Nucleic Acid Immunization for Rapid and Cost-Effective Antibody Discovery
Introduction
Over the past few decades, monoclonal and polyclonal antibodies have transformed medicine, diagnostics, and life science research. Two core platforms, animal immunization and in vitro display technologies, have driven antibody generation, each with distinct advantages and limitations (1). Yet, as therapeutic targets grow more complex, so must our discovery tools.
Animal immunization has stood the test of time for over a century (1). It offers the biological advantage of in vivo affinity maturation and lineage diversity, producing antibodies with naturally high affinity and stability. In addition, transgenic animals and humanization techniques have further advanced this method for therapeutic applications (1). However, the need to produce, purify, and quality-control protein immunogens can be a barrier in time-sensitive or structurally sensitive programs (1, 2, 3).
Enter Genetic Immunization: Simpler, Smarter and Cheaper
Genetic immunization circumvents these challenges by enabling in vivo expression of antigens from nucleic acids either DNA or RNA (1, 2, 3). Once delivered into the host, these molecules are translated into the target protein, which is then recognized by the immune system, triggering an antibody response. Because the antigen is produced inside the animal, this method preserves its native folding, post-translational modifications, and membrane context factors critical for targeting conformational epitopes(1, 2, 3, 4).
Key Advantages:
Bypass protein production: eliminates the need for antigen purification
Maintain native conformation: ideal for membrane-bound or unstable proteins
Initiate discovery from sequence alone: accelerate early-stage programs
Highly adaptable: supports targets from viral, bacterial, and human sources
Many Benefits, but Challenges Persist
Despite its potential, efficient genetic immunization depends on three critical factors: nucleic acid design, purity, and delivery. Poorly optimized plasmid DNA (pDNA) or mRNA sequences can reduce antigen expression or alter protein conformation, undermining the immune response. Likewise, impurities from plasmid or IVT mRNA production, such as residual host DNA, dsRNA, or endotoxins, can interfere with transgene expression or trigger unwanted immune activation linked to inhibition of protein synthesis (5). Delivery is another key hurdle (2). While naked DNA/RNA injection is simple, it often yields weak responses. Alternatives like in vivo electroporation or gene gun techniques improve uptake but require specialized equipment, are time-consuming, and may negatively impact animal welfare. Lipid nanoparticles (LNPs), particularly for mRNA, offer efficient delivery and have been widely used in vaccines but they also come with significant investment costs associated with formulation instruments, consumable requirements, and formulation challenges. Indeed, LNP development demands technical expertise, iterative optimization for each payload or target, and often results in significant nucleic acid loss during formulation(6).
These limitations underscore the importance of building a streamlined, well-integrated workflow for successful implementation of genetic immunization.
Building a Robust Genetic Immunization Workflow
To unlock the full potential of genetic immunization, it is essential to have a reliable, optimized upstream and downstream process. This includes:
High-Quality DNA Design and Production
The foundation of any genetic immunization strategy is the plasmid. Sartorius offers cutting-edge plasmid design and manufacturing services, featuring e-Zyvec®, a unique and innovative DNA assembly technology. Through our user-friendly plasmid platform, you can freely design your plasmids, focusing on your sequences of interest. Our experts will then review your design and advise you, when required, relevant ways to improve your molecules.
Our innovative DNA assembly technology empowers you to obtain the precise plasmid sequence you need, no matter how complex, to express the desired protein. We provide codon optimization to help maximize the potential for improved antigen expression and immunization. Furthermore, if you plan to in vitro transcribe your pDNA into mRNA, you can customize your pDNA sequence by selecting a promoter of interest (e.g., T7, SP6, or T3), adding (5) and (3) UTR regions of your choice, or incorporating a PolyA tail of up to 120 base pairs. All of this is efficiently delivered with the support of our expert team.
Advanced Purification of DNA and RNA
Nucleic acid purity directly impacts expression efficacy, and thus, the immune response. CIMmultus® ready-to-use chromatographic columns are ideal for the rapid and scalable purification of both plasmid DNA and mRNA, offering high resolution and throughput in bioprocessing environments. CIMmultus® columns range in sizes suited for milligram purification, all the way to commercial multi-gram production scale under GMP conditions. Scaling down to microgram production is also easy using CIM® multi-well plates (24-well or 96-well plate). Read More
Molecules | pDNA | pDNA | mRNA | mRNA | mRNA |
|---|---|---|---|---|---|
Application | Capture | Polishing | Capture | Capture | Polishing |
Column Chemistry | DEAE | SDVB | |||
Type of Chromatography | Anion Exchange | Hydrophobic | Affinity | Multimodal | Reversed Phase |
*Purification of pDNA: CIM® DEAE and CIM® C4 HLD are available in pack, see HiP² Plasmid Process Pack.
Optimized In Vitro Transcription (IVT) Process Development Services
Production of high-quality RNA is the basis of successful RNA immunization. Cornerstone® process development services can accelerate the use of RNA for immunization by developing optimized IVT protocols and purification workflows. This service is especially beneficial for organizations planning to manufacture DNA and RNA in-house but facing constraints in time, expertise, or personnel.
In addition, PATfix mRNA enables characterization of RNA purity and integrity, as well as reliable at-line insight during process development and production of mRNA, with emphasis on IVT reaction components (nucleotides and capping reagents).
Rapid and Cost-Efficient In Vivo Delivery of Nucleic Acids
Genetic immunization is effective only when nucleic acids successfully reach their cellular targets. With over 650 peer-reviewed publications, Sartorius' ready-to-use delivery reagents, in vivo-jetRNA+® for RNA (including mRNA, circRNA, saRNA) and in vivo-jetPEI® for DNA, provide reliable solutions for the critical step of in vivo delivery. With robust in vivo transfection efficiency, they have demonstrated validated performance across a diverse array of animal models (mouse, rat, rabbit, chicken, alpaca, etc.) and through diverse routes of administration (intramuscular, subcutaneous, intravenous, intraperitoneal, etc.), making them ideal for genetic immunization purposes. These reagents feature a simple 2-step formulation protocol, eliminating the need for specialized formulation and delivery equipment or expertise. Their very high encapsulation efficiency ensures minimal loss of nucleic acids, addressing one of the primary challenges associated with lipid nanoparticles (LNPs) and thereby offering higher cost-efficiency.
Advanced BioAnalytical Instruments for Antibody Discovery
A key determinant of success in antibody discovery is understanding biomolecular interactions at every step. With Sartorius technologies integrated across the workflow, researchers can move from hypothesis to validated antibody candidates with greater confidence, clarity, and speed.
The journey begins with characterizing how, and how well, an antibody binds to its target. Octet® Biolayer Interferometry (BLI) Systems enable high-throughput, label-free analysis of binding kinetics, affinity ranking, and epitope binning—all critical data for identifying the most promising candidates early in the pipeline.
Next comes deeper efficacy studies. The iQue® High-Throughput Screening (HTS) by Cytometry Platform accelerates this phase with rapid multiplexed analysis of immune cell function, ADCC activity, and comprehensive cell health metrics.
To further refine selection, the CellCelector® System automates the screening and isolation of individual cells. This streamlines the identification of clones with optimal antibody characteristics, replacing labor-intensive manual processes with precision automation.
Finally, the Incucyte® Live-Cell Analysis System delivers physiologically relevant insights into cell health and morphology. With real-time, non-perturbing monitoring, researchers can ensure their antibody-producing cells are thriving under optimal conditions.
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Final Thoughts: Transforming Discovery Pipelines
As biologics R&D continues to evolve, genetic immunization represents a compelling path forward, particularly for targets that are poorly suited to traditional platforms. Whether you are dealing with challenging-to-express membrane proteins or require a swift transition from sequence to antibody, this method provides unparalleled flexibility and speed.
By leveraging Sartorius’ plasmid engineering and manufacturing services, DNA/RNA purification and IVT tools and expertise, along with cost-efficient in vivo delivery reagents like in vivo-jetRNA+® or in vivo-jetPEI® you can accelerate your antibody discovery processes, reduce costs, and access antibodies that were previously unattainable.
Furthermore, post-immunization, Sartorius' advanced bioanalytical instruments facilitate the rapid identification and characterization of candidate molecules, ensuring superior target reactivity and optimized functionality.
References:
1:Laustsen AH, Greiff V, Karatt-Vellatt A, Muyldermans S, Jenkins TP. Animal immunization, in vitro display technologies, and machine learning for antibody discovery. Trends Biotechnol. 2021 Dec;39(12):1263-1273. doi: 10.1016/j.tibtech.2021.03.003. Epub 2021 Mar 25. PMID: 33775449.
2: Dodd RB, Wilkinson T, Schofield DJ. Therapeutic monoclonal antibodies to complex membrane protein targets: antigen generation and antibody discovery strategies. BioDrugs. 2018 Aug;32(4):339-355. doi: 10.1007/s40259-018-0289-y. PMID: 2993475 (2).
3: Ren P, Peng L, Yang L, Suzuki K, Fang Z, Renauer PA, Lin Q, Bai M, Li T, Clark P, Klein D, Chen S. RAMIHM generates fully human monoclonal antibodies by rapid mRNA immunization of humanized mice and BCR-seq. Cell Chem Biol. 2023 Jan 19;30(1):85-96.e6. doi: 10.1016/j.chembiol.202 (2).1 (2).005. Epub 2023 Jan 13. PMID: 36640761; PMCID: PMC9868106.
4: Ozdilek A, Avci FY. Glycosylation as a key parameter in the design of nucleic acid vaccines. Curr Opin Struct Biol. 2022 Apr;73:102348. doi: 10.1016/j.sbi.202 (2).102348. Epub 2022 Mar 4. PMID: 35255387; PMCID: PMC8957583.
5: Krušič A, Mencin N, Leban M, Nett E, Perković M, Sahin U, Megušar P, Štrancar A. Reverse-phase chromatography removes double-stranded RNA, fragments, and residual template to decrease immunogenicity and increase cell potency of mRNA and saRNA. Mol Ther Nucleic Acids. 2025 Feb 22;36(2):102491. doi: 10.1016/j.omtn.2025.102491. PMID: 40166612; PMCID: PMC11957593.
6: Schober GB, Story S, Arya DP. A careful look at lipid nanoparticle characterization: analysis of benchmark formulations for encapsulation of RNA cargo size gradient. Sci Rep. 2024 Jan 29;14(1):2403. doi: 10.1038/s41598-024-52685-1. PMID: 38287070; PMCID: PMC10824725.
