How to boost cell culture reproducibility and cut cytokine costs  

  • Activity-based cytokine dosing allows for reproducible product yields
  • Standardized, calibrated cytokine activities enable reliable manufacturing conditions
  • Optimal cytokine dosing and transparent activity data optimize cost calculations

During ex vivo cell processing, similar to in vivo cell-to-cell communication, the cellular response to a cytokine stimulus depends on the concentration of active cytokine. However, due to the production process of these recombinant proteins, the concentration of biologically active recombinant cytokine deviates from the total protein concentration.  

The consequences of this are striking for both, your research and your cost calculations. Find out below, why you should know all the facts about the differences between total and active cytokine concentrations, their impact on the cellular response the cytokine product elicits, and how standard lot-to-lot testing of our MACS® GMP Cytokines increases reliability of cell manufacturing conditions.   

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Cytokine activity

What is "biological activity" or "cytokine activity"?

The biological activity of a recombinant cytokine (cytokine activity) is a measure for its effectiveness in inducing a particular biological response. 

Biologically active cytokines induce a cellular response.
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In this example of dendritic cell differentiation, 10 µg of IL-4 and GM-CSF of different biological activities yield different amounts of dendritic cell product.

Why do recombinant cytokines have variable biological activities?

Processes that occur during recombinant protein manufacturing significantly influence cytokine activity. Proteins expressed in E.Coli, for example, are subjected to misfolding and aggregation; and protein production in mammalian systems depends on glycosylation patterns.

Protein refolding during recombinant protein production.
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Many proteins are expressed in E.Coli in the form of insoluble inclusion bodies, which are aggregates of denatured protein. In the production process, these protein aggregates are denatured and refolded into active cytokines. However, during refolding some proteins may misfold or reaggregate.

Variable ratio of active cytokine per production batch.
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Only correctly refolded cytokines are biologically active and their ratio in the total cytokine amount can vary. Thus, protein production can result in different biological activities of identical amounts of cytokines.

How is the biological activity calculated?

Biological responses induced by cytokines follow a sigmoidal-shaped dose-response curve in which the concentration of the cytokine (x-axis) is plotted against its biological effect (y-axis). These dose-response curves can be used to calculate the biological activity of a cytokine batch. 

Cytokine activity determination.
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Based on the dose-response curve, the cytokine concentration necessary to induce half of the maximum effect (ED50) is identified. With this ED50 value, the biological activity is calculated following an easy formula.

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Download our brochure to get a detailed introduction into calculation of cytokine activities.

MACS GMP Cytokines are provided with the lot-specific biological activity

The specific activity for each lot of MACS GMP Cytokines is stated on the respective product quality certificate. Knowing the lot-specific activity, you can always apply the same concentration of active cytokine and create reproducible conditions for ex vivo cell culture processing. 

View the complete product list of MACS GMP Cytokines.

Cytokine standardization

What is calibrated cytokine activity?

Bioassays used for the determination of cytokine activities yield only relative values. These values are not comparable between different assays, between different laboratories performing the same assay, or between the same assay performed in the same laboratory on different days. 

Therefore, bioassays used for activity determination are calibrated with cytokine standards. The resulting cytokine activities measured with calibrated bioassays are then comparable to each other.

What are internationally recognized cytokine standards?

To achieve comparability of cytokine activities between different suppliers and laboratories, bioassays must be calibrated with an internationally recognized standard defined by the WHO. Such standards are available for a large number of cytokines and are distributed by the National Institute for Biological Standards and Control (NIBSC). 

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Get a detailed introduction into calibration of cytokine activities within this PDF.

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Download a table of bioassays and calibration reagents used for calibration of MACS GMP Cytokines.

MACS GMP Cytokine activities are calibrated with internationally recognized standards 

All MACS GMP Cytokines are calibrated using NIBSC international standards or reference reagents whenever these are available. The specific activity for each cytokine lot is stated on the respective product quality certificate. This allows for reliable cytokine usage and maximizes comparability of your results with other laboratories and other publications. 

View the complete product list of MACS GMP Cytokines.

Preferring activity-based over weight-based cytokine dosing

What are the disadvantages of weight-based cytokine dosing? 

Using the same weight-based amounts of cytokine batches of different biological activities results in variable cellular responses.

Weight-based cytokine dosing introduces variability.
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The dose-response curve indicates that applying the same cytokine amount (10 µg IL-2) of different production batches may induce significantly different cellular responses. In this example, batch A would induce no significant response, batch B ~50% of the maximum response, and batch C would induce the maximum cellular response.

Without knowing the lot-specific activity of a cytokine, researchers may (1) add excessive cytokine to reliably reach saturation or (2) add cytokines in a reasonable amount and accept the resulting variability. However, both possibilities have significant drawbacks.

Excessive cytokine doses increase expenses.
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Adding excessive cytokine amounts in order to reliably reach saturation with weight-based cytokine dosing results in higher reagent expenses (up to 10-fold cost increase).   

Furthermore, high cytokine doses can be detrimental to cells or alter cell behavior, leading to variable results: 

  • High IL-2 doses induce differentiation of effector CAR T cells, while low IL-2 doses preferentially induce differentiation of early memory T cells, reducing the efficacy of the CAR T cell product (Kaartinen et al.).
  • Monocytes/macrophages generated with different doses of GM-CSF differ in their function (Sun et al.).
Insufficient cytokine doses increase experimental variability.
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Working with reduced weight-based cytokine concentrations will result in a high inter-experimental variability. This will not only lower data quality, it will likely also result in an indirect cost increase, as repetition of experiments and possible troubleshooting will be necessary. 

What are the advantages of activity-based cytokine dosing?

Activity-based cytokine dosing ensures high experimental reproducibility: (1) The same amount of active cytokine can be added every time, resulting in a significant reduction of experimental variability. (2) Oversaturation can be avoided, circumventing detrimental effects to the cells. (3) Both, cytokine amount and experimental retesting can be reduced, resulting in significant cost and time savings. 

Activity-based cytokine dosing allows for experimental reproducibility.
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Applying cytokines based on their biological activity allows for reliable and exact cytokine dosing. Variations in cellular responses due to variable cytokine activities or excessive amounts of cytokines can be circumvented, increasing cell culture reproducibility.

The graphs below show the significant reduction in experimental variability that can be achieved when combining activity-based dosing and calibration with international standards. The data was generated in different laboratories that either used cytokines calibrated with their own in-house standards (left) or cytokines calibrated with an international standard (right).

MACS GMP Cytokines allow for reliable activity-based cytokine dosing and cost calculations

Knowing the lot-specific activity of MACS GMP Cytokines allows for convenient activity-based dosing. You can easily apply the same amount of active cytokine and use optimal cytokine concentrations. This maximizes reproducibility of ex vivo cell culture processing, saves time and reagent expenses, and even makes cost calculations for future projects more reliable. 

If you want to learn more about what implications activity-based dosing has for cost calculations, for the logistics of larger projects, and what caveats to avoid, find more information here.  

Scroll through our comprehensive list of MACS GMP Cytokines and find detailed product information on the respective product pages

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