Tools | Technologies | Trends January 1, 2013Volume 33, number 1

Drug Discovery

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36

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Bioprocessing

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BiobusinessCancer Biomarker Hotspot Analysis

Novel Strategies for Cell-Based Assays

Genomic Analysis Drives Biomarker Discovery

Twisting and Turning for Better Protein Expression

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Cancer Biomarker 14-3-3 1 2 5 3 0 4 1 1 3 7 4 3 9 0 2 8 8 0 3 2 2 1 5 4 6 1 6 0 8 4 0 10 0 11 19 2 8 6 14

Cancer Biomarker ALPHA-FETOPROTEIN 0 170 0 17 1 4 13 8 6 8 4 7 0 0 9 12 0 1 3 8 2 2 0 110 1 2 15 2 2 0 2 10 2 8 4 9 0 17 13 3 1 9 18 4

Cancer Biomarker BLADDER TUMOR ANITGEN 0 0 0 0 0 3 4 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Cancer Biomarker B-TYPE NATRIURETIC PEPTIDE 0 0 0 4 0 0 2 3 5 4 6 0 0 0 0 0 0 4 6 0 0 0 0 0 4 0 1 0 0 0 0 3 1 0 9 0 0

Cancer Biomarker CA 125 0 14 3 0 9 2 7 8 5 7 56 1 2 2 12 0 0 1 0 0 1 9 0 2 0 5 1 0 6 1 12 5 4 1 3 53

Cancer Biomarker CA 15_3 0 2 0 0 0 0 2 2 1 1 4 3 5 0 0 1 6 0 0 0 0 0 0 2 0 0 0 2 0 0 1 0 0 1 7 1 3 3

Cancer Biomarker CA 19-9 1 18 1 0 3 0 2 9 3 9 4 15 0 0 15 14 7 0 0 9 0 0 0 10 3 0 5 0 4 0 0 2 2 0 5 3 2 7 2 6 4

Cancer Biomarker CD98 0 1 0 1 0 0 0 0 4 0 0 0 0 0 0 1 0 0 0 0 1 0 2 2 0 0 0 0 5 2 5 0 1

Cancer Biomarker CEA 0 8 0 5 1 15 1 58 152 4 8 6 2 7 0 1 3 13 12 6 6 0 3 2 7 0 1 4 4 59 5 7 0 17 2 6 8 8 0 3 1 14 58 8 51 4 4 9

Cancer Biomarker FASCIN 0 2 2 1 4 2 3 6 2 11 0 0 2 6 0 2 0 2 3 1 3 5 0 0 3 0 0 4 0 3 1 5 5 2 0

Cancer Biomarker HER2_NEU 1 2 6 4 1 78 1 53 2 4 1 4 7 54 4 5 0 2 2 1 4 6 4 1 7 14 13 7 153 3 0 3 0 14 0 0 19 0 2 0 3 3 2 3 1 4 4 2 8 7

Cancer Biomarker IGF-11 0 3 2 9 10 0 2 14 14 13 7 0 0 3 0 2 7 0 0 9 2 0 0 0 10 6 9 3 11 0 1 8 0 0 14 2 1 2 3 6 14

Cancer Biomarker LEPTIN 3 2 4 3 0 71 2 1 119 6 2 19 5 0 1 3 2 3 9 0 1 12 79 1 2 1 12 5 4 7 15 2 2 1 0 2 5 0 52 7 4 3 18 16 2 3

Cancer Biomarker OSTEOPONTIN 2 2 6 4 55 2 1 10 1 2 12 10 7 0 0 9 2 3 0 0 4 9 0 6 0 18 9 14 4 3 1 1 1 6 5 0 154 19 4 3 4 3 19 4

Cancer Biomarker PROLACTIN 2 55 3 4 54 1 1 56 2 4 6 6 8 1 0 2 9 0 3 6 19 5 3 2 1 1 3 11 6 3 3 6 0 7 0 3 8 3 6 7 3 2 8

Cancer Biomarker PSA 1 56 9 9 51 1 3 0 2 9 9 3 9 9 9 0 1 6 14 0 4 4 0 9 6 2 3 2 6 1 4 2 0 6 0 10 2 2 3 2 6 6 2 13

Cancer Biomarker THYROGLOBULIN 0 3 5 4 3 1 1 10 12 9 1 0 2 2 0 14 116 0 2 0 0 0 3 5 0 1 1 0 5 0 10 12 5 7 10 0

Cancer Biomarker TROPONIN I 1 0 0 15 1 0 2 10 4 4 14 0 0 0 1 0 0 2 0 1 0 0 0 0 2 0 7 0 0 0 0 6 0 4 1 1 011

OMICS

Translational Medicine

Lab Automation Sparks Creativity

Tackling Transfection TasksThe discovery in the early 1990s that administration in mice of plasmid DNA encoding both viral and nonviral antigens induced antibody responses held out the prospect of achieving broad immunogenicity using DNA vaccines without the safety issues associated with a replicating pathogen.

Despite this promise, the efficacy of first-generation DNA vaccines against HIV, HPV, and hepatitis was compromised by low an-tibody titers and sporadic immune cell responses. In addition, DNA vaccines face broader challenges encountered by other classes of vaccines such as manufacturing, scale-up, and purification, as well as vaccine resistance.

A number of companies are developing processes and technical platforms designed see page 22

Primary mouse embryonic fibroblast strain Dba/2J cells imaged using olympus 40X/0.9na air objective lens on the bD Pathway 435 High Content Imager. Cells were labeled with Hoechst 33342 (blue), yoyo-1 (green), mitoTracker orange (red), and Cytochrome C – alexaFluor 647 (cyan). Hamner Institute

HOW EXTRAORDINARY HAPPENS

INTEGRATION

COMPATIBILITY

MODULARITY

ONE TOUCHTEACHING

FLEXIBILITY

SAMPLE PREP

SPEED CONTROL

RELIABILITY

OPTIMIZATION

PARALLELPROCESSING

CAPABILITY MULTISPANCAPABILITY

SENSING

SCREENING

HIGH THROUGHPUT

ACCURACY

NORMALIZATION

CONFIDENCE

WALKAWAY TIME

HOW EXTRAORDINARY HAPPENS IntroducIng the Agilent encore MultispAn liquid HAndling systeMwww.agilent.com/lifesciences/see-it-happen

aldevron’s Genetic Immunization and antibody (GIa™) technology is used to test new Dna vaccine techniques in animal models.

Just two decades ago we were only beginning to recog-nize the potential of automated technologies to enhance throughput in drug discovery research. Today, it is difficult to imagine a modern laboratory without robotic equipment.

“We see continuous trends toward increased reliability of robots, partially driven by the introduction of new types of motors,” reports Malcolm Crook, Ph.D., CTO, Process Analysis & Automation (PAA). “Another trend is toward smaller targeted systems, that still flexibly accommodate peripherals as needed.”

PAA will be presenting the “automate.it harmony,” its user interface for software inte-gration, at the Society for Laboratory Automation and Screening (SLAS) meeting later this month. see page 16

Kate Marusina, Ph.D.

Neil McKenna, Ph.D.

Necessary Liaisons: CROs Gain Clout &Responsibility

www.genengnews.com

22 | January 1, 2013 | genengnews.com | Genetic Engineering & Biotechnology News

to circumvent these issues, with particular emphasis on improving the efficiency of de-livery and uptake of vaccines by target cells, referred to as transfection, which can be con-founded by differences in tissue and cell type accessibility between individuals, in addition to other factors.

Many of these companies were present at last month’s DNA Vaccines conference in San Diego. The meeting was sponsored by International Society of DNA Vaccines and organized by BioConferences International, a Mary Ann Liebert company.

“Vaccines have saved more lives than any other invention in human history”, ex-plained J. Joseph Kim, Ph.D., president and CEO of Inovio Pharmaceuticals. “Conven-tional vaccines have been successfully used against the lower hanging fruit in terms of disease, and more complex diseases such as cancer represent both opportunities and challenges for DNA vaccines.

According to Dr. Kim, one of the biggest hurdles for DNA vaccines has been induc-ing sufficiently high immune responses for effective vaccination. “There are two issues at stake,” he said. “The first is to make plas-mids more ‘people-friendly’ to optimize their expression once in the cell, and the second is to optimize the rapid transfer of the DNA

across the cell membrane to avoid degrada-tion by endonucleases.

Inovio’s approach in this area includes informatically assisted optimization of the coding sequence to expand immunogenici-ty across multiple targets and boost expres-sion levels of immunogens in target cells, and to improve the delivery of the plasmid DNA into host cells to minimize exposure to extracellular nucleases. The development of one of Inovio’s candidate high-grade cervical dysplasia vaccines, VGX-3100, encoding the HPV16 and HPV18 E6/E7 antigens, incorporated a combination of these features to increase vaccine antigen immune potency.

“The VGX-3100 vaccine was developed using our SynCon platform and includes highly efficient leader and Kozak sequenc-es,” said Dr. Kim. “We also introduced an endoproteolytic cleavage site to improve protein folding and cytotoxic T lymphocyte processing.”

In addition, Inovio has developed an elec-troporation-based delivery system that, ac-cording to Dr. Kim, allows for more efficient and safe targeting of the antigenic sequence to the target cell. “A low voltage electrical field is applied at the site of vaccine injec-tion, causing the cell membranes to tran-

siently realign into a more porous state,” he explained, adding that electroporation is the most efficient and safe mode of delivery of nucleic acids, requiring no additional chemi-cals, preservatives, or adjuvants.

Looking to the future, “Electroporation is not a static technology,” continued Dr. Kim. “We are developing new devices, referred to as surface electroporators, that sit on the

OMICS

NEWS Genomics & Proteomics

> CLC bio Contributes Expertise to STATegra

CLC bio is the latest company to par-ticipate in the European FP7 project, STAT-egra. It will receive $1.3 million out of the total $7.8 million budget for its efforts. The STATegra project aims to develop an inte-gral analysis platform for different omics data that is capable of providing a more ef-ficient use of genomics technologies. This project will generate and integrate data obtained in proteomic, metabolomics, and epigenomic experiments.

“One of the big challenges today is go-ing past the raw sequence data and getting to grips with the complexity of epigenetic

processes—how, when, and why are cer-tain genes switched off or expressed?” states Michael Lappe, Ph.D., senior bioin-formatics scientist at CLC bio. “We aim to leverage a framework which makes this extremely complex network biology un-derstandable. Ultimately our tools will fa-cilitate the translation of massive amounts of data into useful insights that can be ap-plied in clinical settings.”

Within the overall project, methods for the validation and design of follow-up studies are implemented. Utilizing and val-idating existing datasets, STATegra is also going to feed its experimental results back into the public domain.

> EMD Millipore Institutes Personalized Medicine Services

EMD Millipore’s discovery and devel-opment solutions business is now offer-ing genomic biomarker services. Com-pany officials say the purpose is to aid drug companies in targeting patient sub-groups for improved clinical outcomes, understanding drug safety and efficacy, and characterizing tumors.

Researchers at EMD Millipore recently profiled transcription in gefitinib-treated non-small cell lung cancer cells by qRT-PCR. The genotypic and mRNA expression data were then cross-validated with protein

expression using flow cytometry and mul-tiplex assays, solidifying the connection between the genotype and phenotype of interest. These types of connections pro-vide confidence to preclinical and clinical decision-makers regarding drug safety and efficacy, says Christina Shasserre, vp of dis-covery and development.

> Agilent and SRI Combine Technologies for New Offering

Agilent Technologies and SRI Interna-tional signed a licensing agreement to of-fer laboratory research customers a pack-age that combines Agilent’s latest release of GeneSpring GX, GeneSpring NGS, Mass Profiler Professional, and Pathway Archi-tect version 12.5 with SRI International’s

complete BioCyc Pathway Database Col-lection. The integrated package will allow scientists to access, combine, visualize, and analyze biological datasets across multiple “omics” experiments (genomics, transcrip-tomics, proteomics, and metabolomics).

The new software package will enable re-searchers to display biological pathway data generated by Agilent’s suite of instruments, software, and reagents. Agilent products include microarrays and next-generation se-quencing technology, as well as LC/MS, GC/MS, ICP/MS, and NMR systems.

> Cancer Genome Consortium Expands China Research

The International Cancer Genome Con-sortium is expanding its research in China, where new projects seeking genomic factors in four cancer types common to the world’s most populous nation will be launched.

The projects—focusing on colorectal, esophageal, liver, and nasopharyngeal cancers—join the consortium’s two-year-old and still-ongoing Chinese project fo-cused on gastric cancer, which made new data from sequenced tumors public.

The new projects are expected to take less time than the typical four years, since the consortium began collecting tissue samples from patients with the diseases over the past two years. n

Vaccine delivery using electroporation (>10–100x enhancement in immune responses). Inovio

Transfection Continued from page 1

See Transfection on page 24

rendering of the nucleosome (Dna blue, histone proteins green) highlighting positions of known epigenetic marks in spherical representation. CLC Bio

Full-scale version of methionine metabolism pathway for Saccharomyces C., using bioCyc content and rendered visually in “simple” mode in mPP/Pathway architect 12.5; also shows average, normalized differential abundances for four conditions in the HeatStrips for some of the metabolites.

24 | January 1, 2013 | genengnews.com | Genetic Engineering & Biotechnology News

surface of the skin, and a piezo-electric-based system that doesn’t even require contact with the skin.”

Immune Escape

Annie DeGroot, M.D., CEO and CSO of EpiVax, highlighted immune escape as an important issue in the optimizing the amount of information that DNA vaccines deliver. Immune escape is thought to arise when an infectious agent or, in the case of cancer, a tu-mor, acquires a genetic profile distinct from that toward which a DNA vaccine was ini-tially targeted, allowing it to escape the im-mune pressure of the vaccine.

“In response to the problem of immune escape, our approach at EpiVax has been to combine multiple key antigens or specific epitopes within these antigens, in a single re-combinant vaccine,” noted Dr. DeGroot.

“We use in silico methods to optimize a gene sequence for improved vaccine expres-sion,” added Lenny Moise, Ph.D., director of vaccine research at the company. “They in-clude codon optimization and gene analysis, including 5´ end secondary structure, cryptic splice sites, human genome homology, bac-terial promoters, eukaryotic promoters, in-verted repeats, palindromes, tandem repeats, and nucleosome positioning.”

Similar to Inovio, Epivax’ transfection method is to rely on electroporation to en-hance uptake to improve vaccine immuno-genicity. “There are about 30 Phase I and II clinical trials using electroporation of DNA vaccines, more than any other delivery tech-nology. It represents the most promising DNA vaccine delivery method with a path to the clinic,” said Dr. Moise.

Range of Technologies

Aldevron was the first company to pro-duce a DNA vaccine used outside an ex-perimental setting, a West Nile Virus DNA vaccine that has been used to protect endan-gered species.

“There are many reasons why so few cells are transfected by DNA vaccines,” said Michael Chambers, president and CEO of Aldevron. “One potential hurdle related to DNA vaccine manufacturing includes re-sidual E. coli impurities like colanic acid,” referring to an exopolysaccharide common to many enterobacteria.

“These can cause toxicity and overall negative effects that hurt transfection effi-ciency,” he pointed out. In addition to im-purities in the vaccine formulation, plasmid methylation patterns can also play a role in low transfection and expression. Other hur-dles include issues related to vector size, re-gional micro environments around the DNA injection site, and the inherent toxicology of most compaction/transfection reagents.

“Aldevron has developed or in-licensed advanced manufacturing and purification systems to increase in vivo transfection ef-ficiencies,” said Chambers, citing the use of Nature Technology’s HyperGro plasmid fermentation technology that enables them

to produce large amounts of ultrapure DNA vaccines.

“Aldevron is also working with compa-nies like Sekris Biomedical to develop new host strains that enable scientists to fine-tune the methylation patterns of the plastids they use for immunization,” he added.

Finally, Aldevron’s genetic immunization and antibody service allows our clients to try multiple delivery technologies like Ichor’s TriGrid electroporation system and Pharma-jet’s needle-free delivery system to find the optimal way to administer their vaccines,” he noted.

Complex Formulations

Since cells prohibit entry to large mol-ecules like DNA, unique delivery and for-mulation technologies must be fine-tuned to optimize the transfection and delivery of DNA vaccines.

“Technologies for delivering DNA vac-cines include liposomes, polymers, or elec-troporation,” noted Magda Marquet, Ph.D., co-founder and co-chairperson of Althea Technologies. “However, these methods are often cumbersome for patients to receive, and present difficulties in manufacturing and scale up.”

Althea is pursuing complex formulations, such as polymers and liposomes, to over-come the obstacle of DNA vaccine delivery by delivering DNA directly to cells. Some formulations have challenging requirements.

Dr. Marquet cited one case in which asep-tic processing was required throughout the formulation since product could not be ster-ile filtered prior to filling. “Althea successful-ly scaled up the process and brought in GMP formulation equipment that allowed the li-

posomal complex to be processed aseptically and filled into vials,” she explained.

Martin Schleef, Ph.D., CEO of the Plas-midFactory, echoed Chambers’ concerns over the high standard of purity required of DNA for delivery in humans, and cited a recent study (Woodell et al., J. Gene Med), which demonstrated that chromosomal bac-terial DNA, which is typically present in kit-grade plasmid DNA preparations, is a sig-nificant contaminant leading to low efficacy and severe toxic effects.

“We have developed a manufacturing technology to avoid such contamination,” said Dr. Schleef. In addition, he highlighted plasmid topology as an important parameter in determining transfection efficiency. Cova-lently closed circular (ccc), or supercoiled, DNA adopts a compact form due to internal tensions in the DNA molecule that is optimal for transfection efficiency.

“Ideally the proportion of ccc-DNA in the preparation should be 95% or higher,” said Dr. Schleef, “and we use a manufactur-ing technology to obtain pure preparations of this form.”

PlasmidFactory has also developed a capil-lary gel electrophoresis-based analytical tool to quantify the major plasmid topologies in a given plasmid preparation, namely ccc-DNA, oc (open circular)-DNA, and linear DNA. In addition to fine-tuning the conformation of DNA for transfection, stripping plasmids of genes encoding resistance to antibiotics or other selection markers is an important step for preparing DNA for transfection.

“We have designed strategies to remove such genes from the bacterial backbone of the plasmids used, in addition to the origin of replication, resulting in a simply circular

and extremely small DNA for vaccination, the minicircle,” said Dr. Schleef.

According to Dr. Schleef, PlasmidFactory has obtained all relevant patents for this technology and supplies researchers in gene therapy and DNA vaccination worldwide with this safe, nonviral vector. PlasmidFacto-ry is also tackling the issue of scaling of plas-mid preparations to the amounts required for more demanding applications, e.g., large clinical trials.

“We are developing technologies for ultra-large scale production of plasmid DNA (e.g., kg scale) by use of fed-batch and large scale lysis to obtain pure ccc-plasmid-DNA,” he said.

Critical Factor

As both Chambers and Dr. Schleef have pointed out, the purity of a plasmid prepa-ration is a critical determinant of successful vaccine transfection or delivery.

“If you don’t start with pure DNA, you can forget about reproducible and effective transfection,” said Bill Kuhlman, vp North America for BIA Separations. “Removal of endotoxins and genomic DNA is common-ly achievable at small scale, but producing DNA vaccines at commercial scale requires process steps that can efficiently produce tens of grams of highly pure supercoiled plasmid DNA per run.”

BIA Separations specializes in separa-tions, with particular emphasis on monolith-ic HPLC columns. Rather than traditional bead-based separation columns, monolithic columns are composed of an organic or in-organic substrate and multiple highly perme-able and porous channels that afford a large surface area to the stationary phase.

“We’ve achieved homogeneity of greater than 97% supercoiled DNA with greater than 99% removal of host DNA, protein, and RNA at development through industrial scales,” Kuhlman pointed out. BIA Separa-tions has recently introduced large-scale disposable Monolith columns allowing 48 grams of super coiled DNA to be purified in a single run, while maintaining the purity and recovery seen at small scale.

In addition to manufacturing and deliv-ery, another important consideration for DNA vaccines, like any other candidate ther-apeutic, is safety. The potential integration of DNA vaccines into the host cell genome is of concern due to the possibility of insertional mutagenesis resulting in the inactivation of tumor suppressor genes or the activation of oncogenes in the host genome.

According to Dr. Marquet, “DNA vac-cines have been proven to be safe in multiple studies, with no integration of the DNA into the host chromosomes ever being observed.”

Her assessment is reinforced by Dr. Kim from Inovio Pharmaceuticals. “We have vaccinated or treated over 600 patients with good safety and tolerability profiles,” he said. “Adverse events are typically mild to moderate, and any injection sites resolve without sequelae.”

Transfection Continued from page 22

OMICS

CImmultus columns

for Dna plasmid

purification at all

scales

BIA Separations

althea reports that

its plasmid Dna

manufacturing

processes have been

successfully scaled

up to the 1,000 l

level. Plasmid Dna

produced in althea’s

1,000 l fermentor

can yield millions

of vaccine doses,

according to the

company.