1. Introduction

Lipid nanoparticles (LNPs) have been firmly established as a robust delivery platform for nucleic acid therapeutics, most notably mRNA, as evidenced by their successful deployment in COVID-19 vaccines^[1-3]. However, conventional LNPs are subject to significant hepatic sequestration upon systemic administration, which limits their utility in treating diseases of extrahepatic tissues^[4]. To address this constraint and enable active tissue-specific delivery, antibody-targeted lipid nanoparticles (Ab-LNPs) have been developed^[5-7]. Through the conjugation of specific antibodies onto the LNP surface, Ab-LNPs acquire the capacity to actively recognize and bind to antigens on target cells, enabling efficient and precise in vivo delivery of nucleic acid drugs. This targeting strategy shows substantial promise across multiple therapeutic areas, including oncology and in vivo cell engineering.

The two predominant methods for antibody functionalization of LNPs are post-insertion and direct surface conjugation (Scheme 1). Post-insertion entails the integration of antibody-lipid conjugates into pre-formed LNPs via membrane fusion, while direct surface conjugation relies on the formation of stable chemical bonds between antibodies and functional groups on the particle surface^[8,9]. Both techniques preserve antibody binding activity and present distinct advantages and drawbacks in terms of chemistry, manufacturing, and controls (CMC) during scale-up. A pivotal challenge lies in the judicious selection of a conjugation strategy that ensures manufacturing robustness and batch-to-batch reproducibility.

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Scheme 1. The two predominant methods for antibody functionalization of LNPs. (A) Post-insertion; (B) Direct surface conjugation. PEG: polyethylene glycol.

Despite promising preclinical results and the progression of several candidates into clinical trials, such as Capstan’s CPTX2309 for in vivo cell therapy (NCT06917742), the translation of Ab-LNPs from bench to late phage/commercial production remains impeded by considerable CMC obstacles. These include the rationale-driven selection of antibody modification approaches, complexities in process scale-up, and the implementation of comprehensive quality control (QC) systems^[10,11]. This review provides a systematic analysis of the pivotal CMC challenges in scaling up Ab-LNP production using post-insertion and direct surface conjugation strategies and offers a structured framework together with practical perspectives to facilitate their successful industrial translation.

2. Post-Insertion and Direct Surface Conjugation Strategies

Currently, the primary modification methods for Ab-LNPs are post-insertion and direct surface conjugation^[12]. Taking thiol-based chemical conjugation as an example, the post-insertion method modularizes the entire process. As shown in Figure 1A, LNP preparation and conjugation are separated into two modules: antibodies are first reduced and then conjugated with maleimide (Mal) groups on the surface of micelles to form antibody-micelles. These micelles are subsequently fused with mRNA-loaded LNPs via thermodynamically-driven hydrophobic interactions, inserting into the lipid membrane surface to obtain Ab-LNPs^[13,14]. In contrast, direct surface conjugation involves mixing maleimide lipids (functionalized polyethylene glycol (PEG)-lipids) with other lipids (e.g., ionizable cationic lipids and helper lipids) during LNP formulation, resulting in LNPs with maleimide active groups on their surface. Then, reduced antibodies are conjugated to maleimide-functionalized LNPs to yield Ab-LNPs^[15,16], as depicted in Figure 1B.

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Figure 1. Schematic illustration of the two conjugation modification procedures. (A) Post-insertion; (B) Direct surface conjugation strategies. Conjugation strategies based on thiol chemistry. Ab-LNPs: antibody-targeted lipid nanoparticles.

From a CMC perspective, both methods have their own advantages and disadvantages (Table 1). The post-insertion method isolates the conjugation reaction from LNP formation, minimizing potential impacts on mRNA encapsulation within the LNPs. Additionally, by controlling and purifying intermediates such as antibody micelles, the number of antibodies inserted can be effectively managed, leading to good batch-to-batch consistency. However, due to the need for intermediate preparation, this method presents greater challenges during scale-up production, requiring extensive QC measures. Furthermore, fusion efficiency relies on spontaneous thermodynamic processes, which can lead to unpredictable insertion levels. In comparison, direct surface conjugation involves only an additional conjugation step, simplifying the process and facilitating scale-up production. Nevertheless, the conjugation reaction takes place in the presence of LNPs, which may affect the stability of the encapsulated mRNA. Additionally, conjugation efficiency can be influenced by reaction conditions and may be difficult to control.

Regardless of the conjugation approach, antibody modification is an essential step. Besides thiol-based chemistry^[17], click chemistry, such as alkyne-azide reactions^[18], is also commonly used, and the impact of the conjugation chemistry impact on Ab-LNPs may vary considerably. Beyond conjugation methods, Ab-LNPs face additional challenges including robust scale-up, minimization of aggregation, and QC.

3. Antibody Modification Approaches

Antibody modification approaches have evolved from random conjugation to precise site-specific modification using mild aqueous reactions^[19,20]. Traditional modification strategies primarily rely on the non-site-specific conjugation of the ε-amino group on lysine (Lys) residues. This is typically achieved through one of two common routes: either via direct reaction using reagents such as N-hydroxysuccinimide esters, or by first converting the amines to thiols using thiolation agents like N-hydroxysuccinimidyl-S-acetylthioacetate, followed by conjugation with Mal reagents^[21-23]. While simple and requiring no antibody engineering, this method has significant inherent drawbacks. Due to the abundance of surface-exposed Lys residues, conjugation occurs randomly, leading to heterogeneous products^[24]. While further studies are needed for Ab-LNP, heterogeneous products result in a non-uniform drug-to-antibody ratio (DAR) and potential masking of binding sites as in the frequently-compared modality, antibody-drug conjugates (ADCs)^[25]. This heterogeneity directly impacts the pharmacokinetics, potency, and safety of the final product, limiting its therapeutic application.

To overcome the limitations of non-specific conjugation, site-specific strategies have emerged. Among these, cysteine (Cys)-based thiol chemistry and azide-alkyne cycloaddition click chemistry are two mainstream approaches, exhibiting fundamental differences in stoichiometric control and product uniformity. Early thiol chemistry predominantly employed maleimide reagents, which react with thiols with high selectivity and rapid kinetics (second-order rate constants up to 10²-10⁴ M^-1·s^-1) under physiological conditions^[26,27]. However, the maleimide-thioether linkage suffers from in vivo stability issues, including retro-Michael addition and thiol exchange, particularly in the presence of high concentrations of plasma glutathione. In ADCs, this can lead to premature payload release and increased off-target toxicity, while in Ab-LNPs, it poses a risk of targeting loss^[28]. Next-generation thiol-selective reagents, such as benzoylacrylamides, cyclopropenyl ketones, and vinyl sulfonamides, have been developed, offering improved plasma stability while maintaining high reactivity^[29]. Nevertheless, even with these advanced reagents, controlling reaction stoichiometry remains a challenge for thiol chemistry. As shown in Figure 2A, inadequate control over the antibody reduction process in thiol-based conjugation may readily lead to the unintended cleavage of internal disulfide bonds. For instance, when conjugating targeting ligands (e.g., nanobodies like variable domain of heavy-chain antibodies (VHH)) to nanoparticles, even with optimized maleimide-to-thiol molar ratios, the reaction tends to produce heterogeneous by-products with multiple ligands attached to a single nanoparticle. This necessitates careful control over the reduction level of antibodies to limit the exposure of free thiols^[29]. In stark contrast, bioorthogonal reactions represented by dibenzycloether (DBCO)-azide click chemistry offer a core advantage: the ability to achieve strict and predictable one-to-one stoichiometry (Figure 2B). However, realizing this precise control requires the pre-conversion of thiol-based antibodies into alkyne-modified antibodies, followed by purification and QC to obtain a homogeneous alkyne-functionalized intermediate. While this additional step ensures the certainty of a defined 1:1 conjugation outcome, it inevitably adds complexity and extends the timeline of the overall process. Additionally, in an alternative strategy such as sortase A-mediated transpeptidation, a single azide group can be precisely introduced into a specific site of an antibody (e.g., the C-terminus)^[30]. Subsequent reaction with DBCO-modified molecules, due to the uniqueness of the azide group and the high specificity of the reaction, almost invariably yields a single, well-defined conjugate.

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Figure 2. Schematic illustration of the two site-specific conjugation procedures. (A) Thiol chemistry; (B) Click chemistry. VHH: variable domain of heavy-chain antibodies; SH: sulfhydryl group; LNPs: lipid nanoparticles; DBCO: dibenzycloether.

As exemplified by VHH (nanobodies), a class of single-domain antibodies increasingly employed in pharmaceutical development and targeted delivery systems, these proteins are typically produced via expression in systems such as Escherichia coli (E.coli) or Chinese hamster ovary (CHO) cells^[31-35]. Among these, the E.coli system offers distinct practical advantages, including significantly shorter production cycles and lower costs, compared with the CHO platform^[36,37]. Regardless of the expression system used, the resulting VHH requires subsequent functionalization through chemical conjugation. Comparative studies have shown that biotinylated VHH produced via click chemistry exhibits a biotin-to-VHH molar ratio close to the ideal range of 0.6-0.8:1. In contrast, the same VHH conjugated via thiol-maleimide chemistry shows a significantly higher ratio of 1.3-1.8:1, confirming the generation of multi-modified products. Functionally, while both methods generally preserve antigen-binding capability, click chemistry often better maintains or even enhances binding affinity due to its defined linkage site, minimal steric interference, and high product homogeneity, advantages particularly evident when conjugating large molecules like PEG.

However, research indicates that the conjugation chemistry itself can profoundly and unexpectedly influence biological behavior. Both the most popular conjugation chemistries, thiol-maleimide and DBCO-azide click chemistry, can significantly activate the complement system, albeit through distinct mechanisms (Figure 3). For DBCO-azide chemistry, the issue stems from the hydrophobicity of the DBCO moiety^[38]. Studies demonstrate that DBCO-modified antibodies can aggregate on nanoparticle surfaces, and these surface protein aggregates become potent activators of the classical complement pathway. In mouse inflammatory models, this leads to abnormal accumulation of antibody-liposome complexes in the lungs (up to 210% ID/g), an effect completely abolished in complement C3 knockout mice, confirming the critical role of complement activation^[18]. Notably, this aggregation phenomenon occurs specifically at the nanoparticle interface and is not observed in solution, highlighting the unique effects of the nano-bio interface. For thiol-maleimide chemistry, the mechanism of complement activation is different. The problem lies not with the thiol moiety, but with residual free maleimide groups. These highly reactive groups can undergo non-specific conjugation with thiol-rich plasma proteins, primarily albumin. When albumin “decorates” the nanoparticle surface, it creates sites for attack by the alternative complement pathway. Increasing the molar percentage of maleimide-PEG lipids in liposomes directly leads to a dose-dependent increase in the concentration of the complement activation product C3a, an effect not observed with azide groups. This complement activation not only alters biodistribution but also induces significant toxicities, including a 50% decrease in platelet count and increased hematocrit, which are typical manifestations of complement activation-related pseudoallergy. These findings suggest that “bioorthogonality” in the chemical sense (no side reactions with biological molecules) may differ from its meaning in the immunological sense (no activation of the innate immune system). While the DBCO-azide reaction is chemically specific and orthogonal, its hydrophobic nature triggers undesirable biological responses. To address these issues, in click chemistry, trans-cyclooctene (TCO), which is less hydrophobic than DBCO, may be used as a substitute. TCO-modified antibodies show minimal aggregation on nanoparticle surfaces, and the lung accumulation mediated by TCO systems is significantly lower than that of DBCO systems (74% ID/g vs. 133% ID/g). For thiol-maleimide chemistry, a direct and effective solution is to quench excess maleimide groups post-conjugation by adding thiol-containing small molecules such as Cys. This treatment reduces albumin binding and complement activation in a dose-dependent manner, bringing toxicity down to baseline levels. These strategies provide valuable guidance for antibody modification processes in Ab-LNPs.

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Figure 3. Schematic illustration of conjugation strategies for VHH. (A) Thiol-maleimide reaction; (B) Azide-alkyne click chemistry. Created in InDraw. VHH: variable domain of heavy-chain antibodies.

Click chemistry generally offers advantages in conjugation efficiency over thiol chemistry, and optimization of the conjugation process can also significantly enhance efficiency. For instance, maleimide activity decreases by approximately 10% after 7 days of storage at 4 °C, but by up to 40% when stored at 20 °C for the same duration. This indicates that optimizing conjugation temperature and duration can improve the efficiency of thiol chemistry. Similarly, for different types of protein antibodies, optimization of reaction stoichiometry can enhance conjugation efficiency. For example, in the conjugation of the small peptide cRGDfK with maleimide, the optimal maleimide-to-thiol molar ratio was 2:1, achieving 84% efficiency, while for the larger nanobody 11A4, the optimal ratio was 5:1, with 58% efficiency. Ratios below these optima lead to significantly reduced efficiency, while excessive ligand amounts cause waste without increasing surface loading, suggesting a steric saturation effect, where larger antibody molecules require more maleimide reaction sites. It is noteworthy that introducing site-specific Cys residues at antibody termini (e.g., Thiomab^TM technology) enables more uniform and controllable modification. In ADCs, such site-specific conjugation yields more homogeneous DAR distributions. However, engineered cysteines face challenges such as oxidation (forming disulfide bonds or conjugating with glutathione) and potential interference with proper protein folding, necessitating optimization of the Cys-introduced sequences^[20,39]. Based on this, more sophisticated strategies have been developed. For example, “p-Clamp” peptide sequences can dramatically promote the arylation of a neighboring Cys via specific interactions with perfluoroaromatic reagents, increasing the reaction rate by several thousand-fold. This proximity-driven catalytic approach offers new avenues for site-specific modification. The polyhistidine tag (His-tag) is a common tool for antibody purification. Its rational introduction can improve the efficiency of site-specific conjugation. For instance, introducing a short His₂-tag into interferon α2-a enabled site-specific PEGylation via a bis-alkylation reaction, resulting in highly homogeneous modified products while maintaining good bioactivity (up to 74% of the unmodified protein). However, the introduction of His-tags requires consideration of their potential impacts on protein conformation, stability, and activity, especially near enzymatic active sites or receptor-binding domains. Additionally, histidine residues can act as nucleophiles under neutral to weakly acidic conditions and may compete with thiol-directed reagents (e.g., maleimides), affecting conjugation efficiency and selectivity. Therefore, a comprehensive assessment of the sequence design is essential.

In summary, antibody modification has evolved from simple sequence-based synthesis into an interdisciplinary field integrating protein engineering, materials science, immunology, and pharmacology. The choice of modification strategy for antibody conjugation onto lipid nanoparticles is no longer solely focused on reaction rate and specificity. It now requires a careful balance, considering the overall system stability, scalability, and potential biological consequences.

4. The Hurdles of The Scale-Up Process

The scale-up production of Ab-LNPs fundamentally revolves around resolving the core conflict between translating the highly controlled, precision manual operations, essentially “customized chemistry”, in the laboratory, into a robust, uniform, and reproducible “continuous production process” within large-scale equipment. Beyond the universal scaling challenges shared with conventional LNPs, such as mixing and purification, Ab-LNPs present a series of unique, interrelated, and complex difficulties inherent to their specific antibody conjugation and subsequent antibody purification steps during scale-up. The most immediate manifestation is that, as batch sizes increase, the efficiency of successfully linking target antibodies to the lipid nanoparticle surface (conjugation efficiency) often decreases rather than increases. Furthermore, the proportion of free antibodies, aggregates, and even visible precipitates in the post-reaction system significantly rises, leading to low yield of the active ingredient and inconsistent product quality^[40,41]. In actual scale-up production, aggregation must be avoided, while ensuring that the conjugation efficiency loss remains within 5-15%. These issues are not isolated events, but stem from fundamental changes in the physicochemical environment during scale-up. These changes include uneven mixing, decreased mass transfer efficiency, differences in shear force distribution, and accumulation of side reactions due to prolonged reaction times. These factors collectively cause process failure upon scale-up^[42,43]. For instance, in the GMP production of anti-EGFR immunoliposomes reported by Wicki et al., while ten clinical trial batches were successfully prepared, the process still relied on multiple chromatography purification steps, and its scalability was noted to be limited^[40]. The report indicated that chromatography steps could become a major bottleneck during scale-up. This underscores the importance of embedding “manufacturability” criteria early in molecular and formulation design, implementing precise parameter control, and equipment compatibility during process development, and achieving a balance between high-resolution purification and product stability.

In laboratory research on Ab-LNPs, the pursuit often focuses on the ultimate target affinity of the modified antibody or the highest in vitro cell-killing data. However, an antibody candidate molecule that is difficult to stabilize in large-scale chemical modification and is prone to aggregation becomes a significant obstacle on the path to late phase and commercial productions, regardless of its impressive in vitro performance. As noted in some studies, an scFv with strong bioactivity but low thermal stability (melting temperature Tm of only 65 °C) and weak binding to Protein A resin (recovery rate < 20%) cannot serve as a viable therapeutic targeting module^[44]. It must undergo engineering to improve its stability and purifiability. Therefore, at the antibody engineering stage, priority should be given to formats that are structurally more compact (e.g., scFv, Fab), have more stable disulfide bond networks, and exhibit greater tolerance to reduction and conjugation reactions. This is not to negate the value of full-length antibodies but emphasizes the need for “developability” assessment. For example, comparing the aggregation tendency, activity retention rate, and recovery rate of different antibody candidates under simulated stresses of reduction, conjugation, and purification. Similarly, for the core lipid nanoparticle formulation, screening should not focus solely on encapsulation efficiency at small scales but should incorporate “stress tests”. This includes screening the stability of different phospholipids (e.g., DSPC, DODPC, E-SM) under storage conditions to select the most stable lipid, laying the foundation for long-term storage. Formulation screening must also evaluate lipid compatibility with the antibody conjugation process. Research has found that the type and density of PEGylated lipids significantly affect drug loading efficiency; PEG-DSG showed a significant advantage over PEG-DSPE when loading at high drug-to-lipid ratios^[42]. Additionally, the stability of the conjugation chemistry between the antibody and lipid requires focused investigation^[45]. Studies indicate that traditional maleimide-thiol linkages are prone to retro-Michael reactions and thiol exchange, leading to covalent bond instability^[15]. Using maleimides with electron-withdrawing substituents or pre-hydrolyzing the succinimide ring in vitro can significantly improve covalent bond stability, yielding products with a half-life exceeding two years. Incorporating these considerations into formulation and process design is essential to ensure the final product maintains structural integrity and targeting function during storage and in vivo circulation. Furthermore, combining computational simulations with high-throughput experiments to predict antibody-lipid material compatibility under different process conditions can reduce uncertainties in subsequent scale-up^[43].

During the scale-up of the conjugation process, a key challenge is reproducing the “instantaneous” and “uniform” reaction conditions of small-scale operations in large-scale reactors^[40]. In the laboratory, using pipettes or micro-mixers, antibody/reducing agent solutions can achieve molecular-level homogeneous mixing within milliseconds, ensuring each antibody molecule experiences an almost identical, mild reduction environment to expose a predetermined number of thiol groups. However, in reactors of several liters or larger, mixing achieved by top-stirring is macroscopic and relatively slow, inevitably creating concentration and temperature gradients. Localized areas may temporarily experience excessively high reducing agent concentrations, leading to over-reduction of antibodies, disruption of critical disulfide bond structures, exposure of internal hydrophobic regions, and nucleation for subsequent aggregation and precipitation. Conversely, other areas may have insufficient reduction, resulting in inadequate active sites for conjugation and lowering overall conjugation efficiency. Therefore, a core task of process scale-up is the “engineering re-design” of such critical chemical steps. For example, abandoning simple one-time feeding into the reactor in favor of controlled, multi-point, low-speed addition, or even utilizing in-line static mixers for instantaneous, efficient pre-mixing of reactants before entering the main reactor, to maximally simulate small-scale mixing effects. For the post-insertion method of preparing Ab-LNPs (antibody-lipid insertion into LNPs), mixing efficiency is equally critical. Traditional stirred-tank scale-up may no longer be suitable, necessitating exploration of technologies that better ensure nanoscale uniformity. For instance, optimizing the reactor internal structure through computational fluid dynamics simulation to ensure mass transfer efficiency is not compromised upon scale-up. Guidance of the Food and Drug Administration (FDA) on drug products containing nanomaterials also notes that changes in manufacturing processes and production scale for products containing nanomaterials may make bridging between early development batches and large-scale commercial batches difficult, highlighting the importance of establishing robust control strategies^[43]. The goal at this stage is to define a clear, scientifically understood “design space”, specifying the operating ranges for each critical process parameter (CPP) (e.g., mixing energy input, temperature, feed rate, residence time distribution), transforming the process from an “art” into a “controlled science”. Simultaneously, establishing quantitative relationship models between critical quality attributes (CQA) and process parameters aids in precise control and fault prediction during scale-up. This process requires drawing on scale-up principles from chemical engineering, such as maintaining similar power input per unit volume or mixing time, combined with a deep understanding of nanoparticle formation kinetics and conjugation reaction kinetics for scale-dependent parameter adjustment. The scale-up of mixing time should be adjusted based on antibody reduction efficiency or conjugation efficiency to ensure that the overall decrease in conjugation efficiency during scale-up production is controlled within 5-15%.

The scale-up of Ab-LNPs purification is another intricate battle of fine separation within a complex mixture^[41]. The post-reaction crude product is a “cocktail” of coexisting phases, containing the target Ab-LNPs, unreacted free antibodies, excess antibody-lipid conjugates (for post-insertion), chemical by-products, and aggregates of various sizes and compositions. The difficulty in purification scale-up lies in the fact that these impurities are often very similar in physicochemical properties to the target product (e.g., aggregates of similar size to target particles). Furthermore, Ab-LNPs themselves, as lipid nanostructures surface-modified with antibodies, are exceptionally sensitive to shear forces, osmotic pressure changes, and interfacial interactions with solid-phase media (e.g., chromatography resins, tangential flow filtration membranes). Inappropriate purification operations can induce new aggregation or damage. Therefore, purification strategy development must be deeply aligned with product characteristics. For example, compared to size-exclusion chromatography (SEC), which primarily relies on molecular size separation (difficult to scale up and has low capacity), exploration of ion-exchange chromatography based on surface charge differences, or adsorption-elution strategies based on specific affinity interactions, can be considered. However, the key lies in meticulous screening and optimization of chromatography resins and operating conditions to ensure appropriate binding affinity for the target product and enable high recovery elution under gentle conditions, avoiding lipid bilayer disruption or antibody detachment due to overly harsh eluents. Streamlining the process is an important principle; reducing intermediate steps and product transfers lowers scale-up risks^[46]. Each additional centrifugation, filtration, or buffer exchange introduces another risk of product exposure to air-liquid interfaces or shear forces, another opportunity for yield loss and aggregate formation. Exploring integrated, continuous purification schemes, such as direct tangential flow filtration purification of the reaction mixture combined with in-line adjustment of pH and conductivity for stepwise impurity removal, may be a superior direction. Some studies suggest that chromatographic purification methods have limitations in later-stage scale-up and thus recommend replacing chromatography steps with tangential flow purification processes. During the purification step for free antibody removal in scale-up production, the yield of the Ab-LNPs after purification is typically required to be 60-85%.

While deeply optimizing process scale-up, it is crucial to attach high importance to the long-term stability of Ab-LNPs, which directly impacts product shelf life and clinical efficacy. As mentioned, while widely used, traditional maleimide-thiol chemistry may undergo retro-Michael addition or exchange with serum thiols in vivo/in vitro, leading to antibody detachment and loss of targeting function^[15]. As reported in the literature, reagents like DBM-C2 can selectively form stable bridged structures with the exposed dithiols after antibody reduction, significantly improving conjugate homogeneity and in vivo stability. Advances in conjugation chemistry like this need to be adopted not only at the molecular design stage but also considered for the controllability of their reaction conditions and the removability of by-products during process scale-up. Additionally, the physical stability of the lipid nanoparticle core cannot be ignored, influenced by factors such as lipid composition, phase transition temperature, and surface PEG density^[42]. During scale-up, changes in mixing and cooling rates may alter the particle size distribution and nanostructure of the lipid nanoparticles, thereby affecting drug encapsulation efficiency and storage stability. Therefore, formulation optimization must be closely integrated with process development. Through systematic design of experiments, the combined effects of critical material attributes (CMA) and process parameters on the final product’s stability can be identified, establishing a robust operating space. This also includes in-depth study of the final product formulation, such as screening lyoprotectants and developing lyophilization processes, to ensure the long-term stability of Ab-LNPs in solid form.

In summary, the process scale-up of Ab-LNPs is a multi-dimensional challenge. It requires moving beyond the optimization of individual technical points to establish a holistic, systemic capability spanning “molecular design-process development-production realization”. By selecting robust antibody candidates at the design source, gaining a deep understanding and precisely controlling the scaled-up physicochemical microenvironment during process development, pursuing a balance between high resolution and product-friendliness in purification strategies, and simultaneously addressing the long-term stability of conjugation chemistry and formulation systems, can the immense potential of this technology be transformed into stable, reliable, and affordable industrial products. This is the path to truly bridging scientific innovation to benefit patients broadly. Moreover, an increased antibody conjugation density does not necessarily correlate with enhanced targeting efficacy. Excessive surface modification may, in fact, impair delivery efficiency, potentially through the promotion of protein corona formation or the introduction of steric hindrance that limits receptor accessibility. Therefore, the optimal antibody-to-lipid ratio should be empirically optimized via systematic in vitro and in vivo evaluations.

5. QC Considerations

In the scale-up production process of Ab-LNPs, the establishment and implementation of a QC system are core to ensuring product consistency, safety, and efficacy. As a type of functionalized nano-delivery system, Ab-LNPs have a structural complexity far exceeding that of traditional LNPs, mainly reflected in the additional QC dimensions and process challenges introduced by antibody modification. As noted in the literature, comprehensive characterization of complex nano-carriers requires multiple complementary, label-free biophysical techniques (such as dynamic light scattering (DLS), multi-angle DLS, electrophoretic light scattering (ELS), nanoparticle tracking analysis (NTA), SEC combined with static light scattering, and differential scanning calorimetry (DSC), etc.) to obtain CQA, including size, distribution, concentration, charge, and thermal stability^[47,48]. Although Ab-LNPs share common basic QC indicators such as size, distribution, and encapsulation efficiency with traditional LNPs, their unique antibody modification features significantly increase process complexity and QC difficulty. As the key functional component for targeting, the efficiency, stability, activity, and residual levels of antibody modifications directly determine the targeting efficacy and safety of Ab-LNPs^[47]. Therefore, during scale-up, systematic and sensitive monitoring strategies must be established to guide the optimization and standardization of conjugation processes, ensuring batch-to-batch consistency and feasibility for clinical translation (Table 2).

Firstly, monitoring the antibody modification process is the primary QC point in Ab-LNP process scale-up. The conjugation of antibodies to lipid nanoparticles is not a simple physical mixture but involves multiple factors such as chemical bond formation, spatial orientation control, and density optimization. Slight fluctuations in any process parameter may lead to variations in conjugation efficiency, antibody conformation, or surface distribution, thereby affecting targeting performance. During scale-up, it is necessary to monitor key parameters of the conjugation reaction in real-time, such as reaction time, temperature, pH, and material ratios, and to ensure controllability of the conjugation step by establishing appropriate intermediate testing methods (e.g., purity and concentration analysis of antibody-lipid conjugates). For example, ultra-high-performance liquid chromatography coupled with charged aerosol detection (CAD), high-performance liquid chromatography (HPLC) with evaporative light scattering detection, or HPLC-CAD can be used for the quantitative analysis of functionalized lipids and antibody-lipid conjugates (in post-insertion methods), ensuring accurate material input and monitoring of reaction progress^[49]. Furthermore, by optimizing the eluent pH and sample pretreatment diluent, baseline separation and accurate quantification of multi-component lipids can be achieved, providing stable guidance for monitoring lipid degradation and impurities.

Additionally, antibody density after Ab-LNP conjugation is a key metric for evaluating modification effectiveness. Excessively high or low antibody density may both affect targeting efficiency and in vivo behavior. Methods such as quantitative SDS-PAGE combined with silver staining, surface plasmon resonance (SPR), biolayer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), or nano-flow cytometry can be used to determine antibody density, providing data support for process optimization^[50]. Particularly, SPR and BLI technologies can monitor the binding kinetics between antibodies and target receptors in real-time, not only for density assessment but also indirectly reflecting the functional status of antibodies post-conjugation. SDS-PAGE with silver staining has been successfully used to quantify the surface modification amounts of antibodies such as Herceptin on lipid nanoparticles, and its bioactivity was validated through antibody-dependent cell-mediated cytotoxicity, demonstrating the reliability and practicality of this method, and providing a feasible path for standardized antibody density analysis of Ab-LNPs^[12,51,52].

Secondly, the removal of free antibodies is a critical step in the purification process of Ab-LNPs and must be included as a key item in the QC system. Unconjugated free antibodies remaining in the final product can not only competitively inhibit the targeted binding of Ab-LNPs, but may also cause unintended immune responses or pharmacokinetic interference. Therefore, after the conjugation reaction, free antibodies must be effectively removed through methods such as dialysis, ultrafiltration, or chromatography, and sensitive, specific detection methods must be established for quantitative control of residual levels^[50]. Commonly used detection methods include SEC, HPLC coupled with ultraviolet or fluorescence detection, and ELISA based on antigen-antibody interactions. Among these, SEC can separate free antibodies from Ab-LNPs based on molecular weight differences, but care must be taken to avoid nanoparticle adsorption or aggregation in the column^[47]. Literature indicates that SEC coupled with UV, multi-angle light scattering (MALS), and refractive index detectors successfully achieves high-resolution analysis of LNP size distribution, molecular weight distribution, and siRNA loading, demonstrating the powerful separation and quantification capabilities of SEC combined with multiple detectors in complex nano-systems, and providing technical reference for the separation and detection of free antibodies in Ab-LNPs^[53]. ELISA offers high sensitivity and specificity, enabling accurate quantification of residual free antibody levels through detection of the Fc region or idiotypic epitopes^[50]. During scale-up, the detection of free antibody residues should be monitored as a CPP with reasonable acceptance criteria to ensure the robustness of the purification process and product purity.

Furthermore, whether the antibodies retain their structural and functional integrity post-modification is a core element in evaluating the quality of Ab-LNPs. The conjugation process may cause antibody conformational changes, aggregation, degradation, or masking of active sites, leading to loss of targeting function or increased immunogenicity. Therefore, a series of biophysical and functional testing methods must be established to comprehensively assess the activity, stability, and immunogenicity of antibodies after modification. For binding activity, methods such as SPR, BLI, or flow cytometry can be used to measure the affinity and binding specificity of Ab-LNPs to target antigens, ensuring that conjugation does not affect the spatial conformation of the antibody’s complementarity-determining regions^[50,53]. Stability assessment involves thermal stability (via DSC or differential scanning fluorimetry), aggregation propensity (via DLS, SEC, or MALS), and storage stability (monitoring antibody shedding or degradation under different temperature and pH conditions). The literature, by combining DSC and DLS thermal scans, revealed different aggregation and structural transition behaviors of empty and full rAAV5 under thermal stress, emphasizing the importance of orthogonal techniques for understanding the stability of complex nano-carriers^[54]. Additionally, conjugation may expose new epitopes or alter original immunogenicity profiles, requiring assessment of potential immune risks through methods such as anti-drug antibody testing. These functional QC data not only guide the optimization of conjugation processes (such as selecting mild conjugation chemistry or optimizing reaction conditions), but also provide a basis for safety evaluation in subsequent preclinical and clinical studies.

The establishment and validation of QC methods are the “eyes” of process scale-up, and their accuracy, precision, and robustness directly determine the understanding and control of the process. As a complex nano-drug product, QC for Ab-LNPs spans multiple disciplines, covering physical chemistry, biology, and immunology. For example, size and size distribution can be characterized by DLS, NTA, or field-flow fractionation coupled with multi-angle light scattering^[54]. The latter is particularly suitable for high-resolution analysis of polydisperse systems and monitoring size changes in biological media, as literature notes that AF4-MALS-DLS can detect subtle differences between LNP batches and changes in size distribution after serum incubation compared to batch-mode DLS, providing a high-resolution tool for predicting the behavior of Ab-LNPs in complex biological environments^[48]. Surface charge (Zeta potential) can be measured via ELS; encapsulation efficiency and drug loading require appropriate detection methods depending on the payload type (e.g., small molecules, siRNA, mRNA), such as HPLC or Ribogreen/Picogreen fluorescence assays. These traditional LNP QC indicators still require strict monitoring in Ab-LNPs, as they may change due to antibody modification (e.g., antibody introduction may increase size or alter surface charge), thereby affecting in vivo distribution and efficacy. Moreover, for Ab-LNPs with dual-antibody or multi-targeting strategies, QC complexity further escalates, requiring precise analysis of the density, activity, and relative ratios of different antibodies.

In the context of industrial-scale production, QC strategies must also consider the high-throughput, automation, and online monitoring potential of methods to meet the demands of large-scale manufacturing. Simultaneously, the application of the Quality by Design concept is crucial. By identifying CMA, CPP, and CQA, a design space and control strategy can be established to ensure the robustness of product quality during scale-up. Regulatory agencies such as the FDA have issued guidelines for nano-drugs, emphasizing the importance of process controllability and product characterization^[43]. Therefore, the QC system for Ab-LNPs must be comprehensive, systematic, and compliant with regulatory requirements.

In summary, the QC of Ab-LNPs during scale-up is a multi-dimensional systems engineering effort, with its uniqueness primarily stemming from the introduction of antibody modifications. From monitoring the conjugation process and removing free antibodies to verifying antibody functional integrity, each step relies on advanced, sensitive, and well-validated QC methods. These methods often need to draw from and integrate mature characterization strategies in fields such as viral vectors and traditional LNPs, combined with specific analytical tools from antibody engineering. These methods are applied not only in process development and optimization, but also serve as the cornerstone for ensuring product batch-to-batch consistency, safety, and efficacy.

In conclusion, whether using post-insertion or surface modification approaches to prepare Ab-LNPs, it is necessary to evaluate the antibody modification methods, ensure the robustness of unique processes such as conjugation and purification steps, and continuously optimize the analytical methods for key QC items, paving the way for the successful industrialization of this technology.

Authors contribution

Huang R: writing-original draft, writing-review & editing, visualization.

Wang Y, Shi T, Deng C, Xu C: writing-review & editing.

Piao X, Ren K: Supervision, conceptualization, writing-review & editing.

Conflicts of interest

Congcong Xu is a Junior Executive Editor of BME Horizon. Ri Huang, Yifan Wang, Xijun Piao and Keyun Ren are affiliated with CATUG Biotechnology. The other authors declare no conflicts of interest.

Ethical approval

Not applicable.

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