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Advanced Conjugation Processes and PerKit®

CellMosaic’s advanced conjugation processes have been developed and optimized through its custom bioconjugation services. As of March 2023, more than 500 projects, covering a wide range of compounds, have been completed. Most of the conjugates produced for customers were high-quality, with over 90% containing a single ratio conjugate or pure conjugate. These advanced conjugation processes encompass a broad array of technologies:

• Bioconjugation workflow
• Labeling and bioconjugation methods
• Bioanalytical, biopurification, and bioassay methods
• Modification of biopolymers and small molecules

Unlike medicinal chemistry, bioconjugation is a complex process that has not yet been fully integrated into the drug development workflow. Traditional medicinal chemistry techniques—such as optimizing reaction conditions, characterization, purification, and analysis—do not readily apply to bioconjugation. For instance, in a typical chemical reaction, yields are high, the product is simple, and reaction are reproducible. However, in biopolymer conjugation, yields are often low, and products are more complex, typically containing various isoforms and polyconjugated products. A specific reaction condition that works for one biopolymer may not work for another similar biopolymer.

At CellMosaic, we have developed conjugation processes that can be readily applied to a wide range of biopolymers. These processes have been extensively optimized and validated through custom synthesis projects, enabling efficient conjugation. 

Bioconjugation Workflow

CellMosaic has developed an integrated bioconjugation workflow that can be readily applied to any biopolymer. This workflow incorporates various linkers and linking chemistries, enabling the synthesis of stable or releasable conjugates. In addition, purification and analysis methods have been established for a wide variety of molecules. Characterization methods have been developed for antibodies, proteins, peptides, oligos, and enzymes. Essentially, with minimum adjustments, a variety of conjugates can be developed utilizing this model, along with the methods and facilities available at CellMosaic

Labeling and Bioconjugation Methods

At CellMosaic, several strategies are employed to obtain single-labeled or site-specific bioconjugates, including site-directed cysteine mutagenesis, structure characterization, and purification techniques. These strategies significantly enhance the quality of the bioconjugates.

Peptide labeling and conjugation: Peptide–drug conjugates or biopolymer-peptides are prepared based on a unique bioconjugation strategy developed at CellMosaic. Drugs or biopolymers can be conjugated at any position on the peptide (C-terminus, N-terminus, or middle). Typically, a single, and pure bioconjugate is obtained. The advantages of this peptide conjugation strategy include:

• Freedom to attach the drug or molecule of interest at any point on the peptide.
• Other functional groups in the peptide, such as OH, NH₂, and SH, remain unaffected by the conjugation process.
• This strategy can easily be adjusted to accommodate different types of compounds.
• Short development timeline for the conjugation strategy.

Antibody conjugation and antibody fragment-based bioconjugation: CellMosaic has developed unique bioconjugation strategies for site-specific antibody labeling and conjugation. Small drugs or toxins can be conjugated to an antibody at a specific site, away from the antigen binding site. Additionally, a method has been created for introducing a single labeling site into an antibody for bioconjugation. CellMosaic also offer processes and methods for obtaining antibody fragments for bioconjugation.

Oligo-based bioconjugation: Few of the main challenges in developing oligonucleotide analogs and siRNA-based therapeutics is limited cellular uptake, poor in vivo pharmacokinetic, and lack of tissues/cell target specificity. Recent studies show that chemically conjugating oligonucleotide and siRNAs with bioactive molecules, lipids, or polymers can help overcome these limitations. oligos can also be designed to selectively target genes and deliver small-molecule drugs to disease sites. CellMosaic's oligo-based conjugation strategy allows drugs or biopolymers to be conjugated at any position (3', 5', or via a modified base). Conjugation can be performed in either the solid phase or post-synthesis solution phase, typically resulting in a single, pure conjugate. Key advantages of this strategy include:

• Flexibility to attach a drug or molecule of interest at any point on the oligo.
• Functional groups in the molecule, such as OH, NH₂, and SH, remain unaffected by the conjugation process.
• The strategy can easily modified to accommodate different types of compounds.
• Short development timeline for the conjugation strategy.

Protein-based bioconjugation: Recombinant proteins are often unstable in vivo, requiring high doses for clinical use. Chemical modification with water-soluble molecules can improve protein stability, leading to better therapeutic effects. Recombinant proteins can also be conjugated to antibodies, drugs, or toxins for other beneficial effects. Traditional protein-labeling methods typically target multiple Lys groups in the protein. This approach can alter the overall pI of the protein and may produce partially active or inactive conjugates, leading to heterogeneous conjugates with varying pK properties. To0 address these challenges, CellMosaic uses a combination of mutagenesis, purification, and labeling technologies to produce high-quality protein conjugates.

Bioanalytical, Biopurification, and Bioassay Methods

The CellMosaic team has extensive experience in analyzing and purifying a wide range of small organic compounds, biopolymers, and bioconjugates using HPLC with various media and columns, including reverse-phase, size-exclusion, hydrophobic interaction, anion, and cation exchange. CellMosaic has established methods for analyzing peptides, oligos, proteins, antibodies, and their conjugates. A key aspect of CellMosaic’s expertise is the development of biopurification methods to isolate a single, pure bioconjugate based on its specific properties. For instance, most peptide- and oligo-related conjugates are purified by reversed-phase HPLC, while antibody-based conjugates are separated using affinity purification. Other biomacromolecule-based conjugates, such as HRP, are purified by size-exclusion chromatography.

CellMosaic has also developed methods for analyzing molecules using electrospray ionization (EI) MS or MALDI-TOF MS for various bioconjugates.

An essential element of bioconjugation is understanding the characteristics of the biopolymers and quantifying their functional groups. CellMosaic has established methods to quantify functional groups such as thiol, maleimide, and amines during the conjugation, ensuring predictable final loading and maintaining batch-to-batch consistency.

Modification of Biopolymers and Small Molecules

For certain biological applications, it may be necessary to introduce functional groups or linkers into a biopolymer. Over the years, CellMosaic has developed a variety of methods for modifying biopolymers with unique functional groups for labeling or conjugation, such as aldehyde or thiol groups. Introducing aldehyde groups into biopolymers enables specific reactions at the aldehyde site, including reductive amination, oxime formation, and hydrazone formation. CellMosaic also has expertise in preparing fragmented antibodies, such as Fab, Fab', and combination antibody fragments, including bi-specific antibodies, using a combination of chemical and enzymatic approaches.

PerKit^® Products

Many biotechnology/biochemical companies, biochemical and chemical suppliers, and distributors offer “bioconjugation kits”. However, a closer look at these “kits” reveals that they often contain one or two reagents designed for generic biochemical labeling. A classic example is a fluorescent dye labeling kit that includes only a reactive florescent dye, aiming to increase perceived value. Many such kits do not provide a comprehensive solution for customers (i.e., specific examples, analytical data, purification/cleanup components, or a user manual), often resulting in failed or suboptimal downstream experiments and customer frustration with “bioconjugation kits”.

PerKit® (Personalized Bioconjugation Kit) is a high-quality, comprehensive product line created in 2016 and continually refined. Built on CellMosaic’s proprietary advanced bioconjugation processes, it is designed to meet unique bioconjugation needs for various biomolecules. This product line is ideal for customers with limited budgets or those requiring frequent or large-volume conjugation of one or more molecules. PerKit® stands out from other bioconjugation kits on the market in several key ways.

• Tailored to individual requirements (either customer or molecule): Each molecule is unique and a bioconjugation process that works for one may not work for another. PerKit® products are customized for each class of molecules to help customers perform bioconjugation confidently. Some PerKit® products are made-to-order, with kit contents and user manuals adapted based on customer input during the ordering process..
• Validated processes: Most linkers, spacers, and conjugation chemistries in PerKit® have been validated with similar conjugates developed through CellMosaic's Custom Bioconjugation Services. All PerKit® products undergo testing before market release, with sample conjugation data shared with customers.
• High-quality conjugates: Purification accessories are included to ensure the high quality of the final conjugate.
• Simple and easy to use: Kits come with all necessary reagents and procedures (except customer-supplied molecules). Most conjugations can be completed within a single day.
• Superior customer support: Dedicated customer support is available throughout the conjugation processes. Additional support services are offered for customers requiring further analysis of their conjugates.
• Continuous product development and updates: After launch, CellMosaic continuously monitors product performance in customer’s hands, making improvements to the composition or user manual as needed. Any newly developed processes that enhance kit performance are also incorporated into PerKit.

Since launching, customers have quickly adopted PerKit®. As of August 2024, CellMosaic offers around 40 PerKit products. Below are a few examples of the PerKit product launching timeline:

• December 2016: PerKit® HRP-oligo conjugation kit --- single label product for diagnostic
• December 2017: PerKit® Antibody MMAE conjugation kit --- for ADC candidate screening
• July 2019: PerKit® AP-oligo conjugation kit --- single label product for diagnostic
• January 2021: PerKit® Antibody-oligo conjugation kit --- for drug candidate screening.

[Click here for a complete list of PerKit^® products.] Any project beyond the scope of PerKit™ configuration can be handled through CellMosaic's Custom Bioconjugation Service.