An important new area in biologics manufacturing is the development of biologic conjugates, made through genetic engineering of fusion proteins or conjugation of an additional compound to a protein after purification. Although this process adds a layer of complexity to an already complex manufacturing process, biologic conjugates can provide targeted therapy with improved efficacy and safety profiles.
These protein conjugates can be divided roughly into two types—one type is engineered fusion molecules that reduce immunogenicity and target enzyme activity, and the other type is chemically modifying protein drugs that improve biological half-life and targeted cytotoxic effects.
The cost of goods increases significantly with this type of manufacturing because of challenging cell-line development, product purification, and the complex chemistry of conjugating a chemical compound with a fragile protein without diminishing its biological activity. The problematic manufacturing required to produce conjugated biologics provides an opportunity for CMOs familiar with these complex processes.
One of the major issues related to biologics conjugates is the uniqueness of these compounds. “The applicability of platform approaches is limited and each conjugate has specific physicochemical properties, development, and production challenges. The absence of platform approaches makes this kind of development more expensive and scientifically more challenging. Customers may not fully appreciate the scientific and technical challenges and the associated costs of overcoming them,” explained RecipharmCobra’s Fenge.
One exception is the successful application of platform approaches to genetically engineered fusion proteins. Boehringer Ingelheim’s Esch explained that, “for half-life extension some proteins are expressed already at the genetic level as fusion molecules. The advantage of those technologies is the establishment of a lean and cost-effective large-scale manufacturing process, whereby, the molecular size of the fusion partner determines what expression technology might be best.”
Dr. Fenge explained that “for Fc-fusion proteins, RecipharmCobra has built on platform approaches developed for monoclonal antibody purification, particularly with regard to the protein A or G capture step.”
Merck has had similar success expressing fusion proteins. “In our experience, the principal challenge with fusion proteins is in the selection of an appropriate expression system; depending upon the exact nature of the conjugate, we have found that yeast-based systems are generally reliable production platforms for this class of biologic,” said Taylor.
Despite the use of well-characterized expression platforms for genetically engineered fusion proteins, these types of conjugates can be difficult to work with.
“Typically, titers tend to be rather low compared to monoclonal antibodies,” said Fenge. “This might be due to their complex and often ‘foreign’ structure compared to naturally occurring proteins; leading to aggregation, truncation, improper secretion, or other problems in the cellular process, which result in an improper conjugate structure.”
According to Esch, “soluble expression of toxin conjugates in various expression systems accounts for toxication of the respective production organism due to the toxicity of the fusion partner.”
The challenge of manufacturing chemically modified biologics reaches beyond the lack of platform approaches. Esch noted that “the chemical modification after initial expression and purification adds more process complexity and additional product characterization efforts.” Taylor added that “as far as protein-PEG conjugates are concerned, the main challenge is, generally, to ensure that the PEGylation chemistry is compatible with the biologic partner.”
CMC Icos Biologics’ Wheat echoed a similar sentiment with respect to toxic conjugates indicating that, “as chemically conjugated entities are typically cytotoxic and/or radioactive, conjugates present unique operational challenges for biologics CMOs. Typically, separate facilities are required for the chemical conjugation as well as for the subsequent purification of the conjugated molecule.”
Overcoming the challenges of chemically modified conjugates tends to be resource intensive and “requires an intimate collaboration and true partnership between the CMO and the customer,” noted Fenge. “In some cases, it might be necessary to work with the host cell line or embark on a protein-engineering exercise to make a functional conjugate at a reasonable cost of goods.”
Experienced contract manufacturing personnel provide specialized skills needed for complex manufacturing of conjugated biologics. “As a contract manufacturer, one has to keep several different technologies ready to match the technology to the customers’ needs. Boehringer Ingelheim has broad experience with various types of biologics conjugates,” said Esch.
“For example, to circumvent the problem of host toxication, production in recombinant E. coli in insoluble form is potentially a simpler, more cost-effective method that makes the manufacture of such toxic biologics conjugates economically viable.”
Merck has experience in the manufacture of fusion proteins and proteins conjugated with chemical moieties such as PEG, according to Taylor. “We have gained significant experience in this and usually rely on DOE (design of experiments) to identify optimal conjugation conditions.”
RecipharmCobra Biologics is also experienced with a variety of different conjugates including “classical” Fc-fusion proteins, novel two-domain fusion proteins expressed in CHO cells, and (Fab)2-enzyme conjugates expressed in E. coli involving chemical conjugation. Fenge commented that “protein-analytical expertise and broad capabilities are especially important to success, and it is critical early on in development to understand the compound and to be able to monitor potential product degradation or improper processed forms.”
Conjugated biologics are improved drugs. For example, chemically linking toxins to antibodies provides targeted rather than systemic drug activity. For half-life extension, genetic engineering and pegylation, “technologies open opportunities in the field of patient-friendly application forms,” said Esch.