Toyota visits Zymergi(.com)

This is pretty flattering. Someone from Toyota - the creators of Toyota Production System... which is the precursor to Lean... looked up Process Capability (CpK) on Google and read my blog post:


I'm pretty excited. When you study manufacturing and look for the words of truth that apply across industries, Toyota is the leader in quality. Competing with limited resources and applying the principles that Deming taught them, they went from post-World-War 2 clothmaker to the largest automobile company on planet Earth.

Death to Cell Culture Spreadsheets!

by Oliver Yu

I've been around a few cell culture operations in my time... fermentation, too. And ubiquitous to virtually every operation - be it lab, plant or pilot-plant - are these Excel spreadsheets where research assistants or operators input "cell count" data.

A "cell count" is when a sample of the cell culture fluid is taken from the miniferm/bioreactor. This sample prepped with trypan blue, dropped on a hemocytometer and counted by an operator under a microscope.

Pipetting fluids into a hemocytometer by Jacopo Werther

The trypan blue is absorbed by the carcasses of dead cells while an integral cell membrane of the live cell is impervious to trypan blue.

The operator counts the number of blue dots (dead cells), the number of clear dots (live cells) and thus determine the viability (% of live cells).

From the total number of cells and the sample volume, the operator can estimate the total cell population in the bioreactor.

Also while we have this fresh cell culture sample, a small volume is separately sent through machines that determine the concentration of glucose, lactate, sodium and ammonium, pO2, pCO2 as well as the pH and osmolality.

All this cell count and cell culture metabolite data gets inputted into spreadsheets where we can compute the rate of cell growth so that we can figure out when to transfer/harvest the cell cultures.

Because of the detailed data, these spreadsheets become the crown jewels of process understanding. Cell growth and metabolite profiles at large-scale are benchmarked against their small-scale equivalents. New experimental designs are determined from the results of data from the previous experiment.

And all this is possible because of these cell culture spreadsheets.

The thing is, there are severe limitations to running your core enterprise information this way: spreadsheets are not a viable way to manage data: databases are.

And it is for this reason that we are creating the world's first commercial off-the-shelf cell culture database to manage this core data.

Built by cell culture engineers for cell culture engineers.

Sign up for the beta

OSI PI DataLink and Excel 2007 Error

by Oliver Yu

Ugh.

Recently, my Excel 2007 would launch with these annoying errors:

• Compile error in hidden module: Main
• Compile error in hidden module: modAddin
• Object library invalid or contains references to object definitions that could be found

Useful error messages, huh?

After boggling at these gems of clarity, any typical user simply stops using Excel (if possible).

But if Excel is a daily workhorse of productivity, the next path of least resistance is to disable the Add-Ins.

Still using Excel to store Cell Culture Data?

Of course, if access to tabular OSI PI data (DataLink) or bulk tag configuration (Tag Configurator), your only option is to fix this Excel 2007/OSI PI Add-In error.

How to fix

You simply need to delete the Forms directory:

• For Windows XP:

  C:\Documents and Settings\%USERNAME%\Application Data\Microsoft\Forms

• For Windows Vista, Windows 7:

  C:\Users\%USERNAME%\AppData\Roaming\Microsoft\Forms

Did that work? It usually does.

If not, try this.

If you use OSI PI, check out:

Mammalian Cell Culture - Pressure Control Strategy

by Oliver Yu

Pressure control strategy is the last and the least of the cell culture environmental parameters. Mostly, this is because cell culture media is mostly water and water is an incompressible fluid.

The other reason is that the only thing pressure can impact is the solubility of oxygen and carbon dioxide in the media--and not by much:


The main reason to control pressure is to maintain positive pressure in the bioreactor for contamination risk reduction. Positive pressure means that the pressure in the bioreactor is greater than the outside pressure so that if there is a breach in the sterile envelope (e.g. filter, probe port, rupture disk) that the flow is outwardly.

This outwardly-directed flow does not prevent contamination, it gives the operator an opportunity to identify the breach and address it before the situation gets worse.

How it happens in the real world

Pressurizing bioreactors is a well understood, well-engineered process. Pressure is controlled by shutting off all the valves and modulating the flow of sterile-filtered air in the headspace of the bioreactor. Filtered air comes in and moist air goes out and if more pressure is needed, the more the valve is commanded to open.

In large-scale cell culture, however, there is an opportunity for the vent filter to get clogged, in which case the pressure will slowly increase. Efforts to change the vent filter must be weighed against contamination risk; if this happens mid-culture, the risk is worth it. If this happens late culture, people often wait.

Also, the vessel pressure can drop to zero (same as atmospheric pressure) if there are breaches.

Summary

For commercial manufacturing, controlling product formation and product quality for biologics means controlling the cellular environment. Controlling the cellular environment means having pH, dO₂, agitation, temperature and pressure control strategies defined by manufacturing instructions that pH, dO₂, dCO₂, shear forces, mixing, bubble size distribution, temperature and osmolality.

Big ups to: Bob Kiss, Dan Stark, Jesse Bergevin and Reddy for teaching me everything I need to know about large-scale cell culture.

Mammalian Cell Culture - Temperature Control Strategy

by Oliver Yu

Microbes used in cell culture thrive in a narrow band of temperature between 30 to 40 degrees Celsius.

Humans have a deep body temperature of 37 degC so it stands to reason that anyone exploring the optimum temperature of mammalian cells in a bioreactor ought to start here.

Temperature is a measure of the "internal kinetic energy" of a system. It can be said that in a system with hotter temperature, the molecules are moving faster and "bumping into each other more frequently." The opposite is true in colder systems.

The cells in a bioreactor set to 37 degC "bump" into glucose molecule (and other molecules) at a higher frequency than cells in a bioreactor set to 33 degC. Should the cell culture be set to a higher temperature, key biological processes can fall apart - hence an upper and lower bound to the temperature.


Bioreactor temperature is controlled with a resistance temperature device (RTD) that sends the temperature reading to a controller. If the temperature drifts too low, the controller sends more cool water through the jacket; the temperature drifts too high and the controller sends more hot water through the jacket.

Agitation

For this method to work, the heat transferred through the bioreactor wall must be evenly dispersed throughout the cell culture, so agitation mixes the cell culture ensuring that the no volume of fluid stays local to the bioreactor wall for too long.

The agitation, itself, increases the temperature as the work into the system is dissipated as heat. But this temperature increase is negligible.

Gas Solubilities

Temperature is crucial in determining the solubility of gas in the cell culture, which is mostly media...itself mostly water. The lower the temperature, the more carbon dioxide and oxygen the media can hold. So pH control and dO2 control are impacted by temperature control. In general, if you stick with temperatures found in nature, you're going to grow cells like they grow in nature.

How it works in the real world

Temperature control is a well-understood chemical engineering phenomenon long before the advent of biologics manufacturing. For seed/inoculum cultures, temperature is typically set at at fixed number (e.g. 37 degC). For production cultures that need to last, some temperature control strategies involve a temperature reduction based on biomass or time in order to cool the cell culture and stave off late culture viability.

If a temperature reduction happens, the dO2 controller will call for less oxygen because more oxygen is retained in the culture. The solubility effect for pH control is usually not observable in the control instruments because at this point in the culture the cells are evolving the carbon dioxide.

Mammalian Cell Culture - Dissolved Oxygen Control Strategy

by Oliver Yu

Mammalian cells require oxygen to live and to grow. If you simply inoculated a bioreactor filled with media, the cells would fall to the bottom of the tank, suck all the dissolved oxygen (dO₂) out of the media and subsequently die.

Because dissolved oxygen impacts cell growth and viability, a dissolved oxygen strategy is required. Overall, the dO₂ control objective can be summarized as

Don't let the dO₂ of the culture drop below 5% air saturation.

To achieve a dissolved oxygen between 20 and 60% air saturation, we use a combination of sparging, agitation and media ingredients.

Sparging Air/Oxygen

If you decided to oxygenate the cell culture by blowing air or oxygen into the bioreactor (at the bottom since gases tend to bubble to the top), this would be better but not enough as the cells still sit at the bottom of the bioreactor.

Agitation

To get the cells off the bottom, an agitator spins with impellers pushing the fluid downward. The mixing dissipates the cells from the bottom of the bioreactor and suspends the cells in the media. The agitation also ensures that air, oxygen (as well as CO₂ acid, carbonate alkali and media components) are evenly distributed throughout the cell culture.

The downward pumping impellers are to help impede the speed of air/oxygen bubbles to increase their residence time in the culture.

Shear Forces

Now that you've introduced air/oxygen to oxygenate the cell culture (in addition to the CO₂ for pH control), you've added shear forces to which mammalian cells are not accustomed. Between agitation and bubbles, the greater shear force is with the bubbles.

To help the cells cope with shear forces, surfactant is added to the cell culture media.

How dO₂ works in the real world

dO₂ probes are calibrated when the bioreactor is filled with media. The final step of this calibration is to saturate the media with oxygen and span the probes to 95 or 100%. As the bioreactor awaits inoculation, the saturated media will lose oxygen naturally.

Once inoculated, however, the cells begin using dissolved oxygen and the dO₂ of the cell culture drops. When it drops below setpoint, the dO₂ controller will begin sparging air. When the maximum flow rate for air is unable to meet the oxygen demand, the air is supplemented with pure oxygen. The culture will peak begin to slow; as the culture slows, less oxygen is demanded and oxygen-supplementation is withdrawn.

Other reading:

• Cell culture pH control
• Temperature control of cell cultures

Mammalian Cell Culture - Osmolality

by Oliver Yu

Osmolality ("osmo") is a measure of how "salty" the media is and significantly impacts the cellular environment of cell culture.

CHO cells in a low osmo environment are bursting at the proverbial seams and conversely in high osmo environments are shriveled like Ahnold's balls on steroids.

High osmolality can cause delayed cell growth or accelerate cell death (depending on where the culture is).

Osmolality is not a controlled parameter per se. It is designed to be somewhere between 270 - 330 mOsm/kg (mammals have interstitial osmo of 290 mOsm) for the typical media.

Once the media is inoculated, the cell culture pH control strategy will increase the osmo when alkali is demanded; this is due to the sodium (Na+) ion of the carbonate/bicarbonate pH control strategy.


Also, additional media or glucose added to the cultures have high osmolality themselves and will raise the culture osmo.

How it happens in the real world.

Generally, osmolality increases during the course of cell culture. The addition of base to offset the acidic forces of the CO2 evolution increases the osmolality of the culture. In fed batch cultures, the nutrient-packed batch feed will increase the osmolality of the culture.

While there are no formal osmolality controls for the cell culture, there are typically osmolality specifications for the media and batch feed. Typically media targets final (after initial QS, media powder, peptones, base addition, final QS) osmolality near 290 to 300 mOsm/kg so that the cell culture has a fighting chance at staying within biological range for the cells.