Showing posts with label fermentation. Show all posts
Showing posts with label fermentation. Show all posts

Thursday, August 8, 2013

Drain Water vs. Clean Air - Drain Design for Bioreactor Contamination

UPDATE: The point isn't to install air-breaks at all costs.  The point is to use the correct BioSafety Level for your process, recognizing that a lot of facilities are overly-conservative for the processes they run.

On multiple consulting assignments, we are seeing an alarming trend where CIP manifolds and process piping are piped directly to drain.  We have identified direct piping to floor drains as contamination risks.  And our experience mitigating floor drain contamination risk is to cut the piping.

The main objection to this recommendation is that it would compromise the Class 100,000 clean room status of the process space.

With the cut in the piping, the worry is that contaminants from the drain are now able to enter the processing suite and will send your viable airborne particles beyond your environmental monitoring action limits.

But of the unfavorable options available, there's one that's obvious to us.

Your choices are as follows:
  • Keep the bioreactor sipping drain water, but hey, you've got a Class 100,000 processing suite.
  • Cut the pipes and get your bioreactor sipping fresh, 20 air-changes-per-hour filtered air.

bad choices trooper

It turns out that that we aren't the only ones who think this is true.  In a 2006 article on biocontamination control, @GENBio reported the "original views" of chemical engineer, Jim Agalloco:
...Trying too hard to protect the bioreactor environment can adversely affect the ability to sterilize equipment. For example, a steam sterilizer normally requires an atmospheric break between its drain and the facility drain, but some biotech companies object to that layout because it compromises the controlled environment.
Somehow, the viable airborne particles of the environment matter more than the ability to sterilize equipment.  They further state:
Eliminating the atmospheric break introduces more piping and surfaces, which leads to more opportunities for microbes to grow. To protect the outside of the tank, they purposely risk contaminating the inside.
Which is exactly our position on the matter.

We're aware that managing perceived action is as important as managing action.  But taking the action that keeps cell cultures from contamination is always defensible even if it flies in the face of perception.

Zymergi Bioreactor Sterility Consulting

Friday, May 17, 2013

Industrial Centrifugation demonstrated on YouTube

For large-scale cell culture, centrifugation is one method used to harvest the production cell culture.

Before you can purify your active pharmaceutical ingredient, you need to get rid of the cells (yes, after all that work secreting the API, we get rid of the cells).

Note: some APIs are not secreted, in which case you want to keep the cells, so harvest means different things in different processes.

To separate the cells from the cell culture fluid (CCF), you can filter it or centrifugate it. If you need to process 12,000L or 15,000L in hours, you're doing tangential flow filtration (TFF) or you're using a centrifuge.

If you're using an industrial centrifuge to clarify cell culture, chances are, you're using Alfa Laval.

Three minutes of your time is all it takes to see how large-scale centrifugation works.

Nice work, Alfa Laval, Westfalia

Thursday, January 17, 2013

Biotech Manufacturing in Singapore

Note: The original post has been corrected; this post is the result.  OLY/Jan31,2013

Amgen is going to build their first Asia plant in Singapore. Here's the link from Amgen.

The current list of Singapore Biotech Companies includes:
All these plants are clustered in the Tuas Biomedical Park on the west side of Singapore:
biotech companies singapore
(image from Google Maps)

Zooming in, you can see that these plants are basically right next to each other.
biotech companies singapore
... and right next to Malaysia.

I think the Genentech plant cost 290 million and the Roche plant cost 500 million, so if Amgen gets it done for 200 million, that's pretty cheapOLY 1/31/13 par for the course.

Wednesday, September 5, 2012

Cell Culture Database - Batch Information

You work in biopharma. Maybe you're a fermentation guru... or a cell culture hot shot. Whatever the case... This is your process.
We muggles don't have the luxury of waving our wands and having protein fold themselves mid-air. There's usually a container where the process happens. processes happen in a unit
A time-window (starttime to endtime) is when processes happen.

Operators execute process instructions; these procedures is how the process happens.
The execution of process instruction results in an output. The output of the process step is the product and constitutes the what.
Lastly, the process (step) is given a name describing who the batch is.
It stands to reason that the who, what, how, when, where of a batch is characterized by:
  • batchid
  • product
  • procedure
  • starttime - endtime
  • unit
and fully describe batch information for cell cultures and fermentation.

Organize Your Cell Culture Data

Monday, June 11, 2012

Mammalian Cell Culture - Temperature Control Strategy

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.

cell culture temperature control
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.


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

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.

dO<sub>2</sub> control cell culture

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.


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:

Wednesday, June 6, 2012

Mammalian Cell Culture - Osmolality

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.

cell culture ph
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.

Monday, May 28, 2012

Mammalian Cell Culture Environment - pH Control Strategy

A key requirement of a cell culture is to recreate in a bioreactor the cellular environment that the cells experience were they still in their mammalian hosts, so that the cells grow and secrete the active pharmaceutical ingredient.

Cellular Environment

Key parameters of the cellular environment include:
  • pH
  • osmolality
  • shear forces
  • temperature
  • mixing
To control this cellular environment, pH, dO2, Temperature and Pressure Control strategies are developed in the process definition (a.k.a. Manufacturing Formula):

cell culture process parameters
Bioprocess engineering textbooks and modern cell culture scientists all think that these parameters determine cell growth, cell viability, cell metabolism and ultimately product formation and product quality.

These parameters are what the cells "see" and "feel" during the course of their stay in the bioreactor and large-scale cell culture support actually means being hospitable to our guests by controlling these parameters.

The Mac Daddy of all cell culture parameters is arguably pH. The amount of protons (H+) hanging around can change the way proteins fold thereby changing the function of cellular machinery. These changes can speed and slow the rate of reactions tilting the cells to favor one metabolic pathway over another.

Studies claim that pH increments as small as 0.1 units can change glucose consumption and lactate production dynamics... though I know of no pH probe that has an error smaller than 0.1 units of pH.

pH Control Strategy

The pH control strategy is most simply achieved by using carbon dioxide to lower pH (make more acidic) and sodium carbonate to increase pH (more basic).

ph control strategy
Like any carbonated beverage, water with excess carbonation (CO2) is acidic or sour. Incidentally, this is the same mechanism by which global warming alarmists believe that that greenhouse gases kill off our marine life by making our oceans more acidic.

To increase pH, one simply needs to add a base and if carbon dioxide is the acid, the complementary base is carbonate (a.k.a sodium carbonate).

Because pH between 6.8 and 7.4 is the proven acceptable range for cell culture, a buffer is added to the media to make sure the media stays within that range. The buffer is sodium bi-carbonate and acts to ensure that the pH titration does not overshoot in either direction.

One key feature of this pH control strategy is that the acid is gaseous which means it is sparged in ("blown in via pipes") from the bottom of the bioreactor and bubbles its way to the top. It also means that this acid can be removed from the bioreactor by competing gases like air or oxygen.

On the other hand, the base is liquid, which means that it is dripped in from the top of the bioreactor. This also means that once added, the sodium cannot be removed.

How pH works in reality.

During the early culture, CO2 is often demanded to maintain pH because it is constantly being stripped by the air/oxygen sparge. Once there are enough cells evolving their own carbon dioxide, CO2 demand dwindles. During the mid-culture as the cells are growing gangbusters, the culture becomes acidic demanding sodium carbonate. And if cells start to die off towards the end, the culture may demand more carbon dioxide and less sodium carbonate to maintain a fixed pH.

To summarize:
  • Acid (CO2) is consumed during early and late culture.
  • Carbonate is used during mid-culture.
  • CO2 is a gas and is sparged in from the bottom.
  • Sodium carbonate is a liquid and is dripped from the top
  • Alkali, once added, cannot be removed and contributes to increases in osmolality.

Friday, April 6, 2012

SPC - Process Flow Diagram/Pareto Charts

So that little SPC Book goes into 7-tools to use, the next page goes into Process Flow Diagrams and Pareto charts.

Process Flow Diagram

The first tool appears to be the Process Flow Diagram[tm], where one is supposed to draw out the inputs and outputs of each process step. I suppose in the "Lean" world, this is the equivalent of value-stream mapping.

The text of the booklet calls it a

Pictoral display of the movement through a process. It simply shows the various process stages in sequential order.

Normally, I see this on a Powerpoint slide somewhere. And frankly, I've rarely seen it used in practice. More often, if we show this to consultants to get them up to speed.

Pareto Chart

The pareto chart is essentially a pie chart in bar-format. The key difference is that pie charts are for the USA Today readership while pareto charts are for real engineers -- this is to say that if you're putting pie charts in Powerpoint and you're an engineer, you're doing it wrong.

Pareto charts are super useful because they help figure out your most pressing issue. For example, say you're create a table of your fermentation failures:

So you have counted the number of observed failures alongside a weight of how devastating the failure is. Well, in JMP, you can simply create a pareto chart:

and out pops a pareto chart.

What this pareto chart shows you is the most important things to focus your efforts on. If you solve the top 2 items on this pareto chart, you will have solved 80% of your problems - on a weighted scale.

The pareto is a great tool for metering out extremely limited resources and has been proven extremely effective in commercial cell culture/fermentation applications.

Wednesday, April 4, 2012

You Suck at Reducing Cell Culture Contaminations

zymergi bioreactor illustration
You do, you're awful. But that's why you're here.

Reducing cell culture contaminations is a big deal.

Not big but HUGE.

It's huge simply because contemporary contamination rates are typically between five to 15% and can skyrocket at any time.

You know what I'm talking about. You're nervously awaiting the next bioreactor contamination right now.

Chances are that you've sat through a contamination investigation meeting NOT getting to the bottom of why your cell cultures are coming down contaminated.

You're going through the motions of a contamination response procedure that's in an SOP somewhere because it is GMP to have written procedures and this cross-functional team of warm-bodies isn't pulling you out of this tailspin.

I mean, even if it wasn't for having to sit through crappy meetings, we're still talking about a biological process being out-of-control. Being out-of-control means you are not GMP.

Google Genzyme and you'll see their contamination problems and the subsequent FDA beatdown.

Their Allston plant at 500 Soldiers Field Rd (right next to the Harvard Bschool) faced rampant viral contamination of their bioreactors. These contaminations temporarily shuttered their plant, left patients facing drug shortages, brought in a $175 million consent decree and crashed the stock price.

On a personal note, I interviewed for a position at the Allston plant in 1998 and was rejected; in hindsight, that rejection was one of the better things that happened for my career.

The idea is simple: stop having contaminations and the CEO-toppling domino-effect will never happen.

Easier said than done, right? Well, it starts with hiring the industry veterans that have been there and seen it. (That's what Genzyme did to remedy the situation; several former colleagues are there right now).

Not sure which veteran to hire? Read our FREE case study to see if Zymergi's contamination-reduction expert is the right guy for you.

Stop Sucking At Contaminations

Monday, February 20, 2012

Cell Size and Scale

Cell culture and fermentation scientists/engineers deal with the size of things they cannot see. Here's a cool website I came across that helps with the visualization.

cell sizes

It uses Flash - not HTML5 - but it's worth about 30 seconds of your time.

As you zoom in, you'll see an antibody in there; it's crazy how small it is.

Friday, October 28, 2011

Example of Production Culture KPI: Volumetric Productivity

Say you are running a 2g/L product from a ten-day process at your 1 x 6000L plant, with strict orders from management to minimize downtime. This product is selling like gangbusters, which means every gram you make gets sold, which means you've got to make the most of the 80-day campaign allotted for this product.

The volumetric productivity for the process is 2g/L/10days = 0.2 g/L/day.Running a 6000L capacity plant gives you
  • 12 kilos every 10 days.
  • 8 run slots given the 80-day campaign
  • Maximum product is going to be: 96 kg for the campaign.

But suppose your Manufacturing Sciences team ordered in-process titer measurements and found that Day 8 titers were 1.8 grams per liter. in process titersHarvesting at day 8 means:
  • 10.8 kilos every eight days.
  • 10 run slots given the 80-day campaign
  • Maximum product is going to be 108 kg.
By harvesting earlier, you gain two additional run slots... during which time you can make 21.6 kg; but since you lost 1.2 kg/run for 8 runs totalling 9.6 kg, the net gain is 12 kgs.

There are a lot of assumptions here:
  • Your raw material costs are low relative to the price at which you can sell your product
  • Your organization is agnostic to doing more work (ten runs instead of eight).
It is difficult for plant managers to end a culture early to get 10.8 kgs when simply waiting two more days will get you 12 kgs. It quickly becomes easy when you see how two-run slots open up and you have the opportunity to make 21.6 kgs to make up for the lost product from ending the fermentation early, or rather, the point of maximum volumetric productivity.

Wednesday, September 14, 2011

OSI PI Batch Database (BatchDB) for biologics lab and plant - part 1

Biologics manufacturing is a batch process, which means that process steps have a defined starttime and endtime.

CIPs start and end. SIPs start and end. Equipment preparations start and end. Fermentation, Harvest, Chromatography, Filtration, Filling are all process steps that start and end.

Even the lab experiments are executed in a batch manner with defined starts and end.

Like the ModuleDB, OSIsoft has a data structure within PI that describes batch and it is called PI Batch Database (PI Batch). While it comes free, it does cost at least 1 tag per unit (PIUnit) to use.

The most important table is the UnitBatch table. The UnitBatch table contains the following fields:
  • starttime
  • endtime - when the batch happens
  • unit - where the batch happened (with which equipment)
  • batchid - who (name of the batch)
  • product - what was produced?
  • procedure - how was it produced?

PIBatchIn essence, the UnitBatch table describes everything there is to know about a process step that happens on a unit. Remember: units are defined in the PI ModuleDB, which means the PI BatchDB depends on a configured PI ModuleDB.

So why bother configuring yet another part of your PI server? The main reason is to increase the productivity of your PI users. In our experience, up to 50% of the time spent using PI ProcessBook inputting timestamps into the trend dialog. Configuring PI Batch makes it so that your users can change time-windows in ProcessBook with just a click.

We have seen power-users put eyeballs on more trends in even less time than without PI Batch; and the more trends your team seems, the more process experience they gain.

In this dismal economic environment, simply configuring PI Batch on your PI server can make your team up to 400% more productive. This particular modification takes less than a day to accomplish.