Plasmid morphology determines the success or failure of fermentation: an analysis of the process differences between linear and circular plasmids

In the fermentation production of recombinant proteins, R&D teams often focus their attention on strain screening, promoter optimisation and the control of induction conditions, whilst frequently overlooking a key variable—whether the plasmid vector is linear or circular.

When the host and target gene are identical, a difference in vector conformation alone can lead to a series of issues, including significant variations in copy number, polarised stability across generations, increased fluctuations in fermentation yield, and elevated levels of downstream impurities. Many projects yield promising results during the small-scale shake-flask stage, only to experience a sharp drop in yield upon scale-up to fermentation tanks; the root cause often lies in the fundamental differences between linear and circular plasmids.

It must be made clear that the differences between the two are not limited to DNA structural morphology; fermentation processes are also not interchangeable. Circular plasmids are suitable for free-floating expression systems, whilst linear plasmids primarily integrate into the genome; the two differ fundamentally in terms of fermentation logic, seed culture strategies, feed control and loss prevention protocols. Only by thoroughly understanding the underlying characteristics of both types of plasmids and matching them with dedicated fermentation protocols can stable, high-yield, low-impurity industrial production be achieved.

I. Fundamental Differences in Expression Mechanisms

(1) Circular plasmids: Free-floating, autonomous replication, with high copy numbers coexisting with a high risk of loss

Circular plasmids are closed, covalently linked double-stranded DNA molecules that exist independently of the host chromosome. They replicate autonomously within the cell via their own replication origin, with dozens or even hundreds of copies maintained within a single cell.

Their advantages lie in their ease of construction, high transformation efficiency and flexibility in genetic manipulation, enabling rapid small-scale validation. However, their shortcomings are equally pronounced: there is a significant risk of plasmid loss during successive passages, particularly under conditions of high-density fermentation, prolonged cultivation and the stress of high-level expression of exogenous proteins. Plasmid-free cells, owing to their growth advantage, gradually come to dominate the bacterial population, leading to a precipitous decline in overall yield. If antibiotics are relied upon to maintain plasmid retention rates, antibiotic residues will be generated in the fermentation waste liquid, posing a substantial obstacle to the registration of food-grade and pharmaceutical-grade products.

(2) Linear plasmids: Genomic integration, where long-term stability coexists with copy number limitation

Linear plasmids lack a closed circular structure and are unable to replicate autonomously within the cell; their core function is not free amplification, but rather site-specific integration into the host chromosome via homologous recombination mediated by the two homologous arms at either end. Each cell retains only one to several stable copies, which fundamentally eliminates the risk of free plasmid loss.

The advantage of this approach lies in its outstanding stability across successive generations of the strain; expression levels fluctuate minimally even after dozens of fermentation cycles, and there is no need for continuous antibiotic addition for selection, thereby meeting the compliance requirements for pharmaceutical and food-grade raw materials. The limitations are equally evident: the low copy number results in a lower upper limit of expression per cell compared to high-copy circular plasmids; transformation and positive strain screening are labour-intensive, and the construction cycle is longer; yield increases cannot be achieved by increasing the copy number, but must be realised by optimising expression intensity at the level of the promoter, signal peptide and metabolic pathways.

In summary, circular plasmids excel in high-copy free-floating expression, with the risk of plasmid escape and loss; linear plasmids achieve long-term stability through genomic integration, but are limited by their restricted copy number.

II. Comparison of the Entire Fermentation Process: Process Parameters Must Not Be Interchanged Indiscriminately

1. Seed Culture Stage

· Circular plasmid system: A screening antibiotic must be added during seed activation to maintain plasmid retention; the number of activation passages must be strictly controlled, and multiple consecutive passages are prohibited. The standard procedure is: isolation of a single colony from an agar plate → primary shake flask seed culture (containing resistance) → secondary seed culture, with no more than three passages. Excessive passaging will lead to a rapid increase in the proportion of plasmid-free contaminating bacteria, directly jeopardising the entire fermentation batch.

· Linear plasmid (integrated strain) system: As the target gene is fixed on the chromosome, continuous antibiotic selection is unnecessary; seed culture can be passaged continuously, ensuring high strain stability. Operationally, only periodic verification of the integration site via PCR is required, making it suitable for large-scale, continuous, multi-stage expansion; it also offers superior reproducibility in pilot and production batches.

2. Cell proliferation phase in the fermenter

· Circular plasmid system: High-density fermentation is a double-edged sword. The higher the cell concentration, the greater the metabolic burden caused by exogenous expression, and the higher the probability of plasmid loss. Process strategies should aim to maintain a reasonable cell density and avoid excessive proliferation. A staged cultivation approach should be adopted, with rapid cell accumulation in the early phase and a suitably shortened induction period, to alleviate sustained metabolic stress. Typical issues manifest as significant plasmid loss in cells during the later stages when the fermentation cycle extends beyond 70 hours, resulting in a marked decrease in the target protein content in the supernatant and an increase in the proportion of contaminating proteins.

· Linear plasmid-integrated strain system: There is no risk of plasmid loss, and it can tolerate longer fermentation cycles and higher cell densities. This system allows for the full implementation of high-density batch cultivation with feed addition, extending the product accumulation time. There is no need to worry about yield decline caused by prolonged fermentation; the process has a higher tolerance for errors and is more suitable for pilot-scale upscaling and industrial continuous production.

3. Control of Induced Expression (the aspect showing the most significant differences)

· Circular plasmid system: High copy number drives high-intensity expression, resulting in the rapid synthesis of large quantities of recombinant protein within a short time. This is prone to inducing misfolding of the protein, the formation of inclusions, and triggering cellular stress-induced lysis. In practice, it is advisable to adopt mild or low-temperature induction strategies, reduce the inducer concentration, shorten the induction duration, and avoid induction during the senescence phase of the cells to prevent cell lysis and the release of intracellular proteases, which would lead to the degradation of the target protein.

· Linear plasmid-integrated strain system: The low copy number results in a reduced protein synthesis load and lower intracellular stress, leading to better protein folding quality and reduced degradation. The induction window is relatively broad, allowing induction to be initiated in the middle to late logarithmic phase to extend the product accumulation time; the product has a higher proportion of soluble protein and fewer fragments, significantly reducing the burden of downstream purification.

4. Fermentation Endpoint and Downstream Risks

Towards the end of fermentation of circular plasmids, large numbers of empty plasmid-carrying cells and lysed cells tend to appear; levels of host DNA, heterologous proteins and endotoxins (in the E. coli system) in the fermentation broth rise significantly, increasing the risk of HCP and residual DNA exceeding limits, and consequently raising purification costs. In contrast, strains carrying linear plasmids exhibit a more homogeneous population, minimal cell lysis and lower impurity levels. Variation in parameters between batches is minimal, making it easier to meet the quality control standards set by pharmacopoeias and for the registration of new raw materials.

III. Selection for Industrialisation: Applicability Limits of the Two Approaches

Suitable scenarios for circular plasmid fermentation:

1. Laboratory-scale pilot trials for the rapid screening of genes, promoters and signal peptides;

2. Short-cycle batch fermentation not involving prolonged continuous culture;

3. Scenarios without pharmaceutical-grade compliance requirements, where residue controls are lenient and the aim is to achieve high instantaneous expression levels.

Not suitable for long-term continuous fermentation, multi-generation seed culture expansion, or the mass production of food-grade or injectable-grade raw materials.

Suitable applications for linear plasmid integration fermentation:

1. Pilot-scale upscaling and stable industrial-scale mass production requiring batch-to-batch consistency;

2. Production of raw materials for food, aesthetic medicine and pharmaceuticals, where antibiotic residues are prohibited;

3. Long-duration fermentation, multi-stage seed culture expansion and long-term strain preservation;

4. Products with extremely stringent quality control requirements regarding host DNA, contaminating proteins and degradation fragments.

It is important to note its limitations: integrated strains struggle to achieve ultra-high transient expression levels, and yield improvements must rely on molecular engineering and fermentation process optimisation.

IV. Analysis of Common Misconceptions in the Industry

Misconception 1: A low copy number of linear plasmids inevitably results in low yield.

This is not the case. During the later stages of circular plasmid fermentation, a significant amount of plasmid is lost, leading to a continuous decline in the population of effectively expressing cells; in contrast, integration strains exhibit stable expression throughout the process, and their cumulative yield over the long term often exceeds that of systems using high-copy free plasmids.

Misconception 2: The same fermentation SOP can be applied to both types of plasmids.

Applying the established fermentation parameters for circular plasmids directly to integrated strains may result in insufficient product accumulation due to overly conservative induction conditions, or cause cell lysis due to the blind pursuit of extremely high cell densities. As the vector morphology changes, the entire process must be re-optimised.

Misconception 3: Adding antibiotics is sufficient to prevent plasmid loss.

Antibiotics can only inhibit the growth of plasmid-free cells; they cannot prevent spontaneous plasmid loss. Under conditions of high-density, long-term fermentation, the effectiveness of antibiotic selection steadily diminishes; it can only mitigate the problem, not resolve it completely.

V. Conclusion

Circular plasmids are highly efficient tools for rapid research and development at the laboratory stage, offering the advantages of convenience and flexibility, and are suitable for early-stage process exploration; linear plasmid-integrated strains, on the other hand, are a reliable choice for industrial-scale mass production, with stability, regulatory compliance and scalability as their core advantages. Competition in fermentation during the era of synthetic biology has shifted from the mere pursuit of high copy numbers at a single point in time to a comprehensive assessment of batch stability, controllable impurity levels and regulatory compliance. Accelerating R&D iteration with circular plasmids during the small-scale trial phase, and transitioning to linear plasmid-integrated strains for stable mass production during the pilot phase—a seamless integration of these two approaches represents the optimal pathway for the industrialisation of recombinant proteins.