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Advanced Conjugation Processes and PerKit®

CellMosaic’s advanced conjugation processes have been developed and optimized through custom bioconjugation services. More than 500 projects covering a variety of compounds have been completed as of March 2023. Most of the conjugates made for customers were high-quality product containing over 90% single ratio conjugate or pure conjugate. The advanced conjugation processes encompass a wide array of technologies:

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

Unlike medicinal chemistry, conjugation is a complicated process that has not been fully developed and integrated into the drug development process. The usual techniques in medicinal chemistry, such as the optimization of reaction conditions, characterization, purification, and analysis, do not apply to bioconjugation. For example, in a conventional chemical reaction the yield is high, the product is simple, and the reaction is reproducible, but in a biopolymer conjugation the yield is often low and products are complex, usually containing various products and isoforms, including polyconjugated products. A particular reaction condition that works for one biopolymer may not work for another similar biopolymer.

At CellMosaic, we have developed conjugation processes that can be applied readily to any biopolymer. The processes were extensively optimized at CellMosaic and validated by custom synthesis. The process can be completed efficiently. The processes encompass a variety of linkers and linking chemistry that allow stable conjugation or releasable conjugates. Purification and analysis methods have also been developed for a variety of molecules. Characterization methods have been developed for antibodies, proteins, peptides, oligos, and enzymes. Essentially, with little change, we can develop a variety of conjugates utilizing this model and the methods and facilities at CellMosaic.

Bioconjugation Workflow

CellMosaic has developed an integrated bioconjugation workflow that can be applied readily to any biopolymer. The workflow includes a variety of linkers and linking chemistry that allows stable conjugation or releasable conjugates. Purification and analysis methods have also been developed for a variety of molecules. Characterization methods have been developed for antibodies, proteins, peptides, oligos, and enzymes. Essentially, with little change, a variety of conjugates can be developed utilizing this model and the methods and facilities at CellMosaic

Labeling and Bioconjugation Methods

Several strategies are applied at CellMosaic, including site-directed cysteine mutagenesis, structure characterization, and purification techniques, to obtain single-labeled or site-specific labeled bioconjugates. This has a huge impact on 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. Drug or biopolymer can be conjugated anywhere on the peptide (C-terminus, N-terminus, or middle). Usually, a single and pure bioconjugate can be obtained. The advantages of our peptide conjugation strategy are:

• Freedom to attach the drug or molecule of interest at any point in the peptide.
• Other functional groups in the peptide, such as OH, NH₂, and SH, will not be affected by the conjugation step.
• The conjugation strategy can easily be tweaked to couple different categories of compounds.
• Short timeline for the development of a conjugation strategy.

Antibody conjugation and antibody fragment-based bioconjugation: CellMosaic is capable of site-specific antibody labeling and conjugation based on the unique bioconjugation strategy developed at CellMosaic. Small drugs or toxins can be conjugated to an antibody at a specific site away from the antigen binding site. In addition, a method was developed for specifically introducing a single labeling site into an antibody for bioconjugation. CellMosaic also has a process and methods to obtain antibody fragments for bioconjugation.

Oligo-based bioconjugation: Some of the obstacles in developing oligonucleotide analogs and siRNA-based therapeutics include limited cellular uptake, poor in vivo pharmacokinetic properties, and a lack of specificity for the target tissues/cells. New studies have shown that chemically conjugated oligonucleotide analogs and siRNAs with bioactive molecules, lipids, and polymers can overcome some of these limitations. On the other hand, oligos can be designed that selectively target the gene and are great for carrying small-molecule drugs to the disease target. Oligo-based conjugates are prepared using a unique conjugation strategy developed at CellMosaic. Drug or biopolymer can be conjugated anywhere on the oligo (3', 5', or via a modified base). The conjugation can be performed in either the solid phase or post-synthesis solution phase. Usually, a single and pure conjugate can be obtained. The advantages of CellMosaic’s oligo conjugation strategy are:

• Freedom to attach a drug or molecule of interest at any point in the oligo.
• Other functional groups in the molecule, such as OH, NH₂, and SH, will not be affected by the conjugation step.
• The conjugation strategy can easily be tweaked to couple different categories of compounds.
• Short timeline for the development of a conjugation strategy.

Protein-based bioconjugation: Recombinant proteins are not very stable in vivo and high dosages are often required for clinical use. Chemical modification of recombinant proteins with water-soluble molecules may improve the proteomic stability, resulting in better therapeutic effects. Recombinant proteins can also be conjugated to antibodies, drugs, and toxins for other beneficial effects. Traditional protein-labeling technologies usually take advantage of the multiple Lys groups located in the protein. Modification of the Lys groups changes the overall pI of the protein and may produce partially active or inactive protein conjugates. Lys labeling also produces heterogeneous conjugates that have different pK properties. CellMosaic uses a combination of strategies, including mutagenesis, purification, and labeling technologies, to produce high-quality protein conjugates.

Bioanalytical, Biopurification, and Bioassay Methods

The CellMosaic team has tremendous experience in analyzing and purifying a variety of small organic compounds, biopolymers, and bioconjugates by HPLC utilizing different media and columns, such as reverse-phase, size-exclusion, hydrophobic interaction, anion, and cation exchange. CellMosaic has established methods for analyzing peptides, oligos, proteins, antibodies, and their conjugates. One of the key aspects of CellMosaic’s work is the development of biopurification methods for the separation of a single and pure bioconjugate based on its properties. For example, most of the peptide and oligo-related conjugates are separated by reversed-phase HPLC, whereas antibody-based conjugates can be separated by affinity purification. Other biomacromolecule-based conjugates, such as HRP, may be separated by size-exclusion chromatography.

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

One of the key elements of bioconjugation is knowing the characteristics of the biopolymers and quantifying their functional groups. Methods have also been established for quantifying these functional groups, such as thiol, maleimide, and amines, during the conjugation, making the final loading predictable 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 on a biopolymer. Over the years, CellMosaic has established a variety of methods for biopolymer modification with unique functional groups for labeling or conjugation, such as aldehyde or thiol functional groups. The introduction of aldehyde groups into biopolymers allows them to react with other molecules specifically at the aldehyde site through reductive amination, oxime, and hydrazone formation. CellMosaic also has methods to prepare fragmented antibodies, such as Fab, Fab', and combination antibody fragments, including bi-specific antibodies, using a combination of chemical and enzymatic approaches.

PerKit^® Products

To meet customers' individual requirement for bioconjugation, CellMosaic has developed a product line called Personalized™ Bioconjugation Kit (PerKit^®) based on its advanced bioconjugation processes since 2016. This product line is perfect for customers who have either a limited budget for conjugation or need frequent/large volume conjugation of one or multiple molecules. PerKit^® is different from any other bioconjugation kits on the market in many ways.

• Focusing on individual requirements (either customer or molecule): Each molecule is unique, as a bioconjugation process that works for one molecule may not work for another. PerKits™ are tailored for each class of molecule and designed to help customers perform the bioconjugation themselves with confidence. For example, some of the PerKits™ are made to order. Based on the input from customers when placing the order, the content and user manual of the PerKit^® will be modified to accommodate the need. In some cases, customers can request a PerKit^® sheet containing calculations, chemical structure, and MW of the final conjugate, with recommendations for the conjugation if there is some special feature of the molecule.
• Validated processes: Most of the linkers/spacers and conjugation chemistry used for PerKit^® have been validated in similar conjugates made via Custom Bioconjugation Services. All PerKit^® products will be tested before being put on the market and sample conjugation data shared.
• Produce high-quality conjugates: Purification accessory is included in the kit to ensure the quality of the conjugate.
• Simple and easy to use: The kits are complete with all reagents and procedures except customer-supplied molecules. Most of the kit can be done within a day.
• Superior customer support: Customer support is readily available to customers throughout the conjugation processes. Additional support services are also available to customers who need further analysis of their conjugates.

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