Controlling aggregation in monoclonal antibody therapeutics

Monoclonal antibodies (mAbs) have become the most important form of protein therapeutic and represent five of the top ten selling drugs by revenue worldwide. mAbs bind monospecifically to certain cells or proteins. The objective is that this treatment will stimulate the patient’s immune system to attack those cells. Despite the many advantages of antibodies as therapeutics, their development is complicated by the fact that antibodies are large, complex molecules and are subject to a range of degradation pathways.

This might include the product breaking up into fragments or aggregating, which is an issue as products are typically highly concentrated. This is where antibodies will start sticking together either covalently or noncovalently, which can make them lose efficacy or become immunogenic.

A critical aspect of drug development is choosing the correct formulation buffer. These are the liquid solutions that antibodies are stored in. These are often highly specific to the protein of interest. The formulation buffer is chosen primarily to improve the stability of the protein therapeutic toward degradation, which is effected through maintaining the protein at an appropriate pH. The optimal pH varies from molecule to molecule, though a weakly acidic pH is most commonly used.

Light exposure is a critical environmental factor that typically affects a therapeutic at all stages of manufacture from cell culture to patient administration. Once the amount of light that an antibody is exposed to exceeds a certain amount the antibody will start to aggregate. A major pathway of aggregation under light is an exposed histidine side chain on the mAb becomes oxidized. This molecule will ‘float’ around in solution until it interacts with an exposed histidine amino acid on an adjacent antibody. This results in two covalently bonded antibodies, which is the first step in lots of molecules being heavily aggregated, and therefore potentially dangerous.

Histidine’s pKa of ~6 and stabilizing effects make it a buffer of choice for antibody formulation. In addition to histidine, other amino acids are frequently incorporated into mAb formulation buffers. These include glycine, proline, lysine, arginine, glutamate and methionine. Each of these amino acids are added for the purpose of preventing product degradation. For example, methionine as an amino acid that is especially adept at preventing oxidation events on the product, and proline which is important in maintaining the solution viscosity.

In my work, I identify novel reactions where the amino acids present in the formulation buffer covalently bind to the antibody, or ‘cross-link’. My studies indicate that formulation buffer amino acids bind to mAbs preferentially to them binding to each other, thereby preventing the direct aggregation event described above. However, cross-linking of amino acids to a therapeutic mAb has the potential to significantly impact the overall product quality of the drug. For instance, cross-linking within or close to a complementarity determining region (CDR) may result in a loss of binding activity and hence the efficacy of the drug would be impacted. In addition, cross-linking of charged amino acids could result in aggregation of the antibody, through the induction of a non-native conformational state or through the addition of surface charge.

It is important to monitor the amount of each of these undesired reactions to maintain levels within safe amounts. The work I have completed has defined these modifications for the first time and allows them the be monitored throughout the industry.

Outside of biopharmaceutics, I expect these characterised reactions to play increased roles in cosmetic and nutraceutical industries. The addition of key amino acids onto carbohydrate or protein chains in a controlled reaction is something that has been sought after for some time, and our work describes an efficient way for this to be achieved.