Therapeutic ADCC⁺ Biobetter Antibody Production

Glyco-engineering

Glycosylation of the Fc region closely links to therapeutic antibodies properties. It is known that by changing the conformation at the N-glycosylation site of the Fc region or alternation on glycan-glycan interaction, the affinity of IgG to the Fc gamma receptor is directly affected. And ADCC effect is therefore altered. Also, the presence of a glycan-containing core fucose or terminal sialic acid at Fc Asn297 could also reduce IgG affinity to the Fc gamma receptor. Here at Creative Biolabs, our glycosylation technology, however, has increased IgG affinity for FcγRIIIa by more than 100-fold. This is achieved by knocking out the gene encoding α1,6-fucosyltransferase (Fut8). To achieve accurate glycan-engineering modification, we have developed a variety of targeted gene-editing tools to effectively screen mutants from glycosylation.

Lectin-based glycosylation mutant separation

• Glycan-engineering of host cell lines is done by untargeted screening or targeted gene-genomic editing tools.
• A common method is to use cytotoxic lectin to negatively select glycosylation mutants.
• Lectins are carefully chosen from target glycosylation characteristics.

Targeted gene-editing tool

It is quite a challenge to select various glycosylation mutants from the same gene copy. We have developed diverse gene-editing tools that can perform non-lethal knockout by targeting on a single or a set of genes.

• Transcriptional activator-like effector nuclease (TALEN)
• CRISPR-Cas9

Cell Line Development

Creative Biolabs provides mammalian cell lines for the production of recombinant therapeutic proteins, which could produce a large number of consistent quality products to meet current regulatory requirements.

Glyco-engineering host cell lines

Based on our advanced technology, we offer our customers with afucosylated cell lines. Our optimal glycol-engineering host cell line has several advantages: (i) excellent glycosylation ability; (ii) preventing the synthesis of unfavorable glycan structures (iii) possibility to produce optimized glycoforms. These properties are highly desirable for the production of bio-better versions of therapeutic proteins, such as afucosylated mAbs.

Highlighted features

• Glycan engineered CHO cell lines provided by us have extraordinary productivity, stability under long-term production and good overall quality.
• Screening is done to thousands of clones through multiple evaluation stages. Starting with a small initial screening, we use shake flasks and micro bioreactors to identify high-volume clones and gradually, scale them up.
• Comprehensive product quality attributes are characterized including glycosylation profiles, charge variation, aggregation levels and protein sequence variation.

For more details, please click Stable cell line service.

Bioprocess Control

Like other biopharmaceuticals, therapeutic ADCC⁺ antibodies also require extremely high purity and potency. Creative Biolabs monitors parameters from Quality by design (QbD) and glycosylation related critical quality attribute (gCQA) throughout the production. For the glycosylation control in the upstream biological process, QbD is adopted to make sure the product quality is defined during the designing stage. The key to implementing QbD is to pinpoint gCQA. We have established systematic approaches to identifying and ranking gCQAs through safety and effectiveness (SE) analysis and the construction of impact maps.

Glycosylation control in biomanufacturing

Glycosylation is the most common type of post-translational protein modification. Since glycosylation can have an effect on antibody stability, ADCC activity and the pharmacokinetics of the final product, it is often considered a key quality attribute of therapeutic ADCC enhanced antibodies. Therefore, it is important to control glycosylation in the biomanufacturing process to ensure consistent glycosylation of the final product.

Creative Biolabs focuses on controlling the manufacturing processes (upstream and downstream) to achieve the desired product quality for therapeutic ADCC⁺ antibodies, including:

• Glycosylation control of antibody production host
• Glycosylation control of cell culture process conditions
• Glycosylation control of upstream process
• Controlling protein glycosylation

Purification Strategies

Like other biopharmaceuticals, ADCC antibodies require excellent level of purity and potency. Achieving efficient downstream purification is a critical step in the overall process. For monoclonal antibody products, the industry standard for commercial scale purification is Protein A chromatography. However, in the ADCC antibody production environment, the capture step requires a unique lectin or borate affinity chromatography to selectively capture the glycan component.

Lectin-based purification

Lectins are widely used to identify and isolate certain types of carbohydrate residues, e.g. glycoproteins, glycopeptides, glycolipids, and oligosaccharides. The most commonly used lectin is concanavalin A (Con A).

Borate-based purification

An alternative to lectins when separating glycoproteins is the borate columns because they are inexpensive and stable. The most commonly used borate adsorbent is m-aminophenylboronic acid agarose. It has been successfully applied to selectively capture IgG from CHO cell supernatants.

ADCC Analysis

With the help of our proprietary and advanced ADCC platform technology, Creative Biolabs has developed a revolutionary ADCC analysis method that is believed to address all problems associated with the traditional ADCC analysis. Even with novel cell lines, we are able to provide a more optimized method to measure target cell death with varying levels of biological relevance and sensitivity, all of which have unprecedented levels of performance and consistency.

ADCC assays

• Target cell lysis
• Effector cells activation
• Real-time monitoring

Glycan analysis

• Intact proteins
• Glycopeptides
• Released glycans

Fc Receptor Binding (FcγR) assays

We came up with various advanced technologies technology platforms. Their simplicity and utility are demonstrated when determining binding profiles of therapeutic ADCC⁺ antibodies and FcγR.

For more details, please click Therapeutic ADCC⁺ Antibody Analysis.

With decades of experience in the field of antibody development, Creative Biolabs is able to provide our clients with the flexibility to adapt to the evolutionary changes in ADCC⁺ antibody development filed. For more details about our Therapeutic ADCC⁺ Biobetter Antibody Production service, please do not hesitate to contact us.