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I Founded Dna Diagnostics Inc Biology Essay

Additional projects included the investigation of transcriptional and translational control mechanisms using baculovirus-infected cells as a model system, gene isolation and characterization, developing recombinant plasmid and cosmid clones (i.e. libraries), DNA restriction endonuclease mapping using various DNA/DNA hybridization techniques, sequencing, and investigating DNA-protein interactions.

I have also conducted research on the structure and function of the Epstein-Barr virus genome, a herpes virus responsible for several human diseases. Using recombinant DNA technology a genomic library was constructed and used to establish a detailed restriction map for a region encoding the transformation function.

6.

In 1993, I founded DNA Diagnostics, Inc., a consulting firm specializing in the scientific review of DNA test results in criminal and civil cases where such tests have been performed for individual identification and parentage verification. I work closely with the U. S. Federal government and state government agencies as well as private law firms throughout the United States where I have evaluated and provided opinions on many matters where DNA analysis was involved. I have served on the faculty for the pilot DNA training program sponsored by the DNA Legal Assistance Unit of the American Prosecutors Research Institute, where I presented information on evidence collection, handling, and storage, chain of custody issues, and DNA analysis. I also served as a faculty member in a training program sponsored by the Public Defender Service for the District of Columbia where I worked with attorneys on methods to present scientific data/information in a courtroom.

After completing a Postdoctoral Fellowship that focused on the molecular biology of host-pathogen interactions, I was Group Leader/senior Staff Scientist at Digene Diagnostics, Inc. where I directed senior and junior level scientists on the development and use of nonradioactive DNA probes for the detection and diagnosis of specific human pathogens in clinical samples by in situ hybridization. Digene Diagnostics, Inc. was a private start-up company and I was involved in the build-out or design of my laboratory and the facility, staffing, equipment purchase, as well as providing administrative support/management and supervision.

7.

In addition to my work with DNA Diagnostics, Inc., I am currently a faculty member in the Department of Molecular and Microbiology at George Mason University (Fairfax, VA, U.S.A.) where I teach a graduate level methods and applications course in DNA profiling, a seminar in forensic science, and an undergraduate course in microbiology.

Because I teach a forensic DNA analysis workshop and operate a laboratory, I am responsible for the complete set-up of the laboratory and the purchase of necessary reagents, disposables, and equipment.

8.

My CV is attached that includes a list of relevant publications and a list of cases in which I have provided scientific and technical support and/or have testified. [Enclosure 2].

9.

The Project

I have been requested by Phadia to conduct an independent review and analysis of information and materials provided regarding the development and production processes of Phadia's three recombinant peanut allergens (e.g., Ara h 1, Ara h 2, and Ara h 3) and render a scientific opinion as to Genclis' claim that Phadia used Genclis' proprietary technology in the development of Phadia's recombinant Allergens. To this end, I have studied and analysed many materials and gathered additional information from Phadia and other sources, including the Genclis/Phadia Technology Transfer document ("Technology Transfer for Peanut Recombinant Proteins ARAH1, -2, and -3"), a copy of a one page summary chart prepared by Phadia demonstrating the differences in their respective protein production processes, Enclosure 3, witness statements by Drs. Hans Öman and Joans Lidhom, background material regarding the dispute between Thermo Fisher (formerly Phadia) and Genclis, background material on Phadia's ImmunoCAP products, and documents related to Phadia's development of production processes far its three recombinant peanuts allergens.

Basic Principles

Set forth below are my answers to questions regarding some basic principles regarding the design and development of recombinant proteins:

10. Question: Are Ara h 1, Ara h 2 and Ara h 3 protein sequences known publicly and in the art as evidenced by various databases, including Uniprot and Genbank, and journal articles?

Answer. The three major allergens of peanuts, Ara h 1, Ara h 2, and Ara h 3, have been isolated and characterized by scientists for more than a decade. These protein allergens have been purified and sequenced using standard techniques. The resulting amino acid sequences for each protein allergen have been published in the open scientific literature and submitted to public databases providing the scientific community with a comprehensive and accessible resource of the protein sequence and functionality of each protein (e.g., UniprotKB Consortium, Entry P43238 (1995) (Ara h 1), Enclosure 4; European Molecular Biology laboratory (EMBL) Bank: AY581853 (2004/2005) Ara h 2), Enclosure 5; National Center for Biotechnology Information (NCBI) Genbank: AY848698 (Ara h 3), Enclosure 6). Based on this information the Ara h l, Ara h 2, and Ara h 3 protein sequences are available and readily accessible to the public and scientific community. Similarly, the allergen sequences have been determined and published in the peer reviewed scientific literature (Burks et al., 1995. Recombinant peanut allergen Ara h 1 expression and IgE binding in patients with peanut hypersensitivity. J. Clin Invest. 96:1715- 1721, Enclosure 7; Viquez et al., 2001. Isolation and molecular characterization of the first genomic clone of a major peanut allergen, Ara h 2. J. Allergy Clin. lmmunol. 107:713-717, Enclosure 8; Rabjohn et al., 1999. Molecular cloning and epitope analysis of the peanut allergen Ara h 3. J. Clin Invest. 103:535-542. Enclosure 9; Yan et al., 2005. Isolation of peanut genes encoding arachins and conglutins by expressed sequence tags. Plant Science. 169:439-445), Enclosure l 0.

11. Question: Were the protein sequences of the Genclis recombinant proteins Ara h 1, Ara h 2, and Ara h 3 proteins (the "Genclis recombinant proteins") themselves publicly disclosed beginning in 2007 by sales of ImmunoCAP products containing the Genclis recombinant proteins? Answer: Protein-based components that exist in commercially available products can be analyzed and their composition and sequence determined using standard scientific techniques.

Mass spectrometry is such an analytical technique that is commonly used to analyze proteins/peptides to determine their mass and elemental composition, as well as, the amino acid sequence contained in a peptide or other proteineous compound. Thus, scientists are able to isolate and characterize naturally-occuring and recombinant proteins in such commercial products available to the public, medical, research, or diagnostic community using mass spectrometry. In this case, the Genclis recombinant proteins were effectively disclosed to the public once the ImmunoCAP products containing them were sold in the market, which I understand began in 2007. It should also be noted that the results of mass spectrometry analysis provide information on the amino acid sequences of the proteins, but do not provide information regarding the DNA (genomic sequences) or plasmid clones used in the expression system to produce the amino acid protein sequences Question: Can one use different recombinant production technology, such as different DNA sequences, bacteria, clone vectors, fermentation conditions and purification processes, and still produce two "identical" protein sequences, namely, having 100% identity along the entire lengths and can such "identical" protein sequences have similar performance properties?

Answer. Yes. Manufacturers can use different recombinant production technology, such as different DNA sequences, bacteria, clone vectors, fermentation conditions and purification processes, to produce similar, if not identical, proteins with 100% identity along the entire sequence. Genetic engineering and recombinant DNA (rDNA) technology is widely used throughout the world. Recombinant proteins and other products derived from the use of rDNA technology are found in every facet of our life from the local pharmacy, to the medical testing laboratory, to the biological research laboratory. The most common application of rDNA technology is in basic research in the biological and biomedical sciences. Recombinant proteins have been produced in numerous organisms (e.g., bacteria yeast, baculovirus and insect host, and mammalian cells) or cell-free "systems" (e.g., in vitro production of recombinant proteins in solution using the translation machinery extracted from cells) using rDNA technology to produce an assortment of products for commercialization.

Protein composition can be predicted and even determined using information in the DNA code.

The DNA code is often referred to as being "redundant" or degenerate where more than one codon or triplet of bases (i.e., GAA and GAG) encodes a given amino acid (the building blocks of proteins). This degeneracy allows different codons or DNA sequences to produce similar, if not identical, Ara h 1, Ara h 2, and Ara h 3 recombinant proteins. The resulting proteins would be "identical'' in overall structure and similar in functionality. Efficacy, in part, would be determined by fermentation conditions and in the purification of the protein(s) in the production process. Consequently, it is not surprising that two similar, if not identical recombinant proteins, could be designed and produced by two independent manufacturers using substantially different production methods.

Question: Can one correctly conclude that DNA sequences are "identical'' if there is less than 100% identity along their entire lengths? Answer. No. One cannot correctly conclude that DNA sequences are "identical" if there is less than 100% identity or homology along their entire length. The suggestion that DNA sequences are "identical'' or homologous is often incorrectly used when comparing the similarity between constructs or sequences. The terms "sequence similarity" and "percent homology'' are often used interchangeably to refer to similarities between DNA sequences. DNA sequences that are said to be "identical'' refer to the nucleotide composition and "arrangement" of the bases (i.e., A, T, G, and C) without any difference observed at any site along the "stretch" of DNA. DNA is not considered identical if there are regions in the sequence whereby a nucleotide (or base) has been substituted at a specific site or position in the DNA resulting in the production of functionally equivalent proteins. Partial homology may exist between these two non-identical DNA sequences or constructs that demonstrate less than 100% nucleotide similarity along the entire length. Consequently, significant sequence similarity may exist between non-identical DNA sequences with resulting protein products sharing identical physiochemical and functional properties.

14. Question: Is it common and accepted in the scientific community to cite a journal article to reference a theory or conclusion set forth in the journal article? And does such a citation indicate, and is such citation understood by the scientific community to represent any existing business and/or scientific affiliation or association with the authors of the journal article? Answer: Yes, it is quite common and an accepted practice in the scientific community to cite a journal article or reference a theory from the open scientific literature in a published article. An important, if not critical, aspect of the scientific process is the reporting of new results or "findings" in scientific journals. Such reporting disseminates data and new findings to the scientific community while contributing to the "pool of knowledge'' within a specific discipline.

Most journals accept manuscripts for publication only after the submitted article has been peer reviewed by scientists in the same field and who recommend the paper for publication. In all instances, these submitted articles cite references to other research findings and are considered in integral component of any research paper. In practice, scientists will summarize the findings or other information and then cite the source or the reference. Citing references provides a record of the sources used in the area of research and is a form of professional courtesy and integrity.

Citing sources also strengthens the authority of the work by demonstrating that others opinions and ideas have been considered in forming a given hypothesis and conclusion. For many scientists, citing references in an article from peer-rewiewed journals helps to establish credibility as a researcher. Citing a reference of a scientist's research does not indicate any scientific or business affiliation or association with the laboratory. In fact, most references are citations of research findings from independent laboratories with no affiliations to the author(s) of the submitted article.

Opinions

Set forth below are my opinions on the questions presented in this dispute: 15. Question: Did Phadia develop its own recombinant Ara h 1, Ara h 2, and Ara h 3 proteins (''the Phadia recombinant proteins") independent of the Technology Transfer from

Genclis?

Answer: Yes, Phadia developed and produces its own recombinant Ara h 1, Ara h 2, and Ara h 3 proteins independent of the process described in the Technology Transfer document from Genclis. Soon after the Technology Transfer, Phadia initiated the development of its own recombinant proteins, in part, because of inadequate characteristics or properties of the Ara h 1 and Ara h 3 proteins supplied by Genclis in 2008 and because of the substantial differences in the production process used at Genclis (Witness statement of Dr. Jonas Lidholm, Research Manager at Thermo Fisher Scientific, Exhibit C-WS-1; Summary of recombinant production procedures by Dr. Maria Murby dated 30 October 2008: Enclosure 8 to C-WS-1). The properties of the Genclis proteins included satisfactory IgE antibody binding in the ImmunoCAP assay but displayed unexpected precipitation upon receipt from Genclis Another concern was the significant variations in protein concentration observed between different production lots of Ara h 1 and Ara h 3 from Genclis [E-mail from Mats Rilven to Bernard Bilhain dated November 21, 2007, Enclosure 2 to C-WS-1). Concerns over the Genclis production process included excessively long production time (30 days vs. 7 days for a typical Phadia recombinant production process), the overall low protein yield, low storage stability of 3 months vs. Phadia's protein production scheduled every 18-36 months necessitating a significantly longer storage duration without compromising stability, precipitation issues of the Genclis proteins, and inadequate design in the production process to allow any significant scale-up(Witness statement of Dr. Jonas Lidholm, Research Manager at Thermo Fisher Scientific, Exhibit C-WS-1). This last issue included techniques and equipment used by Genclis that were not suitable or practical for use on an industrial scale. Such techniques as high-speed centrifugation and repeated dialysis steps to separate particles from solution vs. filtration and column chromatography (the latter two are preferred techniques) only added excessive time and labor to the Genclis production process (Witness statement of Dr. Jonas Lidholm, Research Manager at Thermo Fisher Scientific, Exhibit C-WS-l). The Phadia files reveal that its team developed a different process based on its own experience in recombinant protein development and production, and it did not use any of the Genclis process or technology.

16. Question: Is Phadia's process of manufacturing the Phadia recombinant proteins different than Genclis' process of manufacturing the Genclis' recombinant proteins? Answer: Yes, Phadia's process of manufacturing its own recombinant Ara h 1, Ara h 2, and Ara h 3 proteins is different than the process described in the Technology Transfer document from Genclis to produce their recombinant proteins. These differences, detailed below for each recombinant protein, include a) the bacterial host strain, the plasmid vector, and final construct used in their expression system, b) the fermentation process, c) the harvest and storage steps and d) the purification process. Details of these processes are described in several redacted documents provided by Phadia. Although redacted, the unredacted portions of these documents contain sufficient data/information to reveal the substantial differences between the Phadia process and the Genclis process.

a). The expression System. The expression system used in the fermentation process for Phadia's Ara h 1 recombinant protein consists of Escherichia coli (E. coli), a common bacterial host used in protein expression systems, and a plasmid vector, pET102. Phadia uses E. coli strain BL21-AI (see pg 1, English translation of "Frozen Design Odling av rAra h 1" "rAra h 1 Frozen Design Fermentation") by Dr. Lars Heinerud dated 20 October 2009, Enclosure l 1 to C-WS-1) whereas Genclis uses E. coli BL21 (DE3) (see pg 2 Technology Transfer document). These two E. coli strains differ in their genetic make-up. One specific difference is that the E. coli BL21 (DE3) strain used by Genclis contains the lacI gene, a repressor or regulatory gene in the lac operon (a functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter) in E. coli. Phadia's E. coli strain BL21 (AI) does not contain the lacI gene and provides for a different mechanism to control gene expression.

The plasmid vector pET102 used by Phadia is a proprietary derivative of pET23, a commercially available bacterial plasmid which is 5310 base pairs (bp) in size. The pET102 plasmid vector is 3606 bp in size and contains a synthetic gene (to produce the Ara h 1 product) insert of 1803 bp for a total size of 5409 bp (see pp 4-10, ''rAra h 1 Production Transfer Document Package'' (''rAra h l Transfer document") approved by Dr. Jonas Lidholm dated 1 September 2009, Enclosure 10 to C-WS-1. Genclis uses pET24a, a commercially available plasmid vector used in E. coli protein expression systems, that is 5237 bp in size with a clone insert (to produce the Ara h 1 protein) of 1812 bp for a total size of 7049 bp (see pp 2 and 14-20, Technology Transfer document). A comparison of the nucleotide sequence of the Phadia clone insert to the nucleotide sequence of Genclis' insert reveals significant differences. Another difference in the expression vectors are the antibiotic selectable markers. Phadia's plasmid vector uses ampicillin as the selectable marker whereas Genclis' vector contains kanamycin as the selectable marker (pg 1, rAra h l Frozen Design Fermentation, and pg 2, Technology Transfer document). The expression system used in the fermentation process for Phadia's Ara h 2 recombinant protein consists of Escherichia coli (E. coli) and a plasmid vector, pET102. Phadia uses E. coli strain BL21 (pT7-POL23) (see pg 1, English translation of "Frozen Design Odling av rAra h 2" (rAra h 2 Frozen Design Fermentation") by Dr, Lars Heinerud dated 26 June 2009, Enclosure 18 to C-WS-1) whereas Genclis uses E. coli BL21 (DE3) (see pg 2 Technology Transfer document). These two E. coli strains differ in their genetic make-up. One specific difference is that the E. coli BL21 (DE3) strain used by Genclis contains the lacI gene (see above), Phadia's E. coli strain BL21 (pT7-POL23) does not contain the lacI gene.

The plasmid vector pET102 used by Phadia is a proprietary derivative of pET23 (see above). The pET102 vector is 3606 bp in size and contains a synthetic gene (to produce the Ara h 2 product) insert of 417 bp for a total size of 4023 bp (see pp 4-8, ''rAra h 2 Production Transfer Document Package'' (''rAra h 2 Transfer document") approved by Dr. Jonas Lidholm dated 28 September 2007, Enclosure 17 to C-WS-1). Genclis uses pET24a, a commercially available plasmid vector used in E. coli protein expression systems, that is 5237 bp in size with a clone insert (to produce the Ara h 2 protein) of 456 bp for a total size of 5693 bp (see pp 2 and 21 -25 of the Technology Transfer document). A comparison of the nucleotide sequence of the Phadia clone insert to the nucleotide sequence of Genclis' insert reveals significant differences. Another difference in the expression vectors are the antibiotic selectable markers. Phadia's plasmid vector uses ampicillin as the selectable marker whereas Genclis' vector contains kanamycin as the selectable marker (pg 1, rAra h 2 Frozen Design Fermentation, and pg 2, Technology Transfer document).

The expression system used in the fermentation process for Phadia's Ara h 3 recombinant protein consists of E|scherichia coli (E. coli) and a plasmid vector, pET23a. Phadia uses E. coli strain BL21-AI (see above) (see pg 1, English translation of ''Frozen Design Odling av rAra h 3'' (''rAra h 3 Frozen Design Fermentation") by Dr. Lars Heinerud dated 7 January 2010, Enclosure 23 to C-WS-1) whereas Genclis uses E. coli BL21 (DE3) (see pg 2 Technology Transfer document). These two E. coli strains differ in their genetic make-up. One specific difference is that the E. coli BL21 (DE3) strain used by Gendis contains the lacI gene (see above). Phadia's E.

coli strain BL21 (AI) does not contain the lacI gene.

The plasmid vector pET23a used by Phadia is 3593 bp in size and contains a synthetic gene (to produce the Ara h 3 product) insert of 975 bp for a total size of 4568 bp (see pp 4-9, "rAra h 3 Production Transfer Document Package" (rAra h 3 Transfer document") approved by Dr. Jonas Lidholm dated 5 June 2009, Enclosure 22 to C-WS-1). Genclis uses pET24a that is 5237 bp in size with a clone insert (to produce the Ara h 3 protein) of 975 bp for a total size of 6212 bp (see pp 2 and 26-31 of the Technology Transfer document). A comparison of the nucleotide sequence of the Phadia clone insert to the nucleotide sequence of Genclis' insert reveals significant differences. Another difference in the expression vectors are the antibiotic selectable markers.

Phadia's plasmid vector uses ampicillin as the selectable marker whereas Genclis' vector contains kanamycin as the selectable marker (pg 1, rAra h 3 Frozen Design Fermentation, and pg 2, Technology Transfer document).

b). The Fermentation Process. The fermentation processes used by Phadia are different from those processes used by Genclis as described in the Technology Transfer document. The Fermentation process used by Phadia uses glucose as the sole carbon source whereas Genclis uses glycerol as the carbon source. Using glucose versus glycerol, coupled with the different vectors and E. coli host strains, may result in the higher expression levels with decreases in background expression. This "enhanced'' expression in the Phadia system appears to reduce the fermentation production time to 5 to 9 hrs for Ara h 1 and Ara h 3. The fermentation time for Phadia's Ara h 2 is from 5 to 8 hrs. The fermentation production time for all 3 recombinant proteins produced by Genclis is 24 hrs (see pg 1 of each of rAra h 1 Frozen Design Fermentation, rAra h 2 Frozen Design Fermentation, and rAra h 3 Frozen Design Fermentation, and pg 3 of the Technology Transfer document).

c). The Harvest and Storage Steps. The harvest and storage steps used by Phadia for all 3 recombinant proteins are different from those processes used by Genclis (see Technology Transfer document). Phadia uses a single centrifugation step (centrifugation speed [x g] and time redacted) whereas the Genclis process uses 2 centrifugation steps (1st step: 50,000 g for 30 min at 4°C; 2nd step: 100,000 g for 30 min at 4°C) to harvest the "inclusion bodies'' for their 3 recombinant proteins (see pg 2 of each of rAra h 1 Frozen Design Fermentation, rAra h 2 Frozen Design Fermentation, and rAra h 3 Frozen Design Fermentation, and page 3 of the Technology Transfer document). Al1 3 of the Phadia products (i.e., Ara h 1, Ara h 2, and Ara h 3) are then stored at less than -60°C (see pg 2 of each of rAra h 1 Frozen Design Fermentation, rAra h 2 Frozen Design Fermentation, and rAra h 3 Frozen Design Fermentation). Genclis' products are stored at -20°C (see pg 3 of the Technology Transfer document). These differences noted by Phadia in the overall harvesting and storage process may have been due to their previous knowledge and years of experience producing numerous recombinant allergens for use in their ImmunoCAP products.

d). The Purification Process. The purification process that Phadia uses for all 3 of their recombinant proteins consists of a homogenization step, a IMAC (immobilize metal ion affinity chromatography) step, an ion exchange chromatography step, and a buffer exchange step (see Frozen Design Purification of rAra h 1, rAra h 2, and rAra h 3). After the last step, where desalting is achieved, the concentration of the products is determined by spectrophotometry and the products stored at < -65°C (English translation of ''Frozen Design Rening av rAra h 1'' ("rAra h 1 Frozen Design Purification") by Dr. Maria Murby dated 6 October 2009, Enclosure 12 to CWS-1, English translation of "Frozen Design Rening av rAra h 2'' (''rAra h 2 Frozen Design Purification") by Dr. Maria Murby dated 4 May 2009, Enclosure 19 to C-WS-1, and English translation of ''Frozen Design Rening av rAra h 3" ("rAra h 3 Frozen Design Purification") by Dr. Maria Murby dated 6 October 2009, Enclosure 24 to C-WS-1).

In the Genclis purification process, the inclusion bodies (the products that were stored from the centrifugation step described above) of all 3 of their products are solubilized in a "solubilization buffer" and incubated at room temperature for 64 hrs (see pg 5 of Technology Transfer document). For Ara h 1, an ammonium sulfate precipitation step is used (not used for Ara h 2 and Ara h 3), which, presumably, aids in purifying the Ara h 1 protein by altering its solubility.

The solubilized products are subjected to several rounds of high speed centrifugation followed by 4 rounds of dialysis at 10 hrs each (see pg 9 Technology Transfer document). Phadia does not use the high speed centrifugation steps in their purification process nor the dialysis process which adds significant to the purification process. Instead, Phadia employs a rapid buffer exchange step using a Sephadex G25 column to aid in their purification process (see rAra h 1 Frozen Design Purification rAra h 2 Frozen Design Purification, and rAra h 3 Frozen Design Purification).

In summary the differences between the two processes are fundamental. It is clear to me that Phadia started from its own experience and developed its own processes, processes that are completely and fundamentally different than the Genclis process. As mentioned before, even though the Phadia process documents are redacted, I have more than adequate data/information to discern the fundamental differences between the Phadia process and the Genclis process.

17. Question: Does the fact that the Phadia recombinant proteins have the same or similar protein sequences as the Genclis recombinant proteins have any significance to your opinions? Answer: No, the mere fact the Phadia recombinant proteins have similar to identical protein sequences does not change my opinion that the development of the Phadia recombinant proteins was independent of the Genclis processes described in the Technology Transfer document. I believe that Phadia processes were developed based on the data and/or information available in the open scientific literature and due to the previous knowledge and years of experience of developing over 50 recombinant proteins for use in their ImmunoCAP products.

As stated previously, manufacturers can use different recombinant production technology to produce similar, if not identical, proteins with 100% identity along the entire sequence. Protein composition can be also be predicted and even determined using information in the DNA code.

The DNA code is often refered to as being "redundant" or degenerate where more than one codon or triplet of bases (i.e., GAA and GAG) encodes a given amino acid (the building blocks of proteins). This degeneracy allows different codons or DNA sequences to produce similar, if not identical, Ara h 1, Ara h 2, and Ara h 3 recombinant proteins. Thus the resulting proteins could be "identical" in overall structure and similar in functionality. Consequently, it is not surprising that two similar, if not identical recombinant proteins, could be designed and produced by two independent manufacturers using substantially different production methods.

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