Scantibodies Laboratory Inc. (Santee, CA)
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Chemists at Scantibodies Laboratory Inc. (Santee, CA) sort and characterize thousands of plasma units.
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Bovine Serum Albumin
One of the most difficult biological materials to find now is BSA. A constituent of most immunoassays, BSA is used as a blocker to prevent nonspecific interaction of active components with the solid phase or the vial itself.
Albumin is a very flexible molecule. It can undergo significant conformational change in response to its environment, and is remarkably robust in resisting denaturation. This ability to change shape allows BSA to conform to and bind with high avidity to many surfaces. In the case of polystyrene, a material commonly used for in vitro assays, albumin can create more than 100 nonspecific bonds.
But albumin polymerizes easily, which decreases its ability to change shape. Since heat precipitation is one of the primary ways to manufacture BSA—heat-shock purification provides good purity at a reasonable cost—and heat causes polymerization, finished lots of BSA are notoriously inconsistent in their blocking ability. Assay manufacturers thus are forced to qualify each new lot on every assay, often approving one lot for just one assay.
Another cause of inconsistency among BSA lots is impurity. The primary biological function of BSA is to serve as a carrier protein. In the in vitro environment, it will release substances such as nonesterified free fatty acids, vitamin B12, T3, and T4 as a result of changes in environmental pH, ionic strength, or other parameters, or because of sequestering by another binding protein such as an antibody in an immunoassay.
Many different extraction techniques are used during BSA purification to remove specific contaminants. However, exogenous contaminants can be introduced owing to inadequate or improper processing. For example, caprylic acid is often used during heat precipitation to stabilize the albumin. Inadequate downstream processing (in this case, charcoal stripping) will result in caprylic acid remaining bound to the albumin until it is subsequently released into the assay. And microbial contamination occurring during processing can introduce the risk of carrying forward not only organisms but also by-products of cell necrosis, such as proteases and ammonia.
Besides the lot-to-lot inconsistency challenges, significant decrease in the supplier base over the past five years has made BSA difficult to source. Several large manufacturers have closed down or stopped producing BSA. This has also increased the cost; some types of BSA run close to $10/g. Another reason BSA is difficult to source is the appearance of new regulations for animal products, especially concerning bovine spongiform encephalopathy (mad cow disease). Most of the USDA-approved production sites are located in either North America, Mexico, or Australia.
IgM-Positive Plasma
A second biochemical raw material that is difficult to obtain is human IgM-positive plasma.
With the advent of infectious-disease assays in the early 1980s, it became important to measure antibodies as well as antigens in order to better diagnose a specific disease. Infectious-disease assays measure two types of antibodies primarily, IgG and IgM. The presence of IgG indicates past exposure and, perhaps, mature infection, while IgM indicates acute infection of recent origin (usually six to eight weeks before).
Manufacturing a control for an antigen detection assay is simple: one needs only to add the purified, usually recombinant, antigen to a stable serum matrix. Manufacturing the control for an antibody detection assay is more difficult, however, because one needs human antibodies (IgG and/or IgM) that until recently could not be manufactured outside of a human being. Most assay manufacturers use positive plasma from infected human donors to make their IgG/IgM controls. Using positive plasma, especially for IgM controls, presents several challenges. First, IgM is rare; high titer appears only for a few weeks. Since an infected person cannot knowingly donate blood, most IgM units are collected by accident. (A few very unusual donors whose IgM titer stays high for several months arrange special donations in order to sell their plasma to assay manufacturers.)
Natural IgM is inherently unstable, especially when manipulated. Viral inactivation, freeze-thaw cycling, lyophilization, concentration, and exposure to a new matrix, all involve manipulation.
Multianalyte controls require very-high-titer positive plasma. For example, in order to manufacture a TORC IgM control containing toxoplasmosis IgM, rubella IgM, and cytomegalovirus IgM, each positive plasma has to be at least triple the titer of the control’s final titer because it will be diluted threefold with the positive plasmas for the other two analytes.
Each lot of positive plasma is small—less than a liter. Some large assay manufacturers use more than 100 L of a single IgM-positive plasma in a year. Thus, they will encounter more than 100 lots of raw material annually, each with unique stability and cross-reactivity characteristics. Maintaining lot-to-lot consistency is very difficult with such raw material diversity.
Immunization programs are reducing the number of people who contract infectious diseases, especially in Europe and America, home to most of the donor pool. High-titer IgM plasma therefore is becoming unnaturally, as well as naturally, rare.
Finally, IgM-positive plasma is often infectious, which means it requires special handling and labeling.
These challenges have motivated many assay manufacturers to consider alternative methods of producing their IgM controls. One option is to immunize animals with the analyte and harvest the early immune sera, hoping it contains high amounts of IgM. The advantage of this method is that the antibody is polyclonal, like native human IgM. However, disadvantages are that the IgM is animal and that animals are constantly being sacrificed (since IgM appears only once, and briefly), increasing both cost and lot-to-lot inconsistencies.
Another alternative is to use a specific monoclonal IgM. This solves the consistency problem. But most monoclonal cell lines still are animal. Newer monoclonal antibodies are being developed, however, that are chimeric, and even all-human. Such a cell line would address all of the current challenges, thus solving one large problem facing IgM control manufacturers.
Heterophilic Panels
Another challenging biochemical raw material is heterophilic plasma. IVD manufacturers have to prevent false-positive immunoassay test results. False-positive results can have negative consequences that cascade in a domino effect—wrong diagnosis, patient anxiety, further testing, unnecessary treatment, and liability. The main cause of such deleterious results is heterophilic antibodies within the patient’s blood sample.
A common method of identifying a heterophilic false-positive result is to treat the specimen with a falsepositive blocker and look for a result dissimilar to the untreated test result, which would indicate heterophilic interference. Or, the specimen can be serially diluted for testing. Nonlinear results will indicate heterophilic interference. The specimen might also be rerun on different assay systems, with dissimilar results again indicating heterophilic interference in one or more of the assays. Unfortunately, although any one of these test methods is able to identify a false positive, an existing false positive may not necessarily be detected by any of them.
Therefore, it is important that the IVD manufacturer obtain a heterophilic false-positive serum or plasma panel of samples—ideally, one consisting of two sample sets. The first would represent the range of antianimal antibody interferences, while the other can help in evaluating assays being used or developed by showing that optimization studies to remove interferences have been successful.
Of course, it can be very difficult to obtain a panel of heterophilic samples that cause interference in the manufacturer’s particular assay. Problematic samples might be procured from clinical laboratories, but in most cases the remaining sample volume will be too small to allow further testing. A good alternative is to purchase panels from companies that have already screened thousands of units from blood collection facilities and determined certain samples to be heterophilic antibody positive.
With this approach, besides certainty that the sample contains heterophilic antibodies, the manufacturer enjoys the bonus that large volumes from the same unit generally are available.
Normal Human Plasma
Perhaps the largest raw-material procurement problem facing IVD manufacturers is the lack of plasma for use in production of human-based diagnostic test kits and controls. These plasma products are the primary components of controls and standards. The plasma supply has changed over the past five years from abundance to a serious shortage.
Many factors are behind the current shortage. The domestic supply has fallen in the wake of recent hurricanes and other natural disasters and the exportation of a portion of the usable supply of blood products to other world regions. In general, the diagnostics industry takes a back seat to patients’ transfusion needs and fractionation for injectable products, which leads to a shortage of diagnostic-grade plasma. But the primary purpose of blood products is, and always should be, the treatment of human patients. In times of scarcity, available blood products go first to fill patient needs.
Source plasma, or plasmapheresis material, is used primarily for fractionation. Blood centers cannot collect enough of this freshly frozen material to support current fractionation demands and are being asked to ramp up donations to meet this need. (According to the American Red Cross, only 1 of every 12 people in the United States eligible to donate blood actually does.) The current shortage has affected the supply of intravenous immunoglobulin (IVIg) and treatment to cancer and hemophilia patients. Consequently, the fractionation industry is getting creative in the use of nonstandard materials in order to continue manufacturing their product lines, which cuts even further into the diagnostic-grade supply.
In the past, more plasma was made available to the IVD industry from materials that fell out of specification for fractionators by failing to meet either dating or temperature requirements. But greater demand among the fractionators, along with tightened regulations and controls, have significantly decreased the availability of this type of plasma to IVD companies.
Several new infectious diseases of humans either have already affected the availability of plasma for the diagnostic industry or will soon. These include West Nile virus, transfusion-related acute lung injury (TRALI), and Chagas’ disease. The likelihood of FDA-mandated testing of donated blood for West Nile virus and Chagas’ is still uncertain, but a backlash regarding TRALI, found primarily in female blood and plasma, appears likely. Some in the IVD industry have suggested that female plasma products be removed from consideration for injectable material. However, since males donate the majority of blood and plasma, any release of the female plasma base to the diagnostics manufacturing industry should not have a large effect. Alternatively, screening could be used to eliminate potential problem donors or earmark the blood obtained for either fractionation or IVD use.
Cost is a huge factor in sourcing plasma. The higher the demand in relation to supply, the higher the price. Plasma is considered a commodity, and pricing today is at an all-time high. If the product that an IVD company is manufacturing has strong competition, then the cost of the raw-material plasma will affect the price structure. Many companies that once used human plasma or serum components have either switched to animal sources or else eliminated the affected product from their offerings because of being unable to maintain competitive pricing.
Although it is not possible to control the plasma market, an IVD manufacturer can do several things to manage its supply of the material. These include:
- Keeping abreast of the current market and knowing the sources.
- Using its sales and operating plan to obtain an accurate forecast of need, because plasma is not always available for purchase.
- Being prepared to purchase material whenever it becomes available, as the opportunity may not soon return.
- Setting up monthly standing orders to meet forecast demand. Most plasma providers want a one-year commitment, so inventory levels will be high at some times but lower at others. It is better to have material always available at a steady and standard price than to try to locate scarce material when needed and pay such a high price that profit is lost or negative.
- Dealing with only a few well-known, well-chosen primary plasma sources that represent a single group of blood centers, or that broker for multiple centers. Making inquiries at every source may drive up prices by creating an impression of heavy demand.
Conclusion
Many biochemical raw materials are difficult to obtain. The four materials discussed here are among the most challenging. One of the keys to success in acquiring any raw material whose availability is problematic is to partner with a vendor of established reliability who is an expert in that particular product and can offer a long-term solution.
