Comparison of purification strategies for antibodies used in a broad spectrum host cell protein immunoassay.

Host cell proteins (HCPs) are a heterogeneous mixture of impurities that should be minimized in bulk preparations of biotechnologically produced medicines. Immunoassays are commonly used to detect and measure HCPs in therapeutic products, and a successful assay is directly dependent on the quality of the polyclonal antibodies (pAbs) used. These pAbs are enriched from antisera of animals immunized with a broad mixture of HCPs, but there is limited information regarding the best strategy for purification of these critical reagents. The use of protein A or protein G affinity chromatography results in purified pAbs that are not entirely HCP-specific, while the use of HCP affinity chromatography results in a more specific pAb population but may be harder to recover fully. In theory, both approaches have advantages and disadvantages for generating optimal reagents. In this study, we compared reagents from these two purification procedures using the same starting material, as well as those from a step-wise combination of the two by evaluating purity, concentration, reagent coverage by Western blotting, and performance in an enzyme-linked immunosorbent assay (ELISA). This study demonstrates that pAbs purified by each of the methods are very similar in terms of sensitivity, the ability to recognize a broad range of HCPs, and overall performance in an ELISA measuring a range of HCPs in upstream process and final drug substance (DS) samples.

Comparison of analytical techniques to quantitate the capsid content of adeno-associated viral vectors.

Adeno-associated virus (AAV) vectors, which contain a DNA transgene packaged into a protein capsid, have shown tremendous therapeutic potential in recent years. An inherent characteristic of the manufacturing process is production of empty capsids that lack the transgene and are therefore unable to provide the intended therapeutic benefit. The effect of empty capsids on clinical outcomes is not well understood, but there are immunogenicity and efficacy concerns, and these empty capsids are considered a product-related impurity. Therefore, empty capsids should be controlled during the manufacturing process and monitored through analytical testing, but there are limited techniques available that are capable of quantifying capsid content and even fewer that are amenable to validation and implementation as registered release tests in a regulated environment. In addition, there is currently not a widely accepted gold standard technique for quantifying capsid content, and the understanding of how the results compare between different orthogonal technologies is limited. The current study utilizes a comprehensive assessment to evaluate diverse analytical techniques for their ability to quantitate capsid content.

Edetate Disodium as a Polysorbate Degradation and Monoclonal Antibody Oxidation Stabilizer.

Polysorbates are frequently used in biotherapeutic formulations. Interest in assessing their stability, in particular the impact of their degradation products on the stability of therapeutic proteins, has been steadily growing in the past decade. The work presented summarizes a case study of a monoclonal antibody formulation that demonstrated a simultaneous loss of polysorbate and an increase in methionine oxidation. Spiking studies were conducted to determine both the cause and a potential mitigation for the monoclonal antibody (mAb) oxidation and polysorbate 80 (PS80) loss. The results indicated that a different source material exhibited different rates of mAb oxidation and PS80 loss and that in all evaluated materials, the addition of edetate disodium to the formulation mitigated both observed issues. The mAb was assessed for the presence of lipases and lipoprotein lipase was detected at low levels. It is proposed that edetate disodium was effective in mitigating the mAb oxidation and PS80 loss by chelating calcium in the formulation and therefore decreasing the activity of the lipases.