ADCC/CDC⁺ Antibody Process Development

Upstream Process Development

Process development and optimization in our upstream process (USP) consists several components: ADCC's proprietary deglycosylation cell line development and cell engineering, cell clone selection, media and feed development, bioprocess development and amplification, bioreactor optimization and design, and cell collection. Process glycosylation control and ADCC/CDC analysis are also part of the optimization process, and they are each optimized in order to achieve synergistic roles.

Fig.1 Optimization areas and parameters in our upstream processing.^{1, 3}

Stable CHO Cell Lines Development

The choice of expression system is influenced by its ability to conform to high productivity and quality standards. For example, Chinese hamster ovary (CHO) cells are widely used in therapeutic protein production because its proper folding rate and true post-translational modifications turnouts. Furthermore, based on Quality by Design (QbD), a comprehensive quality and characteristic analysis of drug candidates should be carried out in the early stages of cell line development, and appropriate quality standards should be established as early as possible. The following concepts of process optimization at cellular level are considered when developing our workflow:

• High productivity
• Post-translational modification
• Cell growth behavior
• Cell adaptive behavior
• Glycosylation profile
• Protein molecular integrity
• Purification recovery

Media Development and Optimization

Early cell culture media has serum supplement with complex mixtures of unknown ingredients. And because of this, Creative Biolabs has developed a number of serum-free media and fucose analog-added media to facilitate the production of ADCC-enhanced antibodies.

Commercially available media lay the foundations to specific media development process. Creative Biolabs has made breakthroughs in reducing the fucose content of the antibody by adding a fucose analog to the medium. This process is achieved by optimizing the factors such as cell line, medium composition, medium composition interaction and metabolic pathway. A significant increase in monoclonal antibody ADCC activity is, therefore, achieved.

Fig.2 Mass spectra of N-glycans of total CHO cell membrane proteins after treatment with fucose analog (B) under serum-free culture conditions; in comparison to an untreated control (A).^{2, 4}

Fc Variants Development and Determination

An engineered Fc protein backbone is commonly used to improve the ability of IgG1 antibodies to trigger CDC process. It is also known that some key amino acids at CH2 domain can bind to the component C1q. Creative Biolabs established advanced surface plasmon resonance (SPR) platform to measure binding affinities of Fc variant antibodies ti component C1q, aiming to screen for the optimal Fc variant.

Case study

We have previously screened a series of variants that bind to C1q. Based on the data obtained from these studies and other reports on C1q binding locations, we screened several single-substitution variants influencing CDC activity. We identified three substituents in the human IgG1 CH2 domain for further examination. The variant Fc domain (including these substitutions) was constructed, expressed, screened, and finally a humanized IgG1 antibody was obtained. We later examined the binding of the Fc variant to human C1q using SPR. The variant showed a tighter C1q affinity, a 47-fold increase over native IgG1.

Optimization of Bioreactor Systems

Creative Biolabs keeps track of the latest trends on bioreactors and already developed and optimized single-use bioreactor systems. The advantages of our optimized bioreactor systems are as follows:

• Lower costs
• Flexible to use
• Improved production scheduling
• Higher chance to achieve process replication
• Applicable for GMP manufacturing
• Available up to 2000 L scale
• Significantly reducing contamination rates
• Used not only in research but also in manufacturing processes in mini- and mid-scale

Downstream Process Development

Downstream process (DSP) development focuses on yield rate, productivity, purity and overall process capability. These attributes call for establishment of purification platform, QbD-based and Design of Experiments (DoE)-based high-throughput methods. Compared to traditional monoclonal antibodies, ADCC/CDC-enhanced antibody molecular structures are more complex, and difficult to obtain uniform monomer products, because the higher chance to degrade and this is believed to be the most significant challenges for downstream processes. However, Creative Biolabs is working hard to address this issued with a goal to effectively remove impurities but also maintain biologics with high recovery rates to meet clinical and market needs.

Fig 3. Optimization fields in downstream processing.^{1, 3}

Complement Source Control

Human complement component C1q, is essential in assessing the function of CDC-enhanced antibodies. The circulating C1q protein is a hexamer with three distinct protein subunits (A, B and C). As part of the C1 complex, C1q is critical for the activation of the classical complement pathway (CDC). In order to accurately measure the CDC activation effect, excellent complement activity and quality of C1q are required.

We use complement components derived from the serum pool of healthy blood donors, following the standards established by the International Complement Standardization Committee and the International Union of Immunological Societies (IUIS). In addition, the functional activities of all reagents are tested by standard lysis or applicable functional assays with a goal to access biochemical purity using SDS-polyacrylamide gel electrophoresis. For more detailed information on functional valence, please contact us for a certificate of analysis.

Analytical Development

Creative Biolabs provides a full range of analytical development services for our clients. Whether it's for the assessments on characterization, identification, elemental impurities, potency, packaging, stability or bioanalysis, our experienced scientists can help to accelerate your development process and minimize overall development time. For every analytical challenge, we use customized approach to tackle it with you and advance product registration process together.

Our services include but are not limited to:

• Glycosylation profile analysis
• Intact and semi-intact protein profiles
• N-terminal and C-terminal protein sequencing
• Post-Translational Modification (PTM) analysis
• Determination of disulfide bonds (heterogeneity)
• Charge variant analysis
• Size variant analysis (aggregates and fragments)
• Impurity testing (chiral purity, elemental impurity, and residual solvent analysis)
• Determination of host cell protein based on CHO
• High-throughput monoclonal antibody sample purification and sample analysis
• Physical and structural chemistry characterization
• Stability testing (forced degradation)
• Method identification and verification

Bioassay Development

The purpose of using bioassays is to determine the efficacy of a pharmaceutically active substance in a formulated product by observing its effect on a living animal (in vivo) or tissues (in vitro). Unlike conventional physical or chemical methods, bioassays deliver detailed information about the biological activity of the drug substances.

Creative Biolabs' expertise in bioassay and the experience from past projects allow us to work closely with our customers in API development process. The development, optimization, validation and performance of our biological assays are in accordance with GMP regulations.

• ADCC (using a proprietary effector cell line)
• FcγR- and FcRn-based binding assays
• Tissue cross-reactivity studies
• Immuno-oncology assays
• Cytokine release assays
• Potency assays
• Immunogenicity assays

Formulation Development

The development of a highly stable and effective antibody drug not only depends on key quality attributes of key drug candidates, but also on the development of a comprehensive formulation process. Formulation ingredients often involve buffers and excipient screening methods, and packaging materials. Creative Biolabs offers a full range of early and late phase formulation development services to help you obtain ADCC enhanced candidates that meet with pharmacopoeia standards and requirements.

Fig.6 End-to-end formulation development services.

Creative Biolabs is dedicated to developing the right bioassays for your ADCC⁺ antibodies research. For more details about our ADCC/CDC⁺ Antibody Process Development service, please do not hesitate to contact us.

Reference

1. Gronemeyer, Petra, Reinhard Ditz, and Jochen Strube. "Trends in upstream and downstream process development for antibody manufacturing." Bioengineering 1.4 (2014): 188-212.
2. Rosenlöcher, Julia, et al. "Applying acylated fucose analogues to metabolic glycoengineering." Bioengineering 2.4 (2015): 213-234.
3. under Open Access License CC BY 4.0, without modification.
4. under Open Access License CC BY 4.0. The original image was modified by extracting and using a part, and the title was changed to “ Mass spectra of N-glycans of total CHO cell membrane proteins after treatment with fucose analog (B) under serum-free culture conditions; in comparison to an untreated control (A) ”.