% of the tumors were eradicated using the POEGMA conjugate. In contrast, none of the mice treated with the Pegasys survived beyond 58 days.

Focus: Tools and Enzymes

Dr. Russell and Carnegie Mellon University (CMU) colleague Krzysztof Matyjaszewski, Ph.D., pioneered this field about a decade ago while researching different ways to create very dense polymer shields or patches for proteins using controlled chemistry. The past three years have been spent proving and perfecting the chemistry and showing the breadth of the development tools.

In 2015, CMU launched the Center for Polymer-Based Protein Engineering, which Dr. Russell and Dr. Matyjaszewski co-direct. In the spring of 2016, they launched Biohybrid Solutions. The young company’s three-pronged strategy is focused on developing tools, working with partners, and developing in-house projects.

Accessing automated tools for protein PEGylation will allow scientists to develop these proteins themselves. Twenty-five years ago, protein engineering was quite difficult, but the field took off “as soon as the tools became available.” With this precedent in mind, the leaders of Biohybrid Solutions are prioritizing the development of tools to automate polymer-based protein engineering.

The company also looks forward to building a device that will operate on a small scale. The device is still some years from being realized. Nonetheless, Dr. Russell is enthusiastic: “All the chemistry will take place in the device. Scientists will be able to load a protein and, 10 to 15 hours later, remove a polymer-coated enzyme.” In situ protein-polymer purification is the current challenge in the device’s development.

Biohybrid Solutions also is establishing collaborative and strategic partnerships with protein-based drug companies to use this “grafted from” approach to enhance enzymes used in drug manufacturing and therapeutics.

“Protein-polymer conjugates typically are used in therapeutic applications, but they also are used in other industries,” Dr. Russell explains. The food, beverage, paper, healthcare, and biofuel industries all rely on enzymes, for example. “We’re interested in fine-tuning enzymes so they function more effectively in those industries.”

Biohybrid Solutions, working with CMU and strategic partners, has used its “grafted from” polymer-engineering approach with more than 35 enzymes and proteins. For example, products have been developed that exhibit high activity, are stable in the presence of stomach enzymes and hydrochloric acid, dissolve in organic solvents, or change in response to temperature fluctuations.

The company won an SBIR grant in June from the Department of Energy to improve the enzyme catalyst for the production of high-quality, inexpensive biodiesel. It also is one of 19 companies making up the Innovation Corps at CMU’s Center for Innovation and Entrepreneurship this year.

BioHybrid Solutions recently signed a collaboration agreement with FLIR Detection to pursue new applications for polymer-protein hybrid materials. The companies have agreed to work on a Department of Defense project that will involve immobilizing enzymes with the aim of making self-decontaminating protective fabrics. “This collaboration,” said Jeremy Walker, Ph.D., manager, enzyme science & technology, FLIR Detection, “enables FLIR to combine our core protein stabilization competence with BioHybrid’s technologies to pursue new applications in applied material science, manufacturing processes, and other high-impact commercial and defense spaces.”

Biohybrid Solutions

Location: 320 William Pitt Way, Pittsburgh, PA 15248
Phone: (412) 219-3414 
Principal: Alan J. Russell, Ph.D., CEO
Number of Employees: 4
Focus: Biohybrid Solutions grows polymers from proteins and peptides and is developing automated tools to make these “grafted from” conjugations easier and faster to develop.
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