The Impact of Nucleic Acid Contamination in Protein Purification

From uncovering how proteins work at the bench to developing therapeutic biologics or scaling up enzyme production for industrial fermentation, protein purification underpins the science and the applications that follow.

But regardless of the scale, common issues prevail:

1. Cell lysis, mechanical, chemical, or enzymatic, releases chromosomal DNA into solution
2. High molecular weight DNA tangles into long chains that transform lysates into sticky, viscous mixtures [1, 2]
3. The denser the culture, the greater the viscosity
4. The greater the viscosity, the more it blocks filters, slows centrifugation, and impairs chromatography [3]

In both academic and industrial labs, these issues translate into clogged spin filters, smeared pellets, wasted reagents, and non-reproducible experiments. In industrial processes, they can mean blocked filters, fouled chromatography columns, and entire batches of valuable product lost.

Nucleases, which digest DNA and RNA, are the best way to deal with this. But there’s a catch: They can be expensive, which tempts some labs to cut corners and can challenge the economics of large-scale processes.

This article explores the problems of nucleic acid contamination in protein purification and bioprocessing workflows. It outlines the causes, the regulatory and economic consequences, and the solutions, both traditional and modern, that make nuclease treatment more accessible for everyone, from academic labs to biopharma and biomanufacturing plants.

Consequences of Nucleic Acid Contamination in Protein Purification

1. Increased Viscosity and Workflow Challenges 

Viscous lysates cause all sorts of practical bottlenecks:

1. Inaccurate and slow pipetting
2. Clogged sterile filters
3. Chromatography runs slower, reducing productivity

2. Reduced Product Quality and Yield

DNA can also reduce yield and quality:

1. It can bind target proteins directly, leading to co-precipitation and poor recovery
2. Its negative charge allows it to occupy anion-exchange columns, crowding out the intended protein
3. And even after purification, residual DNA can promote protein aggregation, reduce enzymatic activity, and cause instability during storage

3. Misleading Experimental Results 

Residual nucleic acids can distort downstream assays by interfering with binding and enzymatic activity studies or skew spectrophotometric readings. And in cell-based experiments, it can even trigger nucleic acid–sensing pathways.

4. Regulatory and Safety Considerations 

For therapeutic proteins and precision fermentation processes within the foodtech sector, DNA contamination can cause severe issues. Residual DNA above regulatory thresholds renders products unusable, due to risks of oncogenic, infectious, or immunogenic effects. [4]

Regulators impose strict limits:

1. 10 pg to 10 ng of DNA per therapeutic dose
2. Fragments limited to 200 bp or smaller [5, 6]
3. Both WHO and FDA stipulate that DNA content must not exceed 10 ng per dose

5. Operational and Economic Impact 

On the processing floor, nucleic acid contamination translates directly into inefficiency and cost by clogging and fouling filters, blocking columns, and slowing flow rates.

A MilliporeSigma study showed that without nuclease treatment, chromatographic flow rates fell to 0.5 mL/min. With nuclease treatment, flow rates improved tenfold and protein recovery rose 15–40%. [7]

The result is inevitable: Longer processing times, higher labor and energy use, more frequent filter replacements, and ultimately higher production costs that can wreck the economics of a process.

Strategies for Mitigation 

Products like Benzonase® and Denarase® have been the weapons of choice against nuclear contamination. They are effective, but their high cost often limits them to critical applications rather than routine workflows. 

Next-generation nucleases, such as enGenes e^xrase®, now provide equivalent performance at a fraction of the cost.

Derived from the well-characterized Serratia marcescens endonuclease, e^xrase® matches traditional nucleases in both breadth and efficacy. It rapidly hydrolyzes DNA and RNA, whether single- or double-stranded, across a wide range of processing conditions.

The cost advantage comes from how it’s made. The enGenes platform uses growth-decoupled expression and facilitates extracellular secretion, which drives markedly higher expression yields. At the same time, it simplifies downstream processing by reducing the number of unit operations. Together, these features deliver a 30-fold cost saving, making nuclease treatment more affordable and practical for both small labs and large-scale operations.

Learn More 

From clogged filters in a protein prep to regulatory barriers in a biopharma and food product pipeline, nucleic acid contamination in protein purification and bioprocessing workflows is a universal problem. Addressing it effectively improves reproducibility, saves time, and safeguards product quality.

Now with the advent of modern, cost-effective nucleases, labs of all sizes can treat nucleic acid contamination as a solvable problem rather than an unavoidable frustration.

If you would like to learn more about nucleic acid removal or are curious about how e^xrase® might fit into your workflow, the team at enGenes is always happy to share more detailed technical information or provide samples for your own evaluation. You can get in touch with them at [email protected].

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.

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