Wednesday, February 18, 2015

Detection of Residual Protein A in Biological Therapeutics

Over the past 10 years the pharmaceutical community has witnessed an explosion in growth of biotherapeutics. An increased focus on developing and marketing antibody based products resulted in a product shift to the point that in 2013 antibody based therapies represented one-third of all biotherapeutics and nearly half of all revenue in the product category. In fact, 2014 saw a new approval record set with nine new antibody products entering the market and many more in the pipeline or under review for approval in 2015.

Treatment of even a small population will require industrial scale production of therapeutic proteins, using a variety of bioprocessing methods including recombinant cell line expression systems, chromatographic purification methods and stringent purity assessment. Purity requirements include minimizing the concentration of host cell proteins and DNA ranging in the parts per million or lower relative to the product. Additionally, the formulation must be sterile insuring no viable microorganisms exist in the final product and void of any residual contaminants from the purification process itself.

A recombinant human monoclonal antibody is commonly produced in a mammalian cell line such as Chinese Hamster Ovary (CHO) cells during large scale manufacturing. Purification typically relies on the use of a three-column chromatography process to meet the stringent purification requirements: 1) Protein A affinity chromatography, 2) Cation exchange (CEX), and Anion Exchange (AEX); a viral filtration (VF) step is also generally used during the final stages of production. Resin with immobilized Staphylococcal Protein A (PA) has a high affinity for the crystallizable fragment (Fc) region present in rhuMAb IgGs allowing capture from the culture media or crude cell lysate of the host cell line. While these resins provide a high capacity and selectivity for the target protein, trace amounts of the PA ligand has been found to leach from the column contaminating the antibody product. Residual PA contamination of a biotherapeutic may result in immunogenic consequences as well as toxicological and/or mitogenic effects. Therefore, reliable, robust methods for the detection and quantification of trace amounts of PA are necessary and mandated in the US by the FDA.

Recently we demonstrated automation of a HTS compatible homogenous proximity assay for the detection of residual PA in biological therapeutics. The application includes screening results for detection of residual PA in a panel of ten (10) human IgG antibodies including samples of the  biologically active antibody components in the therapeutic products Herceptin®, Rituxan®, and Erbitux®.

D Ekers. (2015, Jan. 29) Antibodies: $75 million in sales and no slowing down in sight. [Web log comment] Retrieved from
Mehta, A., et al. 2007. Purifying Therapeutic Monoclonal Antibodies. CEP. SBE Special Section: Bioprocessing. S14-S20.
Zhu-Shimoni, J.; Gunawan, F.; Thomas, A.; Vanderlaan, M.; Stults, J. 2009. Trace level analysis of leached Protein A in bioprocess samples without interference from the large excess of rhMAb IgG. J. Immunol. Methods, 341 (1-2), pp. 59–67.

By: BioTek Instruments, Peter J. Brescia Jr., MSc, MBA

Thursday, February 5, 2015

3D Cell Models Enhance Hepatotoxicity Screening

A few weeks ago I wrote a blog comparing the character "Flat Stanley" to the long-standing, traditional method of cell culture, and also explaining how three dimensional (3D) cell culture methods could help provide a more relevant model for drug discovery. Last week further evidence was seen to support this shift when BioTek's Flat Stanley hit the road with me to attend the High-Content Analysis and Phenotypic Screening Conference in San Diego, CA. This conference, apart from providing a wealth of information for those looking to learn more about cellular imaging, highlights another important shift in drug discovery, which is the re-emphasis of methods to analyze the final phenotypic effect of a test molecule during early screening; away from the target-based approach which has been in place since the 1990s. Here again, a significant amount of attention was placed on the incorporation of physiologically relevant 3D cell models into the screening process during plenary sessions of both conference tracks.

Of interest during the 3D focused talks was the high level of attention given to incorporating 3D cell models into toxicity screening…particularly hepatotoxicity. It has become generally accepted that primary human hepatocytes, when aggregated into a 3D configuration, maintain higher levels of cell viability and functionality compared to 2D cultured hepatocytes. Therefore, inclusion of these “microtissues” provides the ability to generate a clearer picture of the potential toxic effect of lead molecules.

BioTek has partnered with 3D cell culture providers to demonstrate how the combination of in vitro liver models, appropriate assay technologies, and image-based analysis can provide procedures for measuring hepatotoxic effects that are both robust, yet easy to perform.

Application Notes

By: BioTek Instruments, Brad Larson, Principal Scientist

Monday, January 26, 2015

Calling All Cooks!

Yes, you - the scientist reading this article. You see, I liken an experiment to a recipe. You have a list of materials needed and then you have your instructions, detailing how much and when to add a particular component. There might even be some cooking time (a.k.a incubation) at the end of which you get your final product. Of course, then comes the "taste test" or analysis. Did you add too much of this, too little of that... it might even take a few tries for optimization. Remember, you've got to satisfy the critics!

Just like in cooking, a scientist benefits from having the right tools. On television, a popular cooking competition show sabotages the cooks' efforts by substituting necessary kitchen tools like knives, bowls and pans with "unconventional" utensils. Hopefully, you don't have to deal with this kind of subversion, but it does make you realize how the right tools make tasks easier and cleaner.

Scene from BioTek’s Holiday 2014 video : The right tools make tasks easier and cleaner, but we can have fun once in a while, right?

Why do something manually when you can automate it and spend your time doing something else? For example, in cooking you can hand-whip cream or set your electronic mixer to do it while you slice some strawberries. In a lab, automating a task can have multiple benefits such as time, money and reproducibility. Yes, it may get done either way but is it efficient? For advice on automating your lab, check out this article from Lab Manager, with contributions from BioTek's own Jason Greene.

Cooks' and scientists' tools have to be dependable. At BioTek, we take pride in every step of the process -  from research and development to manufacturing, all of which is done in the United States. If you’re a cooking aficionado, you know which brands to buy. But whether you’re a new or seasoned scientist, you should know to contact BioTek Instruments first.

BioTek Instruments: Proudly made in Vermont, USA

By: BioTek Instruments, Ellaine Abueg Ph.D., Product Manager, Specialist

Friday, January 16, 2015

Flat Stanley…a Model for Cell Culture?

I’m sure most, if not all of you are familiar with Flat Stanley. Originally a series of books, he’s now more famous for the projects that are performed in elementary school with him. Students color in a picture, cut it out, and then take him on trips, or mail him to friends or family across the country and the world. The idea is to help kids learn about other people, places, and cultures that they might otherwise not even know existed. A great way to get children to learn in a fun, unique way; wouldn’t you agree?

But would you also agree that Flat Stanley makes a great model of the tissues and tumors that exist within the human body? Preposterous you would most likely say. Yet upon closer examination, drug discovery has been using the “Flat Stanley” method of tissue culture for decades. Cells are added to wells of a slide or microtiter plate, spread out, and attach to the bottom in a flat, two-dimensional manner. Unfortunately this cell culture model has led to numerous clinical failures of lead molecules, as in vitro results do not correlate to what is actually seen in vivo with animal or human subjects. Therefore a new cell culture model is needed. Three dimensional (3D) cell culture is poised to meet this demand. The 3D cell culture methods which now exist, including scaffold-, hanging drop-, or ULA plate-based models, provide a means in which cells aggregate together into structures that have length, width, and depth to them, as they would in the body.

The following links provide information regarding the projects that BioTek has performed, as well as the liquid handling, reading, and imaging instrumentation used to further enhance the results generated using 3D cultured cells.

Application Notes:
Hot Applications:!applications/cjg9

As you will see, BioTek can help you out whether you are new to 3D cell culture, or would like to see what new opportunities exist using cells cultured in this manner.

Move over Flat Stanley…there’s a new cell culture model in town!

By: BioTek Instruments, Brad Larson, Principal Scientist

Thursday, January 8, 2015

Cancer, the Cell Cycle and Bad Luck

The replication of cells has long been known to be responsible for tissue, organ, and species growth and reproduction. This, of course, is a highly regulated and controlled process that results in numerous differentiated cells performing a variety of specific tasks. Cancer on the other hand is uncontrolled growth of cells, where cellular differentiation is often lost. 

Proliferating cells (normal and cancerous) repeatedly transition between cellular duplication (interphase) and cell division (mitosis). This repetitive process has been described as a cycle. The concept of cell cycle was first utilized to describe these repetitive elements. The terminology (G0, G1, S, G2, and M) attempts to place in temporal perspective two dichotomies; mitosis (M), which can be determined by direct observation, and DNA synthesis (S), which can be established by DNA labeling. G1, the post mitotic gap, G2 the post-synthetic phase or pre-mitotic gap; and G0, a phase in which non-dividing cells exist, are terms used for simplicity (Figure 1).

Figure 1. Schematic of the Cell Cycle
As I've been interested in the cell cycle most of my research career, I was quite intrigued by the January 2nd article in the journal Science that provides statistical modeling that links the number of stem cell divisions with cancer risk. The premise is that because cellular DNA replication causes errors (it's hard to replicate 3 billion base pairs correctly) the more laps around the cell cycle (i.e. DNA replication), the more likely that errors (mutations) will occur that could result in cancer. Thus tissues that require more cell divisions will be more likely to become cancerous than tissues that require fewer.  For this reason, cancers of the colon are more common than bone cancer. The article also states that heredity and environment play a significant role as well.  For example individuals with the Familial Adenomatous Polyposis (AFP) gene are at greater risk of developing colorectal cancer than those who do not carry this marker. Likewise smokers are of greater risk of lung cancer than non smokers (Figure 2).  Because of the topic there has been a great deal of commentary in the press regarding the paper.

Figure 2. Plot of Lifetime Cancer risk vs. Total Stem Cell Divisions of Various Organs [1].
Not surprisingly the lay press has taken this paper and run with it; the most common statement being that two-thirds of cancers can be attributed to "bad luck". While it is definitely not that simple (especially the math), there is some truth regarding  "bad luck".  But luck is only one part of the equation; our genetics, environment and personal habits also play substantial role, which the research paper so nicely states. While scientists have long believed that tissues with rapidly proliferating cells would be more prone to cancer, the data did not always fit this hypothesis. However, when STEM cell divisions were used for analysis a high degree of correlation was observed. Only recently has there been enough data regarding stem cell divisions to make these types of analyses, so I’m sure that this is just the beginning of a new avenue of statistical analysis of cancer. 

Cancer has touched all of us in some way. Very few have not had a family member or friend be diagnosed with or die of cancer. While these data suggest that many cancers are not preventable, they reinforce the need for better early diagnosis and effective treatments for cancers. Most of those afflicted want to know why they have cancer and if they could have avoided it somehow. While the 3-pack a day smoker who gets lung cancer has a pretty good idea as to why they were afflicted, for many others the answer really is bad luck.

  1. Tomasetti, C. and B. Vogelstein (2014) Variation in Cancer Risk among Tissues can be explained by the Number of Stem Cell Divisions, Science, 347:78-81.

By: BioTek Instruments, Paul Held Ph.D., Laboratory Manager

Wednesday, December 24, 2014

Summer Breath

Vermont winters have a beauty that spares fragile stems of dead leaves encased in ice from snapping, and a brutality that can bring large, strong branches from healthy trees crashing to the ground. The human body, and the spirit it embodies that enables us to persevere the diseases and burdens that can storm it, encapsulates this same dichotomy. I have lived through decades of Vermont winters, and each year the question “will this be the winter that breaks me?” comes progressively earlier, and shoveling out the answer “no” takes more work as it becomes buried deeper the older I get.

Just before this latest blizzard hit, Conn Carey from Luxcel Biosciences in Cork, Ireland visited the BioTek Applications Lab. We are collaborating to optimize the parameters for detecting a family of time resolved fluorescent probes on various BioTek readers. These probes measure intra- and extracellular oxygen and extracellular pH, all predominately used in the study of cell metabolism. As cancer cells have a code for survival that has yet to be broken, how they breathe, eat, and proliferate to take over an otherwise healthy body are important and ongoing research questions looking for answers. The Luxcel MitoXpress® -Intra Oxygen Consumption probes can be used to measure how cells breathe in response to various stimuli, and work such that increases in intracellular oxygen concentration result in decreased probe signal, while conversely decreases in intracellular oxygen concentration result in higher probe signal. This is demonstrated below by images obtained on the BioTek Cytation 3 using the gas controller to progressively decrease oxygen (O2) levels delivered to HepG2 cells (a human liver carcinoma cell line) over 40 minute kinetic intervals at 12%, 6%, and 3% (left to right, no enhancement).

I’ve developed many survival techniques for making it through Vermont winters, and sometimes the season still wins. Today, I adjust my ear buds and select random shuffle on my winter playlist. The first song is "Summer Breeze" (Seals & Crofts, 1972, Warner Bros. Records). As a typical ‘70s wild child an alternative botanical was blowing through my mind the year that song was released, but now I can anticipate the lighting of a midnight jasmine Yankee Candle that I hope my 14 year old daughter, who loves shopping for aromatics, will present to me on Christmas. I take a deep summer breath, plate my cells, and look into the dark “know”.

By: BioTek Insturments, Wendy Goodrich, Applications Scientist

Wednesday, December 17, 2014

ASCB Show Highlights

Scientifically-speaking (no pun intended with the name of this blog!), we are on the cusp of some amazing breakthroughs in the field of Cell Biology. Over the last decade, cell-based research applications have been growing at a blistering pace, with several sub-application areas leading that charge.  At the ASCB show in Philadelphia last week, this trend was quite evident when walking the Exhibit Hall. The areas of live-cell imaging and 3-dimensional cell biology were strongly represented across the vendor booths. These two areas are growing more quickly than other areas in the Cell Biology market and require some special attention when it comes to application requirements.  Despite a lot of "solutions" advertised at ASCB for these particular areas, not all of them hit the mark.  A company needs to have more than just an in-depth understanding of applications in order to deliver a successful solution. Price, instrument footprint, ease-of-use, software capabilities, modularity, adaptability... there are a host of other factors at play for researchers to consider. 

Some of the most enjoyable aspects of the ASCB show were the numerous conversations I had with existing BioTek customers who voiced how their particular instrument hit the mark for their specific cellular application.  This spanned from imaging to washing to plate reading.  This was a good reminder to me that any instrument is only as strong as the team behind it. 

Another item I thoroughly enjoyed while at ASCB was daydreaming.  Daydreaming?!?  That’s right - I began this blog mentioning how we are on the cusp of some amazing breakthroughs in the field of Cell Biology.  From growing organs for transplant patients to reducing animal testing using 3D cellular models for predicting drug toxicity, novel cellular research capabilities that were only dreamed of half a century ago, are now announced regularly.  The consortium of brainpower driving advancement in this field is staggering.  And contingent on those advancements are research instruments that deliver uncompromising performance, reliability and features.  That is where I had fun daydreaming.  As a Product Manager at BioTek, I can only imagine what innovative new products we will offer over this coming decade -  a decade where cellular research will redefine what is possible, not just in the lab, but all the way to the Clinic. BioTek will keep at the front of researcher's Cell Biology requirements, which will help us continue to launch innovative products such as Cytation 5. Products that will further enable advances in this field and allow performing research you never thought possible. As I stop to consider this, it makes me wonder - what customer stories will we hear at the ASCB show over the coming decade?  Here's an open invitation to stop by our booth at a future ASCB show to let us know how BioTek products have helped your cellular research!      

By: BioTek Instruments, Caleb Foster, Product Manager, Development