Poster 1 CHARACTERIZATION OF A NOVEL PROTEIN ANTIVIRAL

192 Pages·2014·1.42 MB·English

CHARACTERIZATION OF A NOVEL PROTEIN ANTIVIRAL FROM LONOMIA OBLIQUA USING
BIOINFORMATICS TOOLS AND ACTIVITY ANALYSIS BY REAL TIME

Ana Carmo
Laboratory of Parasitology and Entomology, Instituto Butantan, Brazil
e-mail: [email protected]

1Giovanni, D.N.S., 2Yamasaki, L. H. T., 3Figueiredo, C. A., 3Oliveira, M. I., 3Santos, F. C. P., 4Curti, S. P.,
1Tonelotto, M., 2Rahal, P., 5Moraes, D., 1Mendonça, R.Z.

1Laboratory of Parasitology and Entomology, Instituto Butantan, Brazil
2Laboratory of Genomic Studies, IBILCE-Unesp, Brazil,
3Center for Respiratory Diseases, Center for Virology, Instituto Adolfo Lutz, Brazil
4Center for Vector Transmitted Diseases, Center for Virology, Instituto Adolfo Lutz, Brazil
5Faculdade Oswaldo Cruz, Brazil

The control of viruses is of great interest to the public health area. Several studies have been conducted
that show the presence of pharmacologically active substances in the hemolymph of insects. Recently we
have demonstrated the existence of a potent antiviral in the hemolymph of Lonomia obliqua caterpillar.
This protein was able to reduce at 106 times the replication of herpes virus and in 10,000 fold the rubella
virus. Assays using RT-PCR to determine viral RNA present in no treated and rAVLO treated infected
cells also showed a reduction in the same scale. The analysis of this protein by bioinformatics suggests
that this protein is globular, secreted with a signal peptide which is cleaved between amino acids 16 and
17. The studies also allow us to infer that this antiviral protein has the ability to bind to MHC class I. It was
found that there are several protein binding sites on the weak and strong bases with various HLA. The
bioinformatic analysis also shows a strong presence of α-helices in the N-terminal region and allowed to
classify the antiviral protein as α/β type of structure, as we detected the presence of more than 30% α-
helix and 20 % of β-sheet found separately along the protein chain. In the BLAST sequence analysis of
cDNA antiviral protein, no sequence similarity was found in Genbank, suggesting that it is from a novel
protein family. It can be inferred by an analysis of this region that the possible antigenicity region would
be between the 70-110 amino acids, showing high accessibility. This high antigenic region on the surface
can be a possible region to interaction with other proteins.
Financial support: FAPESP (08/57263-5), CAPES.

Poster 1
ADVANTAGES AND CHALLENGES OF DEVELOPING BISPECIFIC ANTIBODY BIOTHERAPEUTICS

Xiaojian (David) Zhao
YZY Biopharma
[email protected] and [email protected]

Bispecific antibody biotherapeutics are on the frontier of biotherapeutic development. By binding multiple
targets and activating therapeutic mechanisms, they present great potential as new biotherapeutics. YZY
has established technology platforms and IPs on developing the pipeline of bispecific antibody
biotherapeutics.

Although many biopharmaceutical companies have spent significant money, resource and time
developing biospecific antibody biotherapeutics, it has been very challenging to develop them for major
medical indications due to their complex structures and multiple functional mechanisms. YZY has
overcome many challenges in developing bioprocess platforms and anticancer biospecific antibodies (Y-
Body®) that interact with specific cancer targets as well as human T lymphocyte target. Our works and
successes on expression, purification and quality improvement on several biospecific antibodies (Y-
Body®) will be presented. These efforts and achievements represent the most advanced technologies
and processes in biotherapeutic development.

Poster 2
OPTIMIZATION OF HIV-1 VIRUS-LIKE PARTICLE PRODUCTION IN CAP-T® CELL SYSTEM

Francesc Gòdia
Departament d’Enginyeria Química
Universitat Autònoma de Barcelona
[email protected]

S. Gutiérrez-Granados, L.Cervera, M.M. Segura
Departament d’Enginyeria Química
Universitat Autònoma de Barcelona

Upon expression, the Gag polyprotein of HIV-1 spontaneously assembles giving rise to enveloped virus-
like particles (VLPs). These particulate immunogens offer great promise as HIV-1 vaccines without any
associated biosafety concerns as they do not contain the viral genome and they are non-infectious. Insect
and mammalian cells are the most used systems for HIV-based VLP production as they provide the
appropriate post-translational modifications. In this work, for the first time, CAP-T cell line has been used
for Gag-based VLP production by transient transfection. CAP-T presents all the desirable features for
therapeutic protein production: human post-translational modifications, suspension and serum-free cell
growth, high cell densities and GMP compliance.

Generation of VLPs was carried out by transient transfection of CAP-T cells using PEI as transfection
reagent. A plasmid coding for Gag polyprotein fused to GFP was used for the generation of fluorescent
VLPs. Gag-GFP VLPs of proper quality and quantity were produced in CAP-T cells. Correct assembly
and budding of VLPs into the cell culture supernatant was observed upon transfection by confocal
fluorescence microscopy. Spherical virus particles surrounded by a lipid envelope with a size consistent
with immature HIV particles (130 nm) were observed by transmission electron microscopy (TEM).
Nanoparticle tracking analysis (NTA) was also used for VLP characterization. Around ~3-5×1010
VLPs/mL were produced in batch culture at 72 hours post-transfection. Further optimization of VLP
production with CAP-T cells platform will be reported. CAP-T is a suitable and promising cell line for the
production of HIV-1-derived VLPs with sufficient quality and quantity for pre-clinical studies.

Poster 3
DEVELOPMENT OF AN EXPRESSION PLATFORM
FOR ALTERNATIVE SCAFFOLD THERAPEUTICS

Kevin D. Smith
Janssen, USA
[email protected]

Fnu Aapjeet, Janssen, USA
Victoria Hull, Janssen, USA
Subinay Ganguly, Janssen, USA
Steven Lang, Janssen, USA

The Biologics Research group at Janssen is developing a novel class of proteins for use as alternative
scaffold therapeutics. These are small, 10-20kDa, highly-stable proteins that have the potential to
develop new drug candidates with superior efficacy and differentiated properties from typical biologics.
They can be engineered with multiple binding specificities, targeted to specific tissues, and used for
diagnostic and therapeutic applications. Additionally, they are simple molecules ideally suited for
expression in E. coli at high levels in a soluble form.

To support the process development and manufacturing of our alternative scaffold therapeutics, we have
developed an in-house proprietary E. coli expression platform. To develop this system, common E. coli
manufacturing strains were first screened to identify a host with high expression, superior product quality
and fermentation performance. Then, chemically-defined batch and feed mediums were developed
through engaging in a collaborative media development process with external partners and internal
fermentation systems. Finally, fermentation process parameters that maximize growth in our E. coli strain
of interest were established. The resulting fed-batch fermentation process supports high cell-density
growth and alternative scaffold expression, reaching peak titers of >8 g/L and OD >200 in a 20h
fermentation run.

The combination of the potential superior efficacy and differentiated properties, coupled with a robust
proprietary production process, makes these alternative scaffold proteins an important therapeutic
platform in the Janssen pipeline. This poster will describe the nature of the alternative scaffold molecules
and the process by which the expression platform was developed.

Poster 4
A SCALABLE IN VITRO SYSTEM FOR THE GENERATION OF LARGE QUANTITIES
OF STEM CELL DERIVED HEPATOCYTES

Ravali Raju
Department of Chemical Engineering and Material Science, University of Minnesota, USA
[email protected]

David Chau1, Catherine Verfaillie2 and Wei-Shou Hu1

1Department of Chemical Engineering and Material Science, University of Minnesota, USA
2Stamcel Instituut, Katholieke Universiteit Leuven, Belgium

Liver failure is a major cause of mortality for which the only cure is self-regeneration or transplant of organ
or primary hepatocytes. The transplant option is severely limited by the shortage of organ donors and
primary hepatocytes. Stem cells have been explored as alternative source of hepatocytes. These stem
cell-derived hepatocytes, in addition to being used for the treatment of liver disease, may also be valuable
for drug discovery and toxicity evaluation. Using growth factors to guide stem cells towards endoderm
and then hepatic fate, we have established a process of deriving hepatocyte-like cells that express key
hepatic markers and functions. The key challenges with this protocol are the presence of heterogeneous
cell populations after differentiation as well as limited functional maturity when compared to primary
hepatocytes.

Clinical applications such as transplantation or bio-artificial liver devices will entail the use of
approximately 109 cells. Therefore, we explored the possibility of expanding progenitor cells arising during
various stages of hepatic differentiation to increase the number of hepatocytes generated. Endodermal
cells were successfully expanded up to 8 fold, then continued to differentiate to hepatocyte-like cells. In
vitro expansion of progenitor cells allow for enrichment of hepatic cells while eliminating undifferentiated
cells. Furthermore, this strategy reduces the reliance on large populations of embryonic stem cells. These
progenitor cells can be expanded to even larger scales by employing suspension-based culture
techniques. Finally, transcriptome data collected during various time points of differentiation has aided in
identifying key genes and pathways changing throughout the process. Such analysis can provide insights
into the liver development process and enable modification of key genes to further increase the functional
maturity of hepatocytes. Thus, this system allows for generation of large quantities of functional stem-cell
derived hepatocytes to meet the demands imposed by various clinical applications in the near future.

Poster 5
IDENTIFICATION OF A NOVEL ANTIVIRAL PROTEIN FROM PHYLLOCAULIS BORACEIENSIS
MUCUS, AND ACTIVITY ANALYSIS BY REAL TIME PCR

Ronaldo Mendonça.
Laboratory of Parasitology and Entomology, Instituto Butantan, Brazil
[email protected]

2Figueiredo, C. A., 2Oliveira, M. I., 3Curti, S. P., 1Ana Rita de Toledo-Piza

1Laboratory of Parasitology and Entomology, Instituto Butantan, São Paulo/SP,
2 Center for Respiratory Diseases, Center for Virology, Instituto Adolfo Lutz, São Paulo/SP, 3Center for
Vector Transmitted Diseases, Center for Virology, Instituto Adolfo Lutz, São Paulo/SP.

The control of human viruses is of high interest in human and animal health. Several works have
demonstrated the presence of bioactive peptides with therapeutic action in hemolymph or extracts of
insect. However, relatively little data are available on molecules from insects with antiviral activities. The
mucus of Phylocaullis boraceiensis, a Brazilian terrestrial slug, has being studied as a natural compound
with several biological activities. This study proposes the bioprospecting of molecules
with antiviral activity in Phyllocaulis boraceiensis mucus. In this study, the effects of mucus in the infected
culture were investigated. The effect on the virus replication was measured on confluent monolayers of
infected cells with measles, influenza, herpes, rubella (enveloped virus) and picornavirus (non enveloped
virus). The cultures were observed daily for evidence of cytopathic effect. The addition of 1% of mucus
significantly decreased the virus titer (p=0.002). The antiviral substance responsible for this activity was
isolated and purified by gel filtration chromatography and further fractionated using an ion exchange
column system. The analyses of the viral replication by mRNA determination also were performed by
qPCR analysis. The results showed that mucus led to a 256-fold reduction of measles virus and a
reduction of 128-fold in influenza virus production. When the mucus was used in a picornavirus, the
reduction observed in virus replication was of 2.187-fold. Assays using RT-PCR to determine viral mRNA
present in infected cells, treated with mucus, showed that purified antiviral substance was able to reduce
at 103 times the replication of herpes and rubella virus. Heating and freezing seem to have no influence
over its antiviral activity. The protein does not display virucidal activity and does not act on receptors on
the cell membrane. The observations suggest the presence of a potent antiviral substance in mucus
which affects the innate antiviral immune response.

Key words: Antiviral, Phyllocaulis boraceiensis, Measles virus, Herpes virus, Rubella virus, Influenza virus

Financial support: FAPESP (12/22555-1 and 12/906-9).

Poster 6
A SERUM-FREE ADAPTED CV-1 CELL LINE AS A POTENTIAL HOST CELL LINE
FOR AN ONCOLYTIC VACCINIA VIRUS PRODUCTION

S. Liu
The Advanced Center for Biochemical Engineering, Department of Biochemical Engineering, University
College London and Centre for Molecular Oncology & Imaging, Institute of Cancer, Barts and the London
School of Medicine and Dentistry, Queen Mary University of London, UK
[email protected]

Y. Wang(2), D. Nesbeth (1), L. Ruban(1), Y. Zhou (1)
(1) The Advanced Center for Biochemical Engineering, Department of Biochemical Engineering,
University College London

(2) Centre for Molecular Oncology & Imaging, Institute of Cancer, Barts and the London School of
Medicine and Dentistry, Queen Mary University of London, UK

Oncolytic virotherapy is a powerful treatment which employs replication-competent viruses to destroy
malignant cells. It has emerged as a new generation therapeutic approach in cancer along with its
accumulated clinical safety data and demonstrated anti-tumoural effects over the past decades. Thus, to
develop an efficient, reproducible and scalable manufacturing process that enables high yield production
has become an issue to be considered in preparation for industrial scale oncolytic viral vaccine mass
production.

In this study, a continuous green monkey fibroblast cell line, CV-1 cell line was found to produce higher
titers of a genetically engineered, tumor-targeting Lister strain vaccinia virus (VVL-15 RFP), at laboratory
scale than Vero cell line which has been extensively characterized and largely used in the manufacture of
viral vaccines. We demonstrated the successful adaptation of CV-1 cells from growth in serum containing
medium (SCM) DMEM supplemented with 5% FBS to growth in two serum free media (SFM), VP-SFM
and OptiPRO SFM. CV-1 cells in OptiPRO SFM exhibited a strong growth performance with the shortest
cell doubling time and highest plating efficiency among cell growth in the three media. High and
comparable recovery ratios were obtained from revitalized cells after being cryopreserved in OptiPRO
and SCM respectively in liquid nitrogen for over 6 months. Furthermore, the virus titres produced in
OptiPRO SFM were found to be 4.6 times higher than the SCM culture and 2.6 times higher than the VP-
SFM culture at 72 hours post-infection. Last but not least, preliminary results have shown that CV-1 cells
adapted to OptiPRO SFM were able to attach to and grow on Cytodex 1 microcarriers under static culture
conditions.

These results suggest that the adapted CV-1 cell line in OptiPRO SFM has the potential in the
development of a large scale microcarrier cell culture for mass production of the oncolytic Lister strain
vaccinia virus.

Key-words: oncolytic vaccinia virus, viral vaccine production, serum free medium, process development,
scale-up

Poster 7
IMPACT OF LC:HC RATIO ON STABLE IGG EXPRESSION AND QUALITY

Steven C.L. Ho
Bioprocessing Technology Institute (A*STAR) and Division of Bioengineering,
Faculty of Engineering, National University of Singapore
[email protected]

Miranda van Beers, Bioprocessing Technology Institute (A*STAR)
Monika Mueller, Bioprocessing Technology Institute (A*STAR)
Zhiwei Song, Bioprocessing Technology Institute (A*STAR)
Muriel Bardor, Bioprocessing Technology Institute (A*STAR) and University of Rouen
Yuansheng Yang, Bioprocessing Technology Institute (A*STAR) and School of Chemical and Biomedical
Engineering, Nanyang Technological University

Key Words: CHO cells, monoclonal antibody, cell line generation

Immunoglobulin G (IgG), the most common class of commercial monoclonal antibodies (mAbs), exists as
multimers of two identical light chains (LC) and two identical heavy chains (HC) assembled together by
disulfide bridges. Due to the kinetics of mAb assembly, having LC and HC in equal amounts is not optimal
for expression.

We designed a set of vectors using internal ribosome entry site elements to control LC: HC expression at
four different ratios of 3.43, 1.24, 1.12, 0.32 in stably transfected mAb expressing Chinese hamster ovary
(CHO) cell pools. Having excess LC aided in the effectiveness of amplification and generated the highest
mAb titers among the transfected pools. Majority of the secreted product were the desired mAb
monomers at all LC:HC ratios above one. N-glycosylation profile and conformational stability were also
similar for those ratios. For pools with low LC: HC ratio 0.32, monomers only constitute half of the product
with the other half being aggregates and incomplete mAb fragments. High mannose type N-glycans
increased while fucosylated and galactosylated glycans decreased significantly at the lowest LC: HC
ratio. Product stability was also adversely affected.

Having LC in excess is the most beneficial for both mAb expression and quality. This report is the first
demonstration that LC: HC ratio can affect mAb production and quality in stably transfected CHO cells.
Observations made in this study should be taken into consideration in future when designing vectors for
mAb cell line generation and quality by design projects.

Poster 8
PARALLELIZED CONSTRUCT SCREENING AND SCALE-UP OF FULL-LENGTH MEMBRANE
PROTEIN EXPRESSION FOR BIOPHYSICAL STUDIES

Georg Schmid
Hoffmann-La Roche, pRED Pharma Research & Early Development, Discovery Technologies,
Switzerland
[email protected]

The production of integral membrane proteins (e.g. GPCRs, transporters, ion channels) suitable for
biophysical studies e.g. crystallization/biostructure or SPR-based assays is still a challenge. It is
mandatory to optimize protein yield and homogeneity during expression and subsequent detergent
solubilization. Membrane proteins have been purified from insect cells, transient transfections and stable
mammalian cell lines.

We have established a parallelized 24-deepwell plate (DWP) based expression platform for performing
construct screening and evaluating expression conditions for membrane proteins in transient
transfections. The platform’s throughput is suitable for handling several 10s to 100s of constructs or
conditions. 24-DWP cultures are compared to spin tube, shake flask and pilot scale fermentations.
Expression and purification scale-up to stirred tank and Wave bioreactors is reproducibly demonstrated.
Coupling our 24-DWP system to a Guava FACS analyzer equipped for handling 96-well plates allows for
quantification of membrane proteins via GFP tag or antibody labelling. Solubilized membrane protein
quality is then ultimately analyzed by fluorescent size-exclusion chromatography (FSEC).

Selected results and solutions to technical challenges will be presented for cell culture and FACS analysis
including the assessment of the following factors (general expression parameters for deepwell plates,
post-transfection conditions (temperature, HDAC inhibitors, etc.), synchronization of cell population at
time of transfection, different host cell / vector combinations, overall reproducibility, cell aggregation
issues, etc.). The predictive value of FACS analyses for membrane protein quantity and quality as
assessed by FSEC will be highlighted.

Poster 9
IMPROVING PRODUCT SAFETY PROFILES: HOST CELL LINES DEFICIENT IN CMP-N-
ACETYLNEURAMINIC ACID HYDROXYLASE (CMAH) AND ALPHA-1-3-
GALACTOSYLTTRANSFERASE (GGTA1)

Joaquina Mascarenhas
Cell Sciences and Development, SAFC, Sigma-Aldrich, USA
[email protected]

Achtien, K., Cell Sciences and Development, SAFC, Sigma-Aldrich, USA
Richardson, S., Cell Sciences and Development, SAFC, Sigma-Aldrich, USA
Sealover, N., Cell Sciences and Development, SAFC, Sigma-Aldrich, USA
Borgschulte, T., Cell Sciences and Development, SAFC, Sigma-Aldrich, USA
George, H., Cell Sciences and Development, SAFC, Sigma-Aldrich, USA
Kayser, K, Cell Sciences and Development, SAFC, Sigma-Aldrich, USA
Lin, N, Cell Sciences and Development, SAFC, Sigma-Aldrich, USA

Post-translational modifications have been shown to affect the bioactivity, clearance rates,
immunogenicity and safety profiles of therapeutic glycoproteins. For e.g. anti- N-glycolylneuraminic acid
(Neu5Gc) antibodies in humans can interact with Neu5Gc sialylated therapeutic proteins (e.g. Cetuximab)
produced in non-human mammalian expression systems causing clinical complications. This is because
of an inactivating mutation in humans of the gene cytidine monophosphate-N-acetylneuraminic acid
hydroxylase (Cmah): an enzyme responsible for Neu5Gc biosynthesis from the N-acetylneuraminic acid
(Neu5Ac) form of sialic acid. Another example of an immunogenic modification on N-glycans is the
addition of a terminal galactose- α-1-3 galactose moiety (or α-Gal) mediated by the α-1,3
galactosyltransferase (GGTA1) gene. r-Proteins that carry this epitope can elicit strong immunogenic or
anaphylactic reactions due to the presence of circulating anti-α Gal antibodies present in most humans.

In this work, we describe the targeted deletion of the genes Cmah and GGTA1 in the CHO host cell line
(SAFC CHOZN® GS-/-), thus allowing for the production of therapeutic proteins lacking the Neu5Gc and α-
Gal immunogenic species respectively. The gene modifications had no significant effect on the growth or
productivity of the cells.

Prior to this, a range of r-protein producing CHOZN® GS-/- clones were tested for the expression of the
Neu5Gc and the α-Gal moieties. Theoretically, the increase in higher order glycosylation structures e.g
G1/G1F or G2/G2F would result in an increase of terminal sialic acid and α-Gal. We treated small scale
fed-batch cultures with a media supplement designed to increase higher order glycosylation structures,
and tested for the presence of Neu5Gc and α-Gal. α-Gal was non-detectable in all CHOZN® GS -/- host
cell line backgrounds. The levels of Neu5Gc were found to be r-protein and clone dependent, varied
depending on the culture and process conditions, and were also found to strongly correlate with the levels
of total sialic acid on the glycosylated protein. Subsequently, the gene encoding Cmah was also knocked
out in a recombinant protein producing cell line and the absence of Neu5Gc in the knock out clones was
confirmed under different process and culture conditions.

While changes in process and culture conditions can be used to manipulate the levels of Neu5Gc and α-
Gal immunogenic species, in this work we have taken the approach of the complete elimination of these
species by knocking out the relevant genes. These results are the first steps towards host cell
engineering to improve product quality specifically the safety profiles of proteins produced in them.

Poster 10