When expressing protein, there are a variety of expression systems to choose from. CHO is seen as the ‘king’ in the industry, but does it meet all needs of rapid production and budget constraints? This meeting will explore Optimizing Protein
Expression through understanding and enhancing expression systems, and will especially focus on CHO cells and other mammalian systems, E.coli and yeasts. Other host systems, such as baculovirus and algae, will be touched on as well. Along
with case studies, the meeting will feature experts who reveal the underlying mechanisms and insights into the varying systems in order to enhance protein expression. Comparing and contrasting systems will also be featured to provide greater understanding
of how these systems function within the context of the results achieved.
WEDNESDAY, MAY 6
7:00 am Registration and Morning Coffee
8:00 Chairperson’s Remarks
Christopher W. Kemp, Ph.D., President, Kempbio, Inc.
8:10 KEYNOTE PRESENTATION
Innovations in Protein Expression Technologies to Deliver New Biotherapeutics
Pranhitha Reddy, Ph.D., Director, BioProcess & Analytical Sciences, Seattle Genetics,
Advances in protein expression and related technologies have enabled the establishment of expression platforms and flexible manufacturing to help deliver high yield, rapid development timelines and desired product quality profile. In addition, protein
and process engineering efforts have led to development of new bio therapeutic modalities, including high potency molecules. Examples of different antibody therapeutics, their development challenges, and innovations to meet these challenges will
be reviewed. The evolving use of new information on genome structure and cell metabolism for expression optimization will be discussed.
8:40 Featured Presentation:
Use of Mammalian Expression Systems for Enhanced Protein Production
Lovisa Holmberg-Schiavone, Ph.D., Team Leader, Reagents
and Assay Development, AstraZeneca R&D
The Reagents and Assay Development group in AstraZeneca is responsible for delivering proteins for all project needs. Several different expression systems such as E. coli, insect cells, Pichia pastoris and different mammalian expression
systems are utilized to cover as many protein targets as possible. Transient expression of heterologous proteins in mammalian systems is a powerful way to generate secreted protein reagents quickly and can also be used for intracellular and membrane
protein targets. To increase success rates with mammalian systems we have recently modified our processes to make robust and economic protocols that could be used at many different scales with minimal change. Improvements included different transfection
protocols, culture conditions, feed of cultures, temperature shifts to maximize yields and protein engineering techniques to increase expression levels and purified protein yields. These changes address licensing costs, scale issues or logistical
challenges and also include the use of factorial experimental screening via a design of experiments (DoE) approach.
9:10 Using the Endoplasmic Reticulum as a Physiological Test Tube: Predicting Poor Solution Behaviors of mAb Clones during Transient Expression in cellulo
Haruki Hasegawa Ph.D., Principal Scientist, Therapeutic Discovery, Amgen, Inc.
Have you picked a lead candidate mAb clone that looked very promising until tested in formulation? How can we avoid selecting physicochemically unfavorable mAb clones unknowingly? In this talk, I will discuss the predictive values of overexpression-induced
cellular phenotypes in assessing the solution behavior properties of individual mAb clones. Our approach paves the way for a preemptive elimination of unfavorable mAb clones from the lead panel from the very beginning.
9:40 Featured Presentation:
MicroRNAs: Targets for Improving CHO Cells as Protein Factories
Colin Clarke, Ph.D., Bioinformatics Research Fellow, National Institute for Cellular Biotechnology (NICB), Dublin City
MicroRNAs (miRNAs) have emerged as an exciting means of engineering the expression of multiple proteins or even entire pathways to build better CHO cells. A number of studies have reported the association of miRNAs with desirable industrial phenotypes
and demonstrated how the manipulation of miRNA expression levels can improve CHO cell culture performance. This presentation will give an overview of the current state-of-the-art in the field.
10:10 GeneOptimizer Program-Assisted cDNA Reengineering Enhances sRAGE Autologous Expression in Chinese Hamster Ovary Cells
Li Lin, Ph.D., Senior Research Fellow, Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health (NIH)
Soluble receptor for advanced glycation end products (sRAGE) functions as a decoy to counter-react RAGE signaling-resultant pathological conditions, and has high therapeutic potential. Our studies showed that recombinant human sRAGE expressed in CHO cells
is modified by specific N-glycosylation, and exhibits higher bioactivity than that expressed in other host systems. We reengineered sRAGE cDNA using the GeneOptimizer program and the resultant cDNA augmented sRAGE expression over 2 fold and maintained
10:40 Coffee Break in the Exhibit Hall with Poster Viewing
11:25 Hijacking E. coli’s Heat-Shock Response Enhances Recombinant Protein Production
Xin Zhang, Ph.D., Burroughs Wellcome–CASI Fellow, Scripps Research Institute
The production of high-yield and high-quality recombinant proteins from Escherichia coli is highly desirable for both academic and industrial settings. In this talk, I will describe a generally applicable method for this purpose, using a
transcriptionally reprogrammed E. coli by overexpressing a heat-shock response transcription factor. Similar strategies of hijacking stress-responsive pathways should be useful to enhance cellular protein folding capacity and improve
recombinant protein production in other cell types.
11:55 CANCELLED TALK: Joseph D. Kittle, Jr., Ph.D., Assistant Professor, Chemistry and Biochemistry, Ohio University; Founder, Molecular Technologies Laboratories LLC
Please visit other conferences.
NOTE: Seats will fill up for the lunches; please come early to get a seat.
Luncheon Presentation I:
Novel Fungal Ultra-High Performance Protein Production Platform for Therapeutic Proteins and Its Application to Site Specific Antibody Drug Conjugation
Juhani Saarinen, CEO, Ilmo Biopharmaceuticals
Novel protein production platform for therapeutic proteins is presented. Current strains are optimized for antibodies, antibody fragments and certain non-glycoproteins. The host yields from several grams/liter to double digit titers in short process
times in simple microbial bioreactors. All production strains are glycoengineered and are devoid of fungal type O-glycosylation. N-glycosylation engineered strains offer humanized N-glycans as well as novel opportunities in efficient site specific
coupling of payloads to antibodies. An ADC candidate is presented.
Luncheon Presentation II:
Superior Protein Yields in CHO and HEK-293 Cells Using a Novel, Highly Efficient Transfection Reagent – FectoPRO
Jelena Vjetrovic, Ph.D., Bioproduction Technical
Support Specialist, Polyplus-transfection
Low transfection efficiency of CHO cells is a major bottleneck hampering Transient Gene Expression (TGE). Polyplus-transfection®, with its 10+ year expertise in transfection, has developed a novel technologically advanced transfection solution
specifically designed for bioproduction. FectoPRO™ outperforms currently available PEI-based and lipid-based transfection reagents. We will present data and protocols leading to unmatched protein and antibody yields in CHO and HEK-293 cells.
1:55 Session Break
2:10 Chairperson’s Remarks
Sam Ellis, Vice President, Thomson Instrument Company
2:15 Baculovirus Expression of HSV-2 Vaccine Antigens: Challenges and Solutions
Rajiv Gangurde, Ph.D., Associate
Director, Protein Production, Genocea Biosciences, Inc.
GEN-003 is a therapeutic HSV-2 subunit vaccine containing two antigens expressed independently in insect cells. The antigens were expressed as Histidine-tagged proteins as part of a successful Phase 1 campaign. For Phase 2 and beyond, we eliminated
Histidine-tags and revised the expression parameters to significantly improve protein titer and control antigen-specific proteolysis. The approaches employed and resulting outcomes will be discussed.
2:45 Production of Antibody Toxin Fusions as a Next-Generation Targeted Therapy Using Algae Chloroplast
Miller Tran, Ph.D., Senior Scientist, Lead Discovery, Verdant Therapeutics, Inc.
Over the last decades, targeted antibody therapies including antibody-drug conjugates and recombinant immuntoxins have garnered increasing attention. However, their production has become increasingly complicated and expensive. To overcome the
challenges associated with the production of targeted therapies, eukaryotic algae are being used to produce recombinant antibody toxin fusions that overcome the shortcomings of established technologies. With media cost in the cents per liter,
the potential of algae are now being realized.
Cell Line Development Tool Box for Expression: E.coli, HEK293, CHO, Insect Cells
Sam Ellis, Vice President, Thomson Instrument Company
The conditions for E.coli, HEK293, CHO and Insect Cell lines need to be maintained at small scale and within fermentation. Data will be presented on techniques and technology that allow for mimicking large scale fermentation with non-controlled
devices from 1mL-3L. All of these techniques are proven technologies for protein production, structural biology, and can lead to successful transfer from different protein groups.
3:45 Refreshment Break in the Exhibit Hall with Poster Viewing
4:45 Problem-Solving Breakout Discussions
Clonality of Production Cell Lines: How are We Addressing Product and Patient Safety to Meet Regulatory Expectations?
Moderator: Pranhitha Reddy, Ph.D., Director, BioProcess & Analytical Sciences, Seattle Genetics, Inc.
- Addressing regulatory concerns in introducing new technologies during cell line development and process development.
- Creating value for patients – contribution of titer and speed in cell line development
- Challenges in defining and controlling critical quality attributes in early upstream development.
Next-Generation Expression Platforms and Production of Complex Multiple Domain Proteins
Moderator: Miller Tran, Ph.D., Senior Scientist, Verdant Therapeutics
Novel multiple domain proteins show a great deal of promise. Recently, proteins such as the Bispecific T-cell engaging proteins (BiTE) antibodies have demonstrated their potential. Still many hurdles remain to bring these next generation proteins
to market. This discussion will center around the specific cellular machinery that is necessary to produce different classes of multi-domain proteins, protein design to help reduce downstream processing, and finally formulations to help stabilize
- biological machinery required to facilitate folding and assembly of complex proteins
- In cell accumulation vs secretion
- preventing protein aggregation in next generation systems
- linker optimization
Choosing the Right mAb Clones as Lead Candidate from the Very Beginning
Moderator: Haruki Hasegawa Ph.D., Principal Scientist, Therapeutic Discovery, Amgen, Inc.
- Individual mAb clones are certainly not created equal in terms of expression level, solution behavior, aggregation propensity, etc.
- What other aspects are not equal?
- Can we choose the lead candidate by the therapeutic efficacy alone?
- Fixing problems by lead optimization vs. choosing the right one to save time and resource. Can we do that?
- The signs of biosynthetic difficulties for certain mAb clone (secretion level, cell viability, cell morphology, etc.)
- Any relationships between expression level and unfavorable physicochemical properties
- Ensuring the fitness for high concentration liquid formulation platform
- What types of solution behaviors can pose challenges in liquid formulation?
- What criteria we should pay attention during the protein expression stage?
- Any alternative formulation strategies to circumvent undesirable solution behaviors?
Host Creation for the Expression of Proteins May Use Auxiliary Proteins. Can We Address Those Auxiliary Proteins as an Extension of the Host Cell Protein Repertoire ?
Moderator: To be Announced
- Can we still use commercially available HCP assays ?
- How to prepare ourselves, what regulatory viewpoint should we prepare for ?
- What if we use auxiliary proteins that are not endogenous present in the host strain ?
- Do we generate a product-specific HCP assay using a mock strain only at phase III or at another stage of development?
- Would it be necessary to provide experimental evidence that the Pichia host containing the expression cassettes for the auxiliary proteins is stable?
The Importance of Selecting a Proper Host Strain
Moderator: To be Announced
Low yields and lack of homogeneity will impact the economics of the production process and costs for the therapeutic. Therefore it is important to carefully evaluate and select a proper host strain for the production of a particular therapeutic
- The production process of a biological will certainly generate unwanted modifications and product related variants.
- Some of these modifications are typically related to the choice of the strain and clone such as the degree of glycosylation and proteolytic degradation.
- Oxidation, carbamylation, deamidation and aggregation are modifications often related to upstream and downstream processes.
- With the ever increasing sensitivity of our analytical techniques we are observing more product related variants. How much effort do we spent to characterize those variants?
Host Cell Characterisation in the Era of Genomics: GMP Cell Bank Testing
Moderator: To be Announced
- Host cell characterisation is typically conservative e.g. sequencing the D2 region of rRNA, Southern blot analysis, RFLP, sequencing of coding region from cellular mRNA and sequencing of flanking regions DNA, …
- Will authorities expect us to perform whole genome sequencing in the near future ?
5:45 Networking Reception in the Exhibit Hall with Poster Viewing
7:00 End of Day
THURSDAY, MAY 7
8:00 am Morning Coffee
8:30 Chairperson’s Remarks
Lovisa Holmberg-Schiavone, Ph.D., Team Leader, Reagents and Assay Development, AstraZeneca R&D
8:35 E. coli and CHO Protein Expression Technology at Genentech
Dorothea E. Reilly, Ph.D.,
Associate Director, Early Stage Cell Culture, Genentech, Inc. – A Member of the Roche Group
Genentech uses both CHO and E. coli to manufacture therapeutic proteins. This talk will provide an overview of cell culture process development at Genentech and some of our more recent work to make complex proteins in E. coli.
9:05 Differences in Clearance of Recombinant Proteins Expressed in HEK and CHO Cells
Mengmeng Wang, Ph.D., Principal Scientist, PDM, Pfizer, Inc.
This investigation used in vitro cell-based uptake assay and in vivo PK studies to study the relationship between glycosylation and clearance of two monomeric versions of antibody that was produced either transiently by HEK293 cells or stably
by CHO cells. We demonstrated that higher clearance of the HEK derived protein was likely due to its higher mannose receptor mediated clearance and co-administration of mannose receptor inhibitor, Mannan, can reduce the clearance.
9:35 A Comparison of Two Methods for the Transient Expression and Purification of Ebola Glycoprotein from HEK-293 and CHO Cells
Christopher W. Kemp, Ph.D.,
President, Kempbio, Inc.
Advances in transient mammalian expression protocols allow proteins to be expressed at levels supportive of large-scale applications. The ability to rapidly produce diagnostic antigens is of particular interest in the area of emerging viral diseases.
This presentation focuses on the comparison of PEI-mediated transient transfection and BacMam transduction for the expression of Ebola glycoprotein. The gene-to-protein approach illustrates the utility of these methods for the rapid production
of diagnostic antigens.
10:05 Coffee Break in the Exhibit Hall with Poster Viewing
11:05 Optimizing the Quality of IgGs Produced in Yeast – Removal of Glycans at a Non-Consensus Asparagine Residue
Juergen Nett, Ph.D., Senior Principal Scientist, Adimab, LLC
Yeast cells offer a facile way to produce milligram quantities of IgGs for screening in high-throughput workflows. In order to avoid the addition of large, high-mannose glycans the consensus asparagine at position N297 is often removed by site
directed mutagenesis. Despite this modification, IgGs produced in yeast often are decorated with additional, high molecular weight glycan structures. This talk will provide an overview on how sequence optimization and purification are able
to remove these unusual post-translational modifications.
11:35 Double Digit-Titers and High Product Quality of Nanobodies® Using Pichia pastoris
Peter Schotte, Ph.D., Section
Head, CMC - Host Creation, Ablynx nv
Pichia pastoris is currently Ablynx’ preferred production host for Nanobodies, a novel class of therapeutic proteins based on single-domain antibody fragments, mainly because of its high expression yields and low amount of secreted
host cell proteins, resulting in short process development timelines. This presentation will address the different aspects of Pichia process development for Nanobody production, from host creation to fermentation and downstream processing,
with the main focus on the optimization of product yield and quality.
Meeting the Need for Rapid Protein Expression and Development with a Cloud-Based Informatics System
Diane Retallack, Ph.D., Director, Molecular
Biology, Pfēnex, Inc.
Pfēnex Expression Technology™ has been developed as a protein production platform to rapidly identify strains that express high titers of soluble, active protein. Employing a combinatorial approach to strain engineering, thousands of unique
expression strains are evaluated in parallel. Core LIMS™ enables tracking results from strain construction/ screening through fermentation and protein purification.
Best of Both Worlds: Innovative Microbial System leverages the Advantages of Both Bacterial and Mammalian Manufacturing
Kristin DeFife, Ph.D., Vice President, Biologics,
Ajinomoto Althea Inc.
Reach high expression using a microbial system and overcome challenges associated with E. coli including lengthy purification, protein aggregation and inefficient refolding processes. The Corynex® system secretes properly folded, biologically
active proteins into the extracellular fermentation broth like mammalian cells, which eliminates multiple recovery and purification steps, lowering cost and speeding time to market.
12:35 End of Conference
5:15 Registration for Dinner Short Courses