Why Is There Antibiotic in Cell Culture Media?

Answer: Antibiotic is there to kill microbes, if any.

That's the short-answer.

Question: Why would there microbes in the cell culture?

Answer: Poor aseptic practices.

So if you have good aseptic procedure, then you ought not have antibiotics in the media, right?

Answer: Right, but we still keep it in just in case.

This type of thinking pervades large-scale cell culture. And ironically, it's backwards.

I'm sure there are smart ways of using antibiotics in cell culture, but I'm not aware of any.

Antibiotic Resistance

Molecular structure of Gentamicin

The first problem with antibiotics is that they select for resistance: over time, the organisms that are susceptible to antibiotics die off leaving the antibiotic resistant organisms to remain. These organisms may become slow-growing, low-level contaminations that are difficult to detect.

Detection

The second problem with antibiotics is that they interfere with detection. QC Microbiology tells us their tests are sensitive to 1 colony-forming unit (CFU) in the sample, which means if there is 1 CFU in a 40mL sample bottle, they'll find it. But 1 CFU/40mL is 25 CFU/L... and for a 12,000L bioreactor, you need a contamination of ~300,000 CFUs in order for QC to detect contamination.

The sooner the culture hits 300,000 CFUs (assuming you have a 12kL), the sooner you know there's a problem, and antibiotics slow things down.

Poor Aseptic Technique

The third problem with antibiotics is that it lets you get away with sloppy procedure. Back in 1973, M.F. Barile's study of mycoplasma contamination in cell culture found that 72% of cultures grown continuously in antibiotics were contaminated versus 7% of cultures without antibiotics. The conclusion was that over-reliance on antibiotics leads to poor aseptic technique.

by Oliver Yu

Happily, we are coming across more and more customers who don't use antibiotics at the production scale, and generally speaking, these contamination investigations are mercifully straightforward.

Those other guys have long weeks of meetings ahead of them.

See also:

• Basic Contamination Probabilities
• If Kryptonite is Root Cause, What's the CAPA?
• Get a FREE Contamination Case Study

Viral Inactivation of Cell Culture Media HTST

by Oliver Yu

For all this talk of bioreactor sterility, the vast majority of this contamination talk refers to bacterial contamination.

What about viral contamination?

Viral contamination is mitigated with HTST treatment of cell culture media. This is where you put the cell culture media in continuous flow while subject to a high temperature for a short period time.

• High-temperature inactivates the virus.
• Short-time ensures that cell culture media components do not denature.

The best time to put the media in continuous flow is when you pump the media from the media prep tank to the bioreactor, so viral inactivation often happens during this transfer. The HTST unit is essentially:

• Heater
• Hold-tube (insulated pipe)
• Cooler

At large-scale, the first plug of media that goes through the HTST may not meet the specification, so this plug of media cannot be permitted to be delivered to the bioreactor. This plug may be sent to drain or recycled through the HTST unit until the HTST unit reaches steady-state.

Once at steady-state, the remainder of the media is pumped (through a sterile-filter and subsequent sterile pipes) into the bioreactor; when batch volume is reached, the remaining media is sent to drain.

Simple enough, right?

The hard part is when your HTST performance begins to degrade:

• Perhaps your sterile filter starts clogging
• Perhaps your heater controller output maxes out
• Perhaps your media-prep post-use is showing problems

As recently as January of this year, Amgen's Drug Product Development published a paper titled, "Identification and root cause analysis of cell culture media precipitates in the viral deactivation treatment with high-temperature/short-time method."

I haven't read the paper, but my manufacturing sciences consulting experience predicts it to say the following:

The calcium phosphate in the cell culture media becomes insoluble at the high temperatures during the HTST. This calcium phosphate precipitate may collect on the surface of the holding-tubes thereby decreasing the heat-transfer coefficient, sporadically causing the HTST to fail.

This calcium phosphate (sandy white stuff) may also clog up the 0.1 micron sterile filter causing a high delta-pressure across the sterile filter, maybe even diminishing the media flow rate.

The problem is that calcium phosphate stays in solution except when both the temperature is high and the pH is high.

Unfortunately, high-temperature is a requirement of HTST; which means the only solution to preventing calcium phosphate precipitation and the ensuing HTST performance degradation and filter clogging is to run the media through at low pH.

See Our Fix

This is a classic multivariate problem where operating in a different range will solve the problem.

See also:

• How to Make Large-Scale Cell Culture Media
• Manufacturing Sciences - Plant Support
• Multivariate Analysis in Biologics Manufacturing

How to Prepare Large-Scale Cell Culture Media

Cell culture media is to mammalian cells what a workout smoothie is to your human cells.


Media's purpose is to make a stainless steel bioreactor hospitable to CHO cells by providing volume where temperature, dissolved oxygen (dO2), and pH can be controlled.

As well, it must provide nutrients intended for cellular uptake as well as a place to absorb metabolic waste. At the start of cell culture, the nutrient supply is defined; by the end of cell culture, the nutrients are depleted. Despite the added nutrients, media is still largely water and can thus be modeled at 1g/mL.

You make cell culture media the same way you'd make a smoothie, except at large-scale, you're making 100 or 500 or even 15,000 liters and so there are differences.

1. Initial QS.
   This is where you add water-for-injection (WFI) into a clean media preparation tank.
2. Add media powder.
   Once the powder touches the water, the media can promote growth. Since the bioreactor is only clean (and not sterile), if you don't proceed quickly, you may have a contamination on your hands.
3. Add bicarbonate powder
   Bicarbonate is the buffer. This whole time, you are agitating, and pH control is OFF.
4. Add peptones (optional).
   Over the past decade, we've seen movement away from bovine (cow) to porcine (pig) peptone. I've read that we now use veggie peptone, but have never seen it.
5. Adjust pH
   Some will dispute the necessity of adjusting the pH in the media prep tank because once the media is transferred to the bioreactor the pH will get adjusted there.
6. Final QS
   This is where you add the rest of the water. If you have an osmolality specification, you'd measure it here as an in-process test before transferring the media to the bioreactor
7. Transfer/sterile filter the media
   While pumping the media over to the bioreactor, there will be sterile filters that remove 0.1 micron particles so that the media that ends up in the bioreactor is free of microbes. In some cases, the media is virally inactivated by passing through a "pasteurizer" that raises the temperature to 121 degC, holds it for 1 minute and cools it down.

by Oliver Yu

It's a bit more than making a smoothie since mixing in a blender is forgiving. But in the preparation of cell culture media where you are making thousands of liters of this stuff at 7 bucks per gallon ($2/liter), the large-scale media preparation procedure has to be written to be highly reproducible.

Credits: Image above is from the greatest movie of all time - The Matrix (1999).

Multivariate Analysis in Biologics Manufacturing

All these tools for data acquisition and trend visualization and search are nice. But at the end of the day, what we really want is process understanding and control of our fermentations, cell cultures and chromatographies.

Whether a process step performs poorly, well or within expectations, put simply, we want to know why. 

For biological systems, the factors that impact process performance are many and there are often interactions between factors for even simple systems such as viral inactivation of media.

One time,  clogged filters with white residue were the result when transferring media from the prep tank to the bioreactor. On several occasions, this clogging put the transfer in hold and stopped production.

After studying the data, we found that pH and Temperature were the two main effects that significantly impacted clogging. If the pH was high AND the temperature was high, the solids would precipitate from the media. But the pH or temperature during the viral inactivation was low, the media would transfer without exception.

After identifying the multiple variables and their interactions, we were able to change the process to eliminate clogging as well as simplify the process.

For even more complex systems like production fermentation, multivariate analysis produces results. In 2007, I co-published a paper with Rob Johnson describing how multivariate data analysis can save production campaigns. From the article is the regression pictured below.



You can see that it isn't even that great a fit. Statisticians shrug all the time at RSquares less than 0.90. But from this simple model, we were able to turn around a lagging production campaign and achieve 104% Adherance To Plan (ATP).

The point is not to run into trouble and use these tools & know-how to fix the problem. Ideally, we understand the process ahead of time by designing in-process capability and then fine tune it at large-scale; we are less fortunate in the real world.

My point in all this is if you are buying tools and assembling a team without process understanding and control,  then you won't know which are the right tools or what is the best training. Keeping your eye on the process understanding/multivariate analysis prize will put you in control of your bioprocesses and out of the spotlight of QA or the FDA.