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U.S. Department of Health and Human Services

Vaccines, Blood & Biologics

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New Ways to Improve Testing of Vaccines for Purity and Safety

Principal Investigator: Vladimir Chizhikov, PhD
Office / Division / Lab: OVRR / DVP / LMD

General Overview

The great promise of 21st Century medicine requires ensuring that vaccines and therapies based on novel scientific technologies are not only effective, but also safe.

One threat to the safety of these products is the risk that they can become contaminated with disease-causing viruses and bacteria (microbes). This can occur during the manufacturing process, which uses living cells of mammals, birds, or insects as tiny biological factories to make these products. Such live factories are called "cell substrates."

There are already many types of tests developed that can detect microbes that contaminate these cell substrates. However, some viruses and bacteria may escape detection by existing tests. That's because these tests generally work by providing a "test-tube environment" in which a sample of the cells is placed or by recognizing unique protein or genetic "signatures" specific for targeted microbes. But some microbes don't grow well enough in this artificial environment to enable the tests to reliably detect them; or they can evolve into variants containing modified "signatures" rendering them unrecognizable by existing tests. As a result, accidental contamination of cell substrates is a significant concern during manufacture of cell-derived biological and therapeutic products. In addition, purity and safety testing using conventional methods usually requires a long time, in some cases exceeding the shelf life of the products. A reduced period for testing is particularly critical for live cell therapies that in some cases require administration prior to availability of test results.

These serious limitations of the current testing technologies have triggered efforts to develop new technologies designed to identify microbes that previously evaded detection by existing tests. Therefore FDA is developing the expertise needed to assess and properly regulate these emerging technologies, which will address an important and urgent public health objective.

Our laboratory is currently focusing on assessing the ability of several new testing technologies to quickly and reliably detect infectious organisms in cell substrates used to make vaccines. Among these technologies are those that identify infectious organisms by isolating, concentrating, and making enough copies of their genetic material so it can be measured; rapidly identifying specific genes by detecting evidence that they are controlling production of the proteins they encode (i.e., micro-array gene expression analysis); and rapidly identifying the specific genes of infectious organisms (massively parallel sequencing).

Scientific Overview

The main difficulty in detecting certain adventitious agents in cell substrates is the "silent" character of the contamination, that is, the absence of apparent turbidity or pH change or noticeable changes in growth and morphology of infected cells, among other obstacles.

In response to this difficulty, we have established a program to develop and evaluate new methods based on new molecular technologies that enable detection of very low levels of contamination with any adventitious agents during the manufacturing process and lot release testing of vaccines (e.g. the smallest known bacterium– mycoplasma--or numerous viral agents of bovine origin).

A major goal of the program is to evaluate several available technical approaches, such as the use of microarrays, mass–spectrometry, and massively parallel sequencing, in order to assess the upper and lower limits of their sensitivity and specificity, and the feasibility of using them to monitor the safety and purity of biologics. To this end, we will collaborate with the companies and research laboratories where these technologies have been developed and used for pathogen detection (e.g., the laboratory of Dr. Joseph DeRisi, University of California, San Francisco and Ibis Biosciences, Inc.).

In order to assess the detection limits and applicability of these new technologies we will use biological samples spiked with different amounts of various adventitious agents. We will compare the results of these tests with results from parallel testing of these samples using techniques currently available in our laboratory.

The program will also study different enrichment methods, including biological enrichment and concentration and capture of adventitious agents using different immunological and genetic-based advanced technologies that enable recognizing germ-specific "signatures" in cell substrates and other biologics.

Our laboratory is also developing biological standards to calibrate existing and new molecular methods in collaboration with the American Type Culture Collection (ATCC), as well as with organizations in Europe involved in developing similar biological standards. This joint effort should simplify development of harmonized guidances for the development, preparation and use of these biological standards.


Biologicals 2015 Jan;43(1):23-30
Use of tangential flow filtration for improving detection of viral adventitious agents in cell substrates.
Furtak VA, Dabrazhynetskaya A, Volokhov DV, Chizhikov V

Genome Announc 2014 Mar 6;2(2):e00127-14
Genome Sequence of Mycoplasma hyorhinis Strain DBS 1050.
Dabrazhynetskaya A, Soika V, Volokhov D, Simonyan V, Chizhikov V

J Appl Microbiol 2014 Jan;116(1):100-8
Preparation of reference stocks suitable for evaluation of alternative NAT-based mycoplasma detection methods.
Dabrazhynetskaya A, Furtak V, Volokhov D, Beck B, Chizhikov V

Biologicals 2013 Nov;41(6):377-83
Collaborative study report: evaluation of the ATCC experimental mycoplasma reference strains panel prepared for comparison of NAT-based and conventional mycoplasma detection methods.
Dabrazhynetskaya A, Volokhov DV, Lin TL, Beck B, Gupta RK, Chizhikov V

Vet Microbiol 2013 Apr 12;163(1-2):149-56
Characterization of Pasteurella multocida strains isolated from geese.
Varga Z, Volokhov DV, Stipkovits L, Thuma A, Sellyei B, Magyar T

Appl Spectrosc 2012 Dec;66(12):1480-6
Enhanced mid-infrared chemical imaging (IRCI) detection of DNA microarrays.
Schoen B, Mossoba MM, Chizhikov V, Rashid A, Martinez-Diaz K, Al-Khaldi SF

Int J Syst Evol Microbiol 2012 Sep;62(Pt 9):2068-76
Lactobacillus brantae sp. nov., isolated from feces of Canada geese (Branta canadensis).
Volokhov DV, Amselle M, Beck BJ, Popham DL, Whittaker P, Wang H, Kerrigan E, Chizhikov VE

J Virol Methods 2012 Aug;183(2):219-23
Application of a full-genome microarray-based assay for the study of genetic variability of West Nile virus.
Grinev A, Lu Z, Chizhikov V, Rios M

J Clin Microbiol 2012 Mar;50(3):1113-7
Eosinophilic Fasciitis Associated with Mycoplasma arginini Infection.
Silló P, Pintér D, Ostorházi E, Mazán M, Wikonkál N, Pónyai K, Volokhov DV, Chizhikov VE, Szathmary S, Stipkovits L, Kárpáti S

Mol Phylogenet Evol 2012 Jan;62(1):515-28
RNA polymerase beta subunit (rpoB) gene and the 16S-23S rRNA intergenic transcribed spacer region (ITS) as complementary molecular markers in addition to the 16S rRNA gene for phylogenetic analysis and identification of the species of the family Mycoplasmataceae.
Volokhov DV, Simonyan V, Davidson MK, Chizhikov VE

J Appl Microbiol 2011 Oct;111(4):904-14
Preparation of reference strains for validation and comparison of mycoplasma testing methods.
Dabrazhynetskaya A, Volokhov DV, Ikonomi P, Brewer A, Chang A, Chizhikov V

J Microbiol Methods 2011 Sep;86(3):383-6
Identification of Mycoplasmas Using a Fluorophore-Free Microarray and Infrared Chemical Imaging (IRCI).
Mossoba MM, Chizhikov V, Volokhov DV, Martinez-Diaz Milians K, Schoen B, Al-Khaldi SF

Am Pharm Rev 2011 May-Jun;14(4):37370
Historical Overview of Mycoplasma Testing for Production of Biologics
Chandler DKF, Volokhov DV, Chizhikov VE

Vet Microbiol 2011 Apr 21;149(1-2):262-8
Novel hemotrophic mycoplasma identified in naturally infected California sea lions (Zalophus californianus).
Volokhov DV, Norris T, Rios C, Davidson MK, Messick JB, Gulland FM, Chizhikov VE

Mol Cell Probes 2011 Apr-Jun;25(2-3):69-77
Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques.
Volokhov DV, Graham LJ, Brorson KA, Chizhikov VE

J Appl Microbiol 2011 Jan;110(1):54-60
Detection of mycoplasma contamination in cell substrates using reverse transcription-PCR assays.
Peredeltchouk M, Wilson David SA, Bhattacharya B, Volokhov DV, Chizhikov V

Methods Mol Biol 2011;671:55-94
Oligonucleotide microarrays for identification of microbial pathogens and detection of their virulence-associated or drug-resistance determinants.
Volokhov DV, Kong H, Herold K, Chizhikov VE, Rasooly A

Appl Environ Microbiol 2010 May;76(9):2718-28
Evaluation of Mycoplasma inactivation during production of biologics: egg-based viral vaccines as a model.
David SA, Volokhov DV, Zhiping Y, Chizhikov V

Methods Mol Biol 2009;465:395-417
Molecular detection of drug-resistant Mycobacterium tuberculosis with a scanning-frame oligonucleotide microarray.
Volokhov DV, Chizhikov VE, Denkin S, Zhang Y

J Virol Methods 2008 Dec;154(1-2):27-40
Microarray-based assay for the detection of genetic variations of structural genes of West Nile virus.
Grinev A, Daniel S, Laassri M, Chumakov K, Chizhikov V, Rios M

Appl Environ Microbiol 2008 Sep;74(17):5383-91
Biological Enrichment of Mycoplasma Agents using Co-Cultivation with Permissive Cell Cultures.
Volokhov DV, Kong H, George J, Anderson C, Chizhikov VE

Appl Microbiol Biotechnol 2007 Nov;77(1):223-32
Application of cell culture enrichment for improving the sensitivity of mycoplasma detection methods based on nucleic acid amplification technology (NAT).
Kong H, Volokhov DV, George J, Ikonomi P, Chandler D, Anderson C, Chizhikov V

Mol Phylogenet Evol 2007 Aug;44(2):699-710
Genetic analysis of housekeeping genes of members of the genus Acholeplasma: Phylogeny and complementary molecular markers to the 16S rRNA gene.
Volokhov DV, Neverov AA, George J, Kong H, Liu SX, Anderson C, Davidson MK, Chizhikov V

J Clin Microbiol 2007 Aug;45(8):2641-8
Development and Validation of DNA Microarray for Genotyping Group A Rotavirus VP4 (P[4], P[6], P[8], P[9] and P[14]) and VP7 (G1-G6, G8-G10 and G12) Genes.
Honma S, Chizhikov V, Santos N, Tatsumi M, Timenetsky MD, Linhares AC, Mascarenhas JD, Ushijima H, Armah GE, Gentsch JR, Hoshino Y

Biochem Biophys Res Commun 2007 May 18;356(4):1017-23
Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples.
Hsia CC, Chizhikov VE, Yang AX, Selvapandiyan A, Hewlett I, Duncan R, Puri RK, Nakhasi HL, Kaplan GG

J Med Virol 2006 Oct;78(10):1325-40
Microarray assay for detection and discrimination of Orthopoxvirus species.
Ryabinin VA, Shundrin LA, Kostina EB, Laassri M, Chizhikov V, Shchelkunov SN, Chumakov K, Sinyakov AN

J Clin Microbiol 2006 Oct;44(10):3752-9
Genotyping of measles virus in clinical specimens on the basis of oligonucleotide microarray hybridization patterns.
Neverov AA, Riddell MA, Moss WJ, Volokhov DV, Rota PA, Lowe LE, Chibo D, Smit SB, Griffin DE, Chumakov KM, Chizhikov VE

J Virol Methods 2006 Sep;136(1-2):8-16
New mosaic subgenotype of varicella-zoster virus in the USA: VZV detection and genotyping by oligonucleotide-microarray.
Sergeev N, Rubtcova E, Chizhikov V, Schmid DS, Loparev VN

Appl Microbiol Biotechnol 2006 Aug;71(5):680-98
Sequencing of the intergenic 16S-23S rRNA spacer (ITS) region of Mollicutes species and their identification using microarray-based assay and DNA sequencing.
Volokhov DV, George J, Liu SX, Ikonomi P, Anderson C, Chizhikov V

J Microbiol Methods 2006 Jun;65(3):488-502
Microarray analysis of Bacillus cereus group virulence factors.
Sergeev N, Distler M, Vargas M, Chizhikov V, Herold KE, Rasooly A

Appl Environ Microbiol 2006 Apr;72(4):2439-48
Discovery of Natural Atypical Nonhemolytic Listeria seeligeri Isolates.
Volokhov D, George J, Anderson C, Duvall RE, Hitchins AD

J Med Microbiol 2005 Dec;54(Pt 12):1127-31
Microarray-based pncA genotyping of pyrazinamide-resistant strains of Mycobacterium tuberculosis.
Denkin S, Volokhov D, Chizhikov V, Zhang Y

J Mol Diagn 2005 Oct;7(4):486-94
A multiplex polymerase chain reaction microarray assay to detect bioterror pathogens in blood.
Tomioka K, Peredelchuk M, Zhu X, Arena R, Volokhov D, Selvapandiyan A, Stabler K, Mellquist-Riemenschneider J, Chizhikov V, Kaplan G, Nakhasi H, Duncan R



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