Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2000-12-20
2003-12-30
Whisenant, Ethan (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06670116
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of Austrian patent application No. A2165/99, filed Dec. 22, 1999, which is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
The present invention related to methods for pathogenic agent detection, and differentiation between infectious and non-infectious forms. Specifically, the present invention relates to pathogen detection using nucleic acid amplification and detection techniques. Moreover, the present invention relates to nucleic acid amplification and detection techniques that permit infectious pathogens to be distinguished from non-infectious forms. More specifically, the preset invention relates to methods used to detect, and distinguishes between, infectious and non-infectious pathogens in biological products and pharmaceutical preparations.
BACKGROUND OF THE INVENTION
In is imperative that biological products intended for in vivo use be free of infectious pathogens. The use of biological products such as, but not limited to, those derived from human plasma are must be carefully tested for infectious pathogens before, during and after processing. The three methods most commonly employed to detect infectious pathogens are serological, cultures and nucleic acid detection assays. Serological methods are highly specific but lack sensitivity and cannot distinguish between living and non-living pathogens. Culturing methods only detect living pathogens and can be exquisitely sensitive if the pathogenic agent to be detected is easily cultivated. However, many pathogens such as hepatitis viruses and human parvovirus B19 cannot presently be cultured and other agents including the human immunodeficiency virus (HIV) are difficult to culture. Nucleic acid detection assays are highly sensitive and specific and can detect both cultivable and non-cultivable pathogens. Consequently, nucleic acid detection systems have become the method of choice for pathogenic agent detection.
Early nucleic acid detection assays used radioactive probes that hybridized with nucleic acid targets that had been extracted from the sample and captured on solid substrates such as filters or membranes. These systems, while extremely specific, lack sensitivity when target nucleic acid in a sample is scarce. However, nucleic acid detection sensitivity was dramatically improved in the late 1980's with the development of the polymerase chain reaction (PCR) assay and related nucleic acid amplification systems. Current nucleic acid amplification assays can detect as little as one copy of pathogen nucleic acid in a biological sample. Moreover, PCR assays have been adapted to automated platforms that permit the screening of hundreds of samples simultaneously. However, present nucleic acid assays, while highly sensitive and specific, cannot distinguish between living and non-living pathogen.
Many biological products are prepared from human plasma sources. These human plasma sources are initially screened for the presence of pathogens using nucleic acid detection systems including PCR. Samples that test negative for selected pathogens are then pooled and processed. Processing steps generally involve pathogen inactivation methods designed to eliminate residual infectious risk should an infectious agent detection assay fail to identify a contaminated sample. Pathogens that present the greatest concern to biological product manufactures are generally viral pathogens, such as, but not limited to hepatitis a virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), human parvovirus B19 (B19), transfusion transmissible virus (TTV), and human retroviruses including HIV, human lymphotropic virus types I and II (HTLV I and HTLV II). Biological product manufactures have developed validated viral inactivation techniques for these agents. Consequently, the risk of transmitting one or more of these agents in a biological product is extremely remote.
Moreover, processed biological materials are tested using nucleic acid detection techniques at various manufacturing stages and again after the final product has been formulated and packaged. On extremely rare occasions pathogen derived nucleic acids (target nucleic acid) may be detected in the final product. The contamination may have come from product supplements such as albumin or may have been missed during initial screening. In some cases, product processing steps may include extraction and concentration procedures that make previously undetectable target nucleic acids detectable. When target nucleic acid is detected in a finished product, the product is generally destroyed. However, in most cases the product would have been entirely safe for its intended use because the target nucleic acid detected was from an inactive, or killed pathogen.
Furthermore, final products derived using recombinant DNA technology intended to replace biological products derived from high risk starting materials may also be contaminated with detectable target nucleic acids. Present regulatory guidelines require that these recombinant DNA technology based biological (recombinant biologicals) must be destroyed even though the contaminate has generally come from product supplements such as albumin that has been subjected to infectious pathogen inactivation processes. Many biological products are extremely scarce and must be derived from plasma sources that present a risk of having transmissible pathogens present. Consequently, the raw materials needed to make many products are in short supply and the costs associated with destroying other wise perfectly safe an effective biologicals is high and can result in shortages of essential therapeutic agents.
Therefore, a pathogenic agent detection assay that combines the sensitivity and specificity of PCR with the ability to discriminate between infectious and non-infectious pathogens would represent a significant advance to biological product development and production.
BRIEF SUMMARY OF THE INVENTION
The present invention provided a solution to this and other problems associated with current nucleic acid detection assays. In one embodiment of the present invention a method is provided wherein a sample is tested using PCR and the total amount of target nucleic acid present is quantified. Next the sample, or an aliquot therefrom, is treated with one or more enzymes that cleaves free nucleic acids and then total target nucleic acid is quantified a second time using PCR. The total amount of target nucleic acid detected in the treated sample or aliquot is compared to the amount present prior to enzyme treatment. Unprotected target nucleic acid present in the sample will be destroyed by the nucleic acid-cleaving enzymes rendering them undetectable using PCR. Unprotected target nucleic acids present in the sample represent nucleic acids remaining after pathogen inactivation procedures have killed the pathogen.
Infectious pathogens, specifically viral pathogens, package their nucleic acids within a protein or protein/lipid/glycolipid structure referred to as a capsid. The viral capsid protects the viral nucleic acid (encapsulated nucleic acids) from nucleic-acid cleaving enzymes such as nucleases including, but not limited to RNase and DNase. However, many viral inactivation protocols effectively kill virus by destroying the integrity of the capsid rendering their nucleic acid susceptible to nuclease digestion. The present invention provides methods that differentiate between protected viral nucleic acid and viral nucleic acids susceptible to nuclease attack. A sample is deemed to have non-infectious viral contamination when nucleic acid detection levels are substantially or completely diminished following nuclease treatment as compared to the untreated sample.
Generally, in one embodiment of the present invention a method for detection, differentiation and quantification of free and encapsulated target nucleic acids in a sample consists of determining a first total target nucleic acid content in the sample (un-digested sample). Next a nuclease is added to the samp
Rieger Manfred
Schwarz Hans-Peter
Turecek Peter
Völkel Dirk
Zimmermann Klaus
Baxter Aktiengesellschaft
Heller Ehrman White & McAuliffe
Whisenant Ethan
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