The Impact of Nucleic Acid Contamination in Bioprocessing and Protein Purification Workflows

Within biotech, it's hard to think of an application that doesn’t involve proteins in some way or another. Whether it's research to identify and characterize proteins of interest, structural biology, development of a therapeutic protein or industrial fermentation, protein purification will be a key part of the workflow.

Many approaches to protein purification exist and one step common to all is the need to eliminate host nucleic acids that are released during the process. While nucleases – enzymes that digest DNA and RNA – are critical for this step, commercial products often come at extremely high prices that limit their use. 

This article outlines why nucleic acid contamination is a major problem in protein purification and presents the modern-day solutions.

So, why should we care about nucleic acid contamination?

DNA and RNA can wreak havoc if not removed during protein purification, and the hidden costs that result – from reduced yields to processing delays – can be devastating. For instance, premature release of nucleic acids during cell lysis can significantly increase viscosity, rendering downstream purification steps such as centrifugation or filtration impossible due to equipment clogging, resulting in complete batch loss.

Before we explore the ways in which nucleic acids can impede protein purification, let's consider the broader implications that extend beyond the laboratory.

Within the biotech and biopharmaceutical industry, excessive nucleic acid contamination can leave companies no choice but to discard or reprocess entire commercial-scale batches worth millions of dollars. The consequences can go far beyond immediate financial losses to the company, and may result in missed regulatory deadlines and delayed patient access to critical therapies.

With such implications, effective nucleic acid removal is not just a technical step to improve the purification workflow; it can be the determining factor in a company’s overall success.

Viscosity: nucleic acids turn cell lysates into syrup

To purify proteins from cell cultures or tissues, the cells and tissues are first lysed using physical, enzymatic or chemical methods, alone or in combination. Regardless of how the cells are lysed, chromosomal DNA will be released into the extracellular space upon lysis. In particular, high molecular weight DNA tends to form tangled chains that dramatically change the viscosity of the lysate making it thick, clumpy, and sticky (1, 2).

The extent of viscosity in the lysate worsens as cell density increases, and this can delay and impede downstream bioprocessing in many ways: by making pipetting and handling difficult, quickly blocking sterile filters used to filter cell lysates thus reducing lysate filtration and centrifugation efficiency, interfering with chromatography flow rates and potentially trapping the target protein leading to reduced overall yield (3).

Reduced product quality and yield

As alluded to earlier, viscous DNA present in a cell lysate can entrap or co-precipitate proteins, leading to reduced yield or complete loss of the target protein. Furthermore, and because of its tendency to be negatively charged, DNA can bind anion-exchange resins used in purification columns and significantly reduce the available binding capacity intended for the target protein.

In addition, residual nucleic acids that remain as contaminants in the protein product after purification can alter protein properties, causing unwanted aggregation, reduced and/or altering biological activity and interference with downstream biochemical assays. Nucleic acid-protein complexes in the final product also create batch-to-batch variability and may lead to instability during storage.

Critically, for therapeutic applications, DNA contamination above regulatory limits renders products unusable for clinical development. This is because regulators recognize the safety risks of residual nucleic acids including the potential for oncogenic, infectious or immunogenic effects when administered to patients (4). For this reason, regulatory agencies have specific guidelines for acceptable limits, typically ranging from 10 pg to 10 ng DNA per therapeutic dose depending on the product and therapeutic modality.

For example, the World Health Organization and U.S. Food and Drug Administration stipulate that DNA content in a final biopharmaceutical product should be less than 10 ng per therapeutic dose, with residual DNA fragments limited to 200 base pairs or smaller (5, 6).

In short, if nucleic acids are present during or after protein purification, protein yield will be greatly impacted with a substantial risk to product quality.

Process, operational, and economic impact

Nucleic acid contamination creates significant bottlenecks that impede processing efficiency and operational performance. As mentioned previously, contaminating nucleic acids slow the protein purification process by causing delays at key steps, promoting cell clumping and reducing filtration and chromatography performance.

A study carried out by MilliporeSigma found that without nuclease treatment to remove nucleic acids during purification, chromatographic flow rates reached as low as 0.5 mL/min, while nuclease treatment improved flow rates nearly ten-fold to ~5 mL/min, with protein recovery increasing by 15–40% (7).

These processing issues can culminate into operational problems including filter clogging and fouling (during sterile or virus filtration), chromatography column blockages, and reduced flow rates that strain equipment and increase maintenance requirements. The cumulative effect extends batch processing times, increases staff costs, drives higher energy usage and may demand more frequent filter replacement.

Ultimately, these bottlenecks, inefficiencies, and operational challenges translate directly into economic losses - from increased labor and raw material costs to equipment maintenance expenses and potential delays in bringing products to market.

New-generation nucleases overcome the cost barriers

Modern nucleases offer a proven solution to all of the issues described in this article, by rapidly fragmenting double-stranded DNA, thereby reducing viscosity, enhancing filter capacity and overall clarification efficiency.

However, while the effectiveness of traditional nucleases such as Benzonase® or Denarase® is undisputed, their high price limits their widespread use in routine applications.

New-generation, cost-effective nucleases such as enGenes eXrase address those economic barriers while maintaining equivalent performance, making nuclease treatment more accessible for routine laboratory use.

For example, while traditional nucleases can cost more than €120,000 per 2000L batch of fermentation/culture, eXrase achieves the same DNA removal efficacy at approximately €4,000 per batch; this represents a 30-fold cost reduction with no compromise to performance. This dramatic cost advantage makes nuclease treatment economically feasible even for large-scale operations and routine laboratory use.

Learn more about eXrase or try it for yourself!

If you would like to learn more about nucleic acid removal or are curious about how eXrase might fit into your workflow, we'd be happy to share more detailed technical information or provide samples for your own evaluation.

Please feel free to get in touch with our team here.

References

1.     Berg MC, Sorz Y, Hahn R, et al. Streamlining process development and scale-up: Risk assessment to reduce workload in primary protein recovery. Biochem Eng J. 2024;212:109513.

2.     Berg MC, Beck J, Karner A, et al. Mass transfer of proteins in chromatographic media: Comparison of pure and crude feed solutions. J Chromatogr A. 2022;1676:463264.

3.     Berg MC, Erdem I, Berger E, et al. Genomic DNA causes membrane fouling during sterile filtration of cell lysates. Separation and Purification Technology. 2023 324:124540.

4.     Wang X, Morgan DM, Wang G, Mozier NM. Residual DNA analysis in biologics development: review of measurement and quantitation technologies and future directions. Biotechnol Bioeng. 2012 Feb;109(2):307-17.

5.     Grachev V, Magrath D, Griffiths E. WHO requirements for the use of animal cells as in vitro substrates for the production of biologicals (Requirements for biological susbstances no. 50). Biologicals. 1998 Sep;26(3):175-93.

6.     FDA, Food and Drug Administration. Center for biologics evaluation and research. Guidance for industry: “Characterization and qualification of cell substrates and other biological materials used in the production of viral vaccines for infectious disease indications.” US Food and Drug Administration, Bethesda, MD. 2010.

7.      Merck KGaA. (undated). Benzonase® Nuclease: Effective removal of nucleic acids and viscosity reduction from protein solutions [Technical Brochure]. Darmstadt, Germany. Accessed 20th June 2025.