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Hollow fiber bioreactors produce highly concentrated secreted proteins from a variety of mammalian cell types in a cost-effective manner. (Photo courtesy of Harlan Bioproducts for Science)
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In 1975, Georges Köhler and César Milstein developed a process for creating monoclonal antibodies. This was the beginning of an immunoreagent-driven healthcare technology march of progress that has become a central part of the growing multi-billion-dollar IVD industry.
Monoclonal Antibodies
Monoclonal antibodies as immunoreagents are critical components of IVD assays. To be sensitive, assays require high-affinity immunoreagents that bind at low concentrations to the analyte of interest. The immunoreagents must also be specific in order to avoid cross-reactivity with other compounds.
The development of these powerful assay tools has traditionally involved hybridoma technology, through which highly specific antibodies are produced in large quantities by the clones of a single hybrid cell that was formed in the laboratory by fusing a lymphoid cell, or B cell, produced in the bone marrow—and that synthesizes antibodies—with a tumor cell. This fusion can result in a large number of B cell hybridoma colonies for screening. A successful fusion will produce 500 to 1000 colonies. However, hybridomas that produce monoclonals of the desired specificity can be as rare as 1 in 500 of these. Or, there may be as many as 5 to 10 growing B cell hybridomas.
Handling the large number of B cell hybrid colonies can be labor-intensive. Therefore, the early identification of monoclonal antibodies with the desired specificity is the most critical step. In addition to the rapid detection of hybridomas producing specific antibodies, the process requires many screening assays. Choosing a reliable screening method is important to success.
Once the antibodies are developed, they are scaled up either in vivo (in animals) or in vitro (via tissue culture).
Advances in in vitro tissue-culture technology have improved the yield and reduced the costs of this scale-up method, which now is more widely used for monoclonal production than the mouse ascites method.
Owing to their specificity and their ability to reproduce the same binding characteristics, monoclonal antibodies, with their chemical-like qualities, are a powerful diagnostic tool. They have been generated against an array of compounds through the years, including cell surface markers, cancer cells, drugs, environmental pollutants, food products, hormones, metals, toxins, bacteria, and viruses.
Monoclonal antibodies are used to create sensitive immunologic assays in such application areas as clinical chemistry, hematology, microbiology, and histopathology because they can detect small amounts of substances in a reproducible way. The biggest areas for bulk sales of monoclonal antibodies are immunoassays ($7.2 billion annually), clinical chemistry ($3.1 billion), hematology ($2.0 billion), and routine microbiology ($1.3 billion). While immunologic assays for nearly every disease exist, diabetes is the largest single disease diagnostic category, accounting for $2.8 billion yearly.
Monoclonal antibodies are also vital research tools for many applications, among them Western blotting, immunohistochemistry, immunocytochemistry, enzyme-linked immunosorbent assay, immunoprecipitation, and flow cytometric analysis.
Assay Trends
Most diagnostic testing is performed in the laboratory, in an immunoassay-based platform. In addition, point-of-care testing devices are used in medical facilities, and self-testing devices are available for home use. Historically, lab testing was thought to be responsible for driving up healthcare costs. As the sensitivity and specificity of IVD tests increase, however, the ultimate healthcare costs will decrease, because earlier detection will mean less reliance on expensive treatments and fewer additional medical procedures due to complications. The demand for IVD tests will only increase as the benefits of wellness and preventative testing become more generally understood.
Antibody-based immunoassays are still the type of diagnostic assay most commonly used for detecting symptoms of disease, but new technologies are emerging. For example, advances in miniaturization and genomics have led to the development of nanotechnology for use in molecular diagnostic techniques. The application of this technology using current diagnostic equipment will allow multiple tests to be run from a single sample. This technology additionally offers the possibility of analyzing entire genomes in minutes. As the IVD and pharmaceutical industries cooperate further in the development and implementation of this technology, physicians’ ability to determine an individual’s predisposition to a specific disease will increase. This is especially important now because of increases in the incidence of such disease states as obesity, hypertension, and diabetes.
These advances have also resulted in the appearance of a vital field that will be the launching point for many of the immunoreagents that will be developed to meet the IVD industry’s future needs: proteomics.
The total number of different proteins in human cells is estimated at between 250,000 and 500,000. Only a small percentage of these have been sequenced or identified. In contrast, the genomes, or the entire sets of genes, for several organisms have been sequenced, including humans. The human genome is estimated to contain about 35,000 protein-encoding genes. One gene can produce as many as 1000 different proteins. However, traveling the road from identifying genes to developing effective diagnostic tests is a challenging journey. As we begin to understand how these cells communicate, and more discoveries are made in the laboratory, improved genetic and genomic diagnostic tests will be developed. This will do more than expand the IVD industry. It will begin to fulfill the promise of personalized medicine with individually targeted treatments.
As the demand for faster and better care grows, the possibilities for codeveloped drugs and diagnostics will further fuel the IVD industry’s own demand for immunoreagents and biomarkers. The extent to which diagnostics identify biomarkers that will improve clinical outcomes is a key issue. That is because, with more industry collaboration and conducive regulatory changes, the value of diagnostics and biomarkers will rise. When this emerging technology is coupled with the current routine test technology, market demand will rise further. In order to meet this demand, many IVD companies will invest in internal assay production and use outside contractors to cover their immunoreagent needs.
Sourcing Immunoreagents
A wide range of technologies are used to develop and manufacture immunoreagents for IVD use. An IVD company’s purchasing group should consider a number of key issues when looking for potential sources. The necessary scale-up is offered in either an in vivo or an in vitro system. The expertise required for the manufacturer’s supply program encompasses cell culture and purification, technical and customer service, and a strong quality management program. The challenge for the IVD manufacturer itself is to contain costs at a maximum level of revenue while maintaining a high-quality immunoreagent supply.
This often can create purchasing challenges in terms of planning and managing inventories. A manufacturer evaluating immunoreagent suppliers has to consider many factors, including each supplier’s capacity, quality standards, service, and understanding of the development process, particularly at the time the order is being set up.
For the IVD manufacturer to avoid back orders and downtime, it is important that the supplier can meet the capacity requirements. The manufacturer should confirm that the required production systems are in place and supported with the expertise and other resources necessary to meet demand. Forecasting is also crucial, to ensure that a continual product supply is maintained. Keeping an ample safety stock inventoried will minimize the risk of the IVD manufacturer falling into a back-order situation.
The immunoreagent supplier should have a strong quality management system in place to ensure that its processes are consistent, reproducible, and accurate. It is critical that the supplier understand the requirements expectations of the IVD company, and agrees to fulfill them prior to initiating the work. The parties often enter into a service, or supplier, agreement that documents the project’s production and business conditions. An important part of this is for the manufacturer to identify its quality expectations for the prospective vendor and make sure that the supplier’s quality systems are in line with expectations. This will go a long way toward minimizing delays when auditing and evaluating prospective vendors.
The evaluation of outside suppliers is a process that should involve the right mix of people. Typically, these would be purchasing, quality, and technical personnel. A coordinated approach will provide a broader understanding of the supplier’s capabilities. Each functional area represented contributes a different perspective. Each should therefore be an integral part of any audit or vendor evaluations. If any functions are not represented initially, the very possible outcome may be costly reaudits or a poor choice of contract supplier, which runs up the ultimate project costs.
While capacity is an important consideration when evaluating suppliers of finished immunoreagents, their quality system is a vital one. Whether the quality program being reviewed is a CGMP program or some other type, it must be one that will meet the IVD company’s manufacturing requirements. Questionnaires can be used as a tool to gather preliminary information, but face-to-face audits are most effective. The quality of the immunoreagents supplied by the contract production provider reflects directly on the IVD manufacturer through its products’ quality. A supplier with a poor quality system is no good.
Once the quality issues are settled, the manufacturer and supplier should define and agree upon technical and purchasing aspects of their arrangement. Technical specifications include not only key requirements such as concentration, purity, and necessary testing, but also small things that need to be clarified, such as details required in the certification of analysis. If any of the manufacturer’s specifications are incorrect or miscommunicated, delays in processing and delivering the supply, as well as additional expense, can result. The technical service capabilities of the vendor also should be considered at this stage. It is vital that the company have the expertise to handle technical problems that will arise during production.
The hybridoma cell lines used to create the immunoreagents of interest are biological systems, so there will be variability from lot to lot. A supplier that understands the process for which a given immunoreagent is supplied, along with the IVD manufacturer’s quality expectations, can minimize that variability. This is especially important when establishing a price structure for immunoreagent production.
IVD purchasing groups often order immunoreagents by volume as either purified or unpurified. Pricing should be based on a specified antibody concentration or product volume to avoid discrepancies in perceptions of what was produced and received. When establishing price, the parties should settle on a methodology for determining antibody concentration if the immunoreagents are taken as bulk raw product. This should be discussed during the negotiation process to avoid any confusion.
Scale-Up
The extensive use of monoclonal antibodies as immunoreagents is a cornerstone of the IVD industry. The amount of immunoreagent varies but is generally gram scale, ranging typically from 100 mg to 100 g. While many antibodies are commercially available, IVD companies tend to use their own clones for the scale-up.
Immunoreagent scale-up amounts to more than culturing large amounts of cells for in vivo or in vitro production. It also requires considerable preproduction work. This includes checking the hybridoma cell line for stability, antibody secretion, cross-reactivity, and reproducibility.
Both in vivo and in vitro production methods for scaling up antibodies have been around for a while. The in vivo method had been the most widely used because it is more economical. However, as in vitro technology has improved and yields have increased, it has become a more cost-effective alternative. In addition, it requires less labor and is flexible. An added advantage is that the reduced or nonexistent animal component ensures minimal extraneous protein in the product, which minimizes downstream purification and processing costs. Maximum environment control also makes this manufacturing process easier to validate, track, and refine.
Many kinds of in vitro production methods are in use. They include roller bottles, tissue culture bags, spinner flasks, fermentation, and hollow-fiber bioreactors. The choice of method varies with need. The tissue culture bags are modular and easy to scale up, but they often produce lower antibody concentrations. Spinner flasks are also easy to scale up, but they can be labor-intensive. Fermentation is generally used in large-scale production operations. Because this method is automated, it is less labor-intensive; but the antibody concentrations are low, and a large capital investment is required.
Finally, there is hollow-fiber production. This method also is easy to scale up. It yields highly concentrated product and is less labor-intensive than some methods. The flow path is disposable; therefore, no cleaning is required. The closed system offers a low contamination risk and no chance of cross-contamination. It does, however, require a skilled technician and cold storage space for the media.
A company looking to scale up has many options to consider. Regardless of the in vitro method chosen, production success is largely based on the nature of the cell line, the media formulation used, and the length of the run. The type of production, the amount of immunoreagent required, and the final format will all be factors in the cost outcome.
Conclusion
Since their introduction as immunoreagents, monoclonal antibodies have propelled the IVD industry. Their use as research tools and diagnostic components has enabled diseases to be tested for and detected that were once thought to be beyond diagnostic reach. The development of novel technologies, increased demand, and the emergence of new markets in the areas of infectious disease, cardiovascular disease, and cancer have also helped drive industry growth. In addition, proteomic research will result in the discovery of new proteins that will be developed as biomarkers that could offer significant predictive value and replace costly invasive procedures. The culmination could be the realization of personalized medicine.
