Tackling Aggregation Challenges in ADC Production

Published on: 
Pharmaceutical Technology, Pharmaceutical Technology-09-01-2018, Volume 2018 Supplement, Issue 4
Pages: s18–s19

Immobilizing the antibodies on a solid-phase support, such as a resin, and carrying out the conjugation of the payload-linker while the antibodies are bound to that support will prevent aggregation at its source.

Antibody-drug conjugates (ADCs) are targeted therapies that combine a specific antibody or antibody fragment linked to a drug. ADCs have revolutionized the treatment of a number of diseases by enabling more specific drug targeting with fewer side effects. Despite the growing ADC pipeline, Charlie Johnson, CEO of ADC Bio, notes that many promising ADCs are being overlooked because of critical aggregation control problems. Pharmaceutical Technology Europe spoke to Johnson about the limitations of conventional manufacturing techniques used in the production of ADCs and why preventing aggregation at its source is the best solution.

Causes of aggregation

PTE: What are the causes of aggregation in ADCs?

Johnson: Aggregation of biomolecules can occur for a variety of reasons. Some antibodies are inherently prone to aggregation, especially when they are maintained in solution under conditions that promote aggregation, for example:

  • Unfavourable buffer conditions where either the salt type and/or salt concentration is too low or too high can cause aggregation.

  • Aggregation can occur if the system is held at a pH that coincides with the isoelectric point of the antibody (this is the point where the biomolecule carries no net charge and represents the point of least aqueous solubility). This pH may be required, for example, when a particular conjugation chemistry requires a specific pH.

  • It is often the case during conjugation that solvents are required to solubilize otherwise poorly soluble payload-linkers, in particular highly hydrophobic ones. Some solvents are known to promote aggregation.

However, the most significant issue that increases the propensity of antibodies to aggregate is the modification of their surfaces through chemical conjugation to hydrophobic payload linkers. Once the payloads are conjugated to the antibody, they present hydrophobic patches that attract hydrophobic areas on other antibodies, thus initiating the aggregation process. Once formed, these low molecular weight aggregates form nuclei sites where further aggregation can occur, eventually leading to high molecular weight aggregates and ultimately precipitation from solution.

Conventional techniques

PTE: What are the limitations of conventional manufacturing techniques used in ADC production and why do they fail in preventing aggregation?

Johnson: Conventional conjugation of ADCs occurs in dilute aqueous buffered solutions. The buffer conditions and pH are adjusted to suit the chemical conjugation conditions for attachment of the payload-linker to the antibody. Historically, ADCs have been prepared by conjugation of either naturally occurring lysine or cysteine residues on the antibody, although more recent developments see the use of ‘site-specific’ residues, some of which focus on engineered-in residues (e.g., thiomabs) or through modification of naturally occurring sites on the antibody (e.g., glycan modification). What is common to all conventional conjugations is that eventually the antibody or modified antibody will be exposed to a payload-linker to produce the ADC, and that is commonly the most unstable point in the process for the antibody, because conditions such as buffer composition, concentration, pH, and addition of co-solvents are selected to optimize the conjugation chemistry, not the stability of the antibody. 

A degree of aggregation is, therefore, always expected, although the degree to which the antibodies aggregate is mainly determined by the inherent hydrophobicity of the payload itself. Efforts have been made to reduce hydrophobicity of payloads either through modification with solubilizing linkers or modification of the payload structure, but these efforts have not fully solved the problems of aggregation. Aggregation of ADCs is not simply an issue of yield inefficiencies and costs, although these issues can be severe and lead to the unviability of a product. 

Aggregates and, in particular, soluble high molecular weight aggregate must be removed as they are immunogenic and can cause severe allergic reactions if administered. Historically, aggregates are removed from ADCs via application of chromatographic methods such as size exclusion or hydrophobic interaction separation techniques. These approaches adds further processing time and cost, and it reduces the yield of the system, which all adds to the cost of producing an ADC. 

Preventing aggregation

PTE: What’s the solution to such aggregation problems? 

Johnson: The best solution to the aggregation problems prevalent with conventional conjugation methodology is to prevent aggregation at its source. If antibodies are held physically separate from each other during the ‘unfavourable’ conditions of conjugation, they cannot aggregate in the first place. 

An obvious way to prevent this aggregation is through immobilization of the antibodies on a solid-phase support, such as a resin, and carry out the conjugation of the payload-linker while the antibodies are bound to that support. ADC Bio’s Lock-Release technology uses this approach and can be performed in either batch or flow mode. It is easily scalable in the same way that other chromatography methods are. 

The initial step is antibody capture onto the resin, which is analogous to the protein A capture step in downstream processing of antibody manufacture. Once immobilized, the antibodies are sequentially exposed to process reagents and the payload-linker to produce the ADC, with intermediary washing steps to remove excess reagents. At the end of the process, the immobilized ADC is released from the resin into a stabilized buffer matrix that prevents any subsequent aggregation. 

Lock-Release processing provides a unique way of preventing aggregation of ADCs at source while ensuring that the ADCs produced are free of contaminants (e.g., free payload-linkers and solvents) that can interfere with in-vitro and in-vivoread-outs and also lead to enhanced toxicity when administered to patients.

ADC manufacturing using Lock-Release is significantly simplified compared to conventional manufacturing, because the process equipment train used is the same irrespective of the ADC being made. Conventional manufacturing, in contrast, will have different requirements in terms of process equipment and hence the manufacturing footprint required depending on the process developed.

Locking mechanism

PTE: Can you tell us more about the locking mechanism?

Johnson: Lock-Release at its most basic level is a conceptual way of constructing ADCs on solid supports, designed to improve both the quality of ADCs, the yield, and as a result, the economics of production. ADC Bio has patented multiple methods of Lock-Release. The favoured technique to date is based on affinity capture, using mimetic resins. The specific resins used depend on the process in question and, to some extent, where on the antibody the conjugation of the payload-linker is required. As with most affinity capture methods, the release method relies on varying pH and salt concentration. The demonstrated capacity of our Lock-Release resins is in the range 5 to 50 mg per mL of resin, but we expect that this range can be enhanced further. 

A significant advantage of using affinity-based capture techniques is that the resin can be re-used over multiple batches, and that the quality of product is extremely high and consistent over multiple batches.

Lock-Release can be run in either stirred batch or column (flow-mode). For manufacturing purposes, flow mode is preferred because it offers predictable linear scale-up as with other chromatography-based techniques. As such, the required manufacturing footprint and equipment train remain similar for all Lock-Release-based processes, which offers significant productivity and capital cost advantages over conventional manufacturing techniques at commercial manufacturing scale.

Article Details

Pharmaceutical Technology
Supplement: APIs, Excipients, & Manufacturing 2018
September 2018
Pages: s18–s19


When referring to this article, please cite it as A. Siew, " Tackling Aggregation Challenges in ADC Production," Pharmaceutical Technology APIs, Excipients, & Manufacturing 2018 (September 2018).