Strategy and Design of Bioconjugation

The strategies used to develop bioconjugates for specific applications can be as varied as the reactions and components used to form such complexes. Bioconjugates can be used for a variety of applications, and in each case the particular bioconjugates that perform best are the result of careful design and are often carefully optimized for their intended use. Each component of the bioconjugate should be considered as an active part of the entire reagent. Each element has its own role, which is critical to the function of the bioconjugate in its intended application. If any one part performs poorly, then the activity or specificity of the entire conjugate may also be affected. Therefore, each biological conjugate must be optimized according to its application. This article provides some general guidelines for the design of bioconjugates, as well as some strategies for selecting the right components and reactions to form final complexes. To get more information about functional groups that can be used, as well as coupling agents, scaffolds, targeting agents, detection ingredients, polymers, particles, antibodies, and any other molecules that may be used.

Application of bioconjugate

The end use of a biological conjugate should be carefully considered in order to properly design its components so that they are suitable for the intended task it must perform. In addition, the conditions under which bioconjugates are used should be considered, as some components may not function properly in the intended medium or environment. Therefore, it is important that when starting the design of any biological conjugate, its final application must be considered first before any experimental procedures to actually manufacture the conjugate are started. For example, if a bioconjugate is used for assay, the most suitable component may consist of a targeted molecule that is coupled to the detection element so that the final complex can bind and interact with its target and then be detected. Alternatively, if the final bioconjugate is used to purify the target molecule, a suitable conjugate may consist of an insoluble support or particle attached to an affinity ligand. Therefore, each bioconjugate application may have completely different conjugate construction requirements.

When designing a new biological bond for a unique application, it is best to first consider the biological bond used by others in similar applications. Table 1 lists some of the main applications of biological coupling and the types of coupling that have been applied to them. The table is not meant to be an exhaustive presentation of every conjugate that has been reported, but merely provides primary examples that can be used as a starting point for the development of new biological conjugates. Because there are so many component choices when designing bioconjugates, and so many reactions that can be used to produce bioconjugates, the options available when creating new conjugates are almost daunting. Therefore, before starting a new design, it is very useful to know the types and structural details of bioconjugates that others have successfully used in similar situations. However, even with this knowledge, for new applications, the bioconjugation-building method may still need to be optimized to function well in the intended application.

Table 1 Bioconjugation components and designs for major applications

In addition, fluorescent dyes for antibody conjugation can be selected based on their relative hydrophobicity or hydrophilicity. In some applications, it is best to choose a dye with more hydrophilic properties to prevent antibody aggregation or reduce the likelihood of non-specific binding of the conjugate in the assay. Most dyes have a highly hydrophobic aromatic core structure that can interact nonspecifically with many hydrophobic regions on biomolecules, or with surfaces used in the determination process. The use of hydrophilic dyes modified with negatively charged sulfonates or neutral PEG chains can significantly reduce non-specific binding and enhance the detection signal-to-noise ratio.

Conversely, if the antibody conjugate is to be used for in vivo imaging or cell-based imaging, where efficient penetration of the membrane structure is required to achieve the desired target, the use of a dye that is slightly less water-soluble may be appropriate. In this case, the use of dyes with fewer sulfonates (for example, there may be no more than two or three negative charges on a macrocyanine type dye) can perform better in the intended application than dyes with more hydrophilic groups or more charges. Therefore, the performance of the bioconjugate in its intended application often determines the best choice for the fluorescent dye derivative used in the conjugate design. For the best results, several potential options should be tested to optimize the performance of dye-antibody bioconjugates.

Another important consideration when preparing conjugates is the relative proportions of each component in the final complex. In the case of dye-labeled antibodies, it is usually important to have more than one dye modification on each antibody molecule. This has the effect of increasing the fluorescence intensity of each antibody, as shown below as it docks at a predetermined target. Each fluorophore adds another potential photon emission to each coupled molecule. However, using more dye to modify antibodies is not always better than using less dye. At certain substitution levels, fluorescent dyes may cause antibodies to aggregate, or begin to quench the potential fluorescent output due to dye-to-dye interactions and energy transfer rather than emission. For a given antibody-dye complex, there is a certain level of dye substitution that will provide the maximum signal in the assay without the possibility of precipitation or quenching. The optimal conjugate can only be determined experimentally by preparing a large number of test dye-antibody bioconjugates in small quantities so that the dye substitution level is slightly different for each preparation. Testing each conjugate for fluorescence intensity, solution stability, and assay performance will determine which one is best suited for the intended application.

Design optimal bioconjugates

One of the most important aspects of successfully forming a bioconjugate involves an adequate understanding of the structural, chemical and active characteristics of the components to be bound together. Designing a conjugation strategy that forms the final complex while still maintaining all the functions of its individual parts is no small task. The successful process involves not only knowledge of the reactive groups, reagent types, and functional groups present on the biomolecule, but also understanding how modifications of the biomolecule may affect its activity. Using current understanding of the 3D structure and function of proteins, including active site components and stability considerations, bioconjugated reaction strategies can be designed to maximize the potential activity and function of the final conjugated substance. With this information, it is often possible to avoid functional groups close to the binding site or active center by carefully selecting the appropriate crosslinker or reaction condition. In some cases, unique functional groups (e.g., biogenic alcohols) or functional groups that occur with limited frequency on biomolecules (e.g., cysteine merethiol groups) can be selected for conjugation, thereby moving the modification or crosslinking away from the active center or binding site and thus helping to maintain the activity of the final bioconjugated product.

As bioconjugation technology advances, some types of bioconjugates that have been in use for years may be redesigned with newer technologies to provide better functionality. For example, one of the earliest types of bioconjugate consists of radiolabeled complexes formed by attaching radioactive atoms to biomolecules. Radioactive iodination involves the covalent modification of certain organic groups within molecules with radioactive iodine-125 atoms (or other radiolabelling) to form detectable complexes, such as modifying tyrosine or histidine residues within proteins or activated aromatic rings within small molecules). Many antibodies are initially labeled in this way to provide a combination of specific antigen binding with radioactive detection. These early bioconjugates are used in many research and diagnostic assays to detect and quantify target molecules. However, beginning in the mid-1970s, with the advent of antiantium-enzyme conjugates, these new detection complexes began to replace radiolabel-labeled conjugates due to stability and safety concerns of radioactive compounds and improved enzyme detection performance and sensitivity. Radioactive bioconjugates are still used in many important applications, but the primary focus of radiolabeling is now not for in vitro research assays or diagnostic tests, but for in vivo therapeutic targeting and imaging of tumor cells.

Over the years, the methods and reactions used to make bioconjugates have evolved in a number of ways. Early methods of forming conjugates often involved the use of homo-bifunctional reactants, which polymerize the components of biological conjugates almost uncontrollably. An example of such a reaction is the formation of an antibody-enzyme conjugate using glutaraldehyde. Glutaraldehyde is an effective crosslinking agent, but the conjugates formed too frequently contain high molecular weight components and are even partially precipitated due to oligomerization of antibodies and enzymes during the reaction. More advanced crosslinkers can now be used to correct this and provide more control during the reaction. As a result, most antibody-enzyme couplings are no longer made using glutaraldehyde. As the science of bioconjugation technology advances, new reagents and reactions continue to provide more options for producing better bioconjugate. Even familiar bioconjugates that have been prepared and applied in research for decades can be improved by redesigning the bioconjugates strategies that were originally used to make them.

Uncontrolled-conjugation and controlled conjugation. Uncontrolled binding can lead to the formation of high molecular weight complexes that may aggregate or precipitate out of the solution over time. (Rad, M. E., 2023)

Under the most controlled and optimized conditions, the bioconjugation process can still produce many different structural species that make up the final composition. When two components react together and at least one of them has multiple covalent linking sites, such as on proteins containing multiple amines, the resulting bioconjugate may have a molecular weight distribution around the average mass. For example, the process of modifying a protein with an amine reactive biotinylation compound can produce an almost Gaussian distribution of the amount of biotin per protein conjugating, the average of which depends on the molar excess of the biotin reagent used relative to the amount of protein added at the beginning of the reaction. For example, if a 10x molar excess of NHS-PEG 4-biotin is added to an antibody, the final bioconjugate may contain an average of 7 biotin per antibody molecule, but the following species will also be present: Each antibody has a range of replacement populations from perhaps 3 biotin to even 12 or more biotin, which at the high end even exceeds the amount of the initially added biotinizing reagent. There are at least two reasons for this: (1) the reaction yield is never 100% efficient (especially in this case, competitive hydrolysis of reactive NHS ester groups on biotinylated compounds), so some molecules will be modified more efficiently than others; (2) local concentration differences caused when the reaction components are first mixed together may cause some antibody molecules to experience a higher molar excess of biotinylation reagents after the solution is fully homogenized than other antibody molecules. Even with such a large distribution of different degrees of biotinylation, reaction conditions that carefully control the amount of modification will help to maximize the activity of the conjugate and make the production of the final complex more reproducible.

Bioconjugates can also take on many different shapes or structures. The simplest form is a bifunctional conjugate produced by the reaction of two different components. However, sometimes bioconjugations are made of more than two components, forming three-functional or even multifunctional complexes that have a combination of three or more different properties. One molecule may provide the targeting ability to bind to a specific biomolecular or cellular component, while the second component may be a biological agent that can be used to treat a disease, and the third group may provide the detection ability. This type of bioconjugate is formed using molecular scaffolds with multiple functional groups that can be used to connect the components of the final complex together. For example, this type of bioconjugate can be built using dendrimer polymers to link together antibodies, chemotherapy drugs, and fluorescent molecules that together provide tumor targeting, therapy, and fluorescence detection functions, all built into the same complexity. In addition, Zaman et al. used a dextran polymer to link the chemotherapy drug doxorubicin and the hepatocellular targeting molecule galactosamine to create a multifunctional bioconjugate for the treatment of cancer.

Examples of multifunctional bioconjugates include the use of molecular scaffolds, such as polymers and dendrimers. Left: Dendritic conjugate containing antibodies to specific epitopes, a chemotherapy agent (methotrexate), and a fluorescent dye for imaging. Right: Illustration of a glucan conjugate containing galactosamine residues for targeting liver cells and the drug doxorubicin for antitumor activity. Polymer scaffolds provide multiple conjugation sites, allowing each conjugate to attach more molecules compared to direct crosslinking. (Rad, M. E., 2023)

The use of appropriate molecular scaffolds can also enhance the activity of one of the components of the bioconjugate. In this regard, polyvalent polymers or dendrimers can be used to pair a large number of copies of the detection molecule or enzyme while additionally linking a small number of targeted molecules. This can result in specific targeting complexes with extremely sensitive detection capabilities. An example of this type of conjugate is a gadolinium chelate dendritic polymer, which can be used as an in vivo contrast agent using magnetic resonance imaging (MRI). The ability to conjugate multiple copies of a gadolinium chelate to the surface of a dendritic polymer significantly enhances the signal beyond the typical signal of directly labeled targeting molecules without the use of scaffolds. Attaching targeted molecules to such modified dendritic macromolecules, such as folic acid taken up by tumor cells, then provides specificity for cancer cell imaging. Kodoma et al. also used a similar dendrimer scaffold to attach polyethylene glycol (PEG) grafts that did not covalently encapsulate the cancer drug methotrexate for in-vivo delivery.

Another important aspect of the design of bioconjugates is the type of connection used to connect the components of the conjugates together. The linker strategy can include multiple reaction groups as well as organic spacer molecules that separate the components and form a bridge between the linking molecules in the biological conjugate. The properties of these connecting arms can have a significant impact on the properties of the final reagent. For example, some crosslinkers contain hydrophobic spacers composed of aliphatic or aromatic groups that are good for forming biological couplings, but may also have harmful effects on the stability or non-specificity of couplings in a given application. Conversely, some linking molecules are hydrophilic, such as PEG-based reagents, and actually increase the solubility of the final biological bond in an aqueous solution. These property differences between hydrophobic and hydrophilic crossbridges also apply to modification reagents, such as biotinylated compounds or thiolated reagents, which can affect the properties of intermediates as well as the final bioconjugates. However, for some applications, an extremely hydrophilic binding may not be appropriate if the biological binding is designed to cross the cell membrane structure, in which case a more hydrophobic linker may actually perform better. Obviously, the design of each part of a biological bond can affect its use and performance in a particular application. This is why there is an emphasis on optimizing and testing multiple bioconjugate designs to determine the optimal structure for the desired application.

Even the length and structure of the span between the two components of a biological conjugate may significantly affect its overall properties. Especially when designing biotherapeutic agents, the chemical structure of the connection can have a huge impact on their efficacy in vivo, sometimes in unexpected ways. For example, Doronina et al. found that different bisamino-acid linkers in the linking arm between anti-tumor monoclonal antibodies and the cytotoxic drug monomyl auristatin F have different effects on the toxicity and effectiveness of the bioconjugate. In this case, the construction of the span also includes an p-amino-benzyl carbamate group that is designed to be cleaved by intracellular proteases, such as cathepsin B. The presence of this group in the linker allows the payload to be released within the tumor cells, which enhances its ability to cause cell death. Finally, the authors found that modifications to the C-terminal amino acid residues of the drug either increased or decreased efficacy. Therefore, depending on the application, the choice of joint structure should be carefully considered in the design of biocouplings, including reaction groups, hydrophilic and hydrophobic span bridges, the possibility of incorporation of cleavage sites, or the use of complex molecular scaffolds to join the components together.

Building bioconjugates using hydrophobic or hydrophilic crosslinkers can also have significant effects on reagent stability and non-specificity, depending on the choice of reagent. Many conjugating strategies involve modifying one protein with a crosslinking agent to provide a secondary reactive group, and modifying another protein to produce the corresponding functional group capable of binding to that protein. The reactive group on the first protein. If the initial reaction on the protein uses hydrophobic compounds with aliphatic or aromatic cross bridges, both proteins will be modified by these modifications. Even before the conjugates are produced, such intermediates can provide considerable hydrophobicity to individual proteins. Once the two modified proteins are mixed together and form couplers, the reactive group or functional group may be blocked at its ends, but the hydrophobic linking arm still protrudes from the protein. Since these initial modifications are usually done by overadding reagents to form a large number of linking arms spread across the protein's surface, these modifications will still be present in the final bioconjugates. In addition, if each modification site does not cause a protein-protein coupling event, then both proteins will have "dead end" modifications stuck to their surface in the final complex. Each of these remaining modification sites may non-covalently interact with hydrophobic pockets on the biomolecules (or with each other), thereby creating a binding for possible non-specific binding of the final complex.

These dead-end hydrophobic modified arms also induce interactions between conjugates, which may lead to larger non-covalent complex formation with significant precipitation potential. Antibodies, in particular, tend to accumulate easily in solution, even if there are no additional hydrophobic linkers on their surfaces. If an antibody conjugate is formed using a hydrophobic modification or crosslinking agent, the result may be enhanced antibody aggregation to the point where stability is significantly reduced. Even the use of a single modification agent to modify an antibody, such as biotinylation, can cause immediate or long-term stability problems in aqueous solutions. For example, a very popular option in biotinylation reactions is the use of NHS-LC-biotin. The reagent has an extended aliphatic spacer arm between the amine reactive NHS ester and the biotin group at the other end of the compound. In principle, the long interval region seems to be beneficial to increase the accessibility of binding of biotinylated antibodies to streptavidin assay conjugates. However, long aliphatic spacer arms actually pose more problems than advantages. If the reaction molar of the biotinylation reagent is too high, the antibody may precipitate almost immediately due to hydrophobic interactions. Even when biotinylation levels are controlled at low levels, biotinylated antibodies typically accumulate slowly in solution over time through interactions between biotinylated antibody chains on individual biotinylated antibody molecules and between the chains and hydrophobic pockets on the antibody, resulting in a sustained loss of activity.

Bioconjugates made of hydrophobic and hydrophilic reagents and their tendency to aggregate or remain in solution. (Rad, M. E., 2023)

Each step in the conjugation process can add hydrophobic or hydrophilic properties to the conjugate. If two proteins are joined together using crosslinkers and modifiers, their water solubility and biocompatibility are greatly affected by the reagents chosen. If hydrophobic aliphatic joints are used, both proteins in the conjugate will display these components on their surfaces, potentially affecting their behavior in solution. If hydrophilic reagents are chosen, such as PEG-based compounds, the molecules are linked by hydrophilic components, and both proteins exhibit these reagents on their surfaces. The presence of hydrophobic modifications on the protein surface may cause conjugates to aggregate or precipitate over time. It may also lead to non-specific binding properties of molecules in biological samples.

In contrast, the use of hydrophilic biotinylation compounds, such as NHS-PEG n-biotin reagents, actually increases the hydrophilicity of the modified antibody after the reaction. Antibodies with PEG-based biotin modifications on their surfaces do not promote hydrophobic interactions and aggregation, but instead become more stable in solution and significantly reduce non-specific binding properties. Therefore, if water solubility and low non-specific binding to biomolecules are important criteria for bioconjugants, it is best to explore the use of hydrophilic crosslinkers and modification agents in design strategies.

Another aspect to consider in the design of biological bindings is the incorporation of modifiers that can have a positive effect on reagent stability and in vivo half-life. One of the most important options in this field is the use of polyethylene glycol (PEG) modifiers or crosslinkers. Modification or PEGylation of existing biological conjugations using linear or branching PEG molecules has been shown to enhance water solubility, increase the hydrodynamic volume of biological conjugations, extend stability and half-life, and reduce the immunogenicity and toxicity of drugs in vivo; PEG modification of biological binding can also significantly improve the water solubility and in vitro stability of the reagent. The presence of PEG groups on the surface of bioconjugates reduces non-specific binding to other biomolecules, thereby improving the signal-to-noise ratio in detection and potentially increasing detection sensitivity.

One of the most important developments in PEG-based reagents in recent years has been new discrete molecules with specific polymer lengths. Early PEG compounds that could be used for protein modification were all based on polydisperse polymer chain lengths where molecular weight distribution was always present in a given reagent preparation. In contrast, discrete PEG compounds are based on pure molecular chain length and can provide deterministic reagents with known qualities and properties. Discrete PEG-based crosslinkers and modification agents are now available to create various types of biological couplings and, in most cases, are a better choice for forming couplings than the corresponding aliphatic linkers.

As seen from the above description, there are many choices involved in preparing bioconjugates, especially when designing new molecules, which often involve major optimizations to determine the ideal conjugation strategy from the initial concept to provide the best performance in the application. Even with experience, it is sometimes not clear from the outset what is the final bioconjugate design that will produce the best activity or efficacy. Therefore, before deciding on design strategies and synthesis routes, multiple bioconjugation reagents and reactions must be investigated, at least in theory, to put several options together. Especially when preparing bioconjugates for potential therapeutic or in vivo use, the characteristics and performance of multiple conjugate designs must be thoroughly investigated before conclusions can be drawn about the optimal structure for a particular application. When used for in vivo diagnosis or therapy, even subtle structural changes can have a significant impact on the performance of biological bonds.

In contrast, for the preparation of bioconjugations designed for more standard "proven" research assays or for research analysis, the choices regarding ingredients to be used and effective potential response strategies are not as daunting as those designed for clinical use. In fact, the rich history of bioconjugate design and application can help guide the preparation of new conjugates that are suitable for almost any intended use. The schemes presented in this paper have been used to create many types of biological conjugates. They provide a viable starting point for building almost all new bioconjugate designs using similar reaction strategies. Ultimately, however, the most viable bioconjugate is thoroughly optimized for optimal performance in its intended application and carefully produced to ensure that the reagent is repeatable and stable.