Strategy for Generating Sequence-Defined Aptamer Reagent Sets for Detecting Protein Contaminants in Biotherapeutics.

Biologic drugs are typically manufactured in mammalian host cells, and it is critical from a drug safety and efficacy perspective to detect and remove host cell proteins (HCPs) during production. This is currently achieved with sets of polyclonal antibodies (pAbs), but these suffer from critical shortcomings because their composition is inherently undefined, and they cannot detect nonimmunogenic HCPs. In this work, we report a high-throughput screening and array-based binding characterization strategy that we employed to generate a set of aptamers that overcomes these limitations to achieve sensitive, broad-spectrum detection of HCPs from the widely used Chinese hamster ovary (CHO) cell line. We identified a set of 32 DNA aptamers that achieve better sensitivity than a commercial pAb reagent set and can detect a comparable number of HCPs over a broad range of isoelectric points and sizes. Importantly, these aptamers detect multiple contaminants that are known to be responsible for therapeutic antibody degradation and toxicity in patients. Because HCP aptamer reagents are sequence-defined and chemically synthesized, we believe they may enable safer production of biologic drugs, and this strategy should be broadly applicable for the generation of HCP detection reagents for other cell lines.

Process-scale purification and analytical characterization of highly gamma-carboxylated recombinant human prothrombin.

Prothrombin (coagulation Factor II) is a complex multidomain glycoprotein that plays a central role in blood coagulation. It is the zymogen precursor to the protease thrombin that catalyzes the formation of the fibrin clot and regulates a multitude of other cellular responses related to coagulation and hemostasis. For the biological activity of prothrombin, the vitamin K dependent posttranslational modification of glutamic acid residues to gamma-carboxylglutamic acid is of crucial importance. Prothrombin can be recombinantly expressed using mammalian cell culture. However, the product is a heterogeneous mixture of variants with different degrees of carboxylation, requiring separation of closely related charge isoforms. A second challenge for purification is the need to remove traces of the product-related impurity thrombin, a protease, to extremely low levels. In this work, we describe a purification strategy that provides solutions to both challenges and results in an efficient and robust process for active recombinant prothrombin. We also describe the analytical characterization of recombinant prothrombin by HPLC, LC-MS/MS, and complementary biochemical assays.

Implementation of parallelism testing for four-parameter logistic model in bioassays.

Parallelism is a prerequisite for the determination of relative potency in bioactivity assays. It involves the testing of similarity between a pair of dose-response curves of reference standard and test sample. The evaluation of parallelism is a requirement listed by both the United States Pharmacopeia (USP) and European Pharmacopeia (EP). The revised USP Chapters 〈1032〉 and 〈1034〉 suggest testing parallelism using an equivalence method. However, implementation of this method can be challenging for laboratories that lack experience in statistical analysis and software development. In this paper we present a customized assay analysis template that is developed based on a fully good manufacturing practice (GMP)-compliant software package. The template allows for automation of the USP-recommended equivalence parallelism testing method for 4PLmodel in bioassays. It makes the implementation of the USP guidance both practical and feasible. Use of the analysis template is illustrated through a practical example. LAY ABSTRACT: Parallelism is a prerequisite for the determination of relative potency in bioactivity assays. It involves the testing of similarity between a pair of dose-response curves of reference standard and test sample. The evaluation of parallelism is a requirement listed by both the United States Pharmacopeia (USP) and European Pharmacopeia (EP). The revised USP Chapters 〈1032〉 and 〈1034〉 suggest testing parallelism using an equivalence method. However, implementation of this method can be challenging for laboratories that lack experience in statistical analysis and software development. In this paper we present a customized assay analysis template that is developed based on a fully good manufacturing practice (GMP)-compliant software package. The template allows for automation of the USP-recommended equivalence parallelism testing method for 4-parameter logistic model in bioassays. It makes the implementation of the USP guidance both practical and feasible. Use of the analysis template is illustrated through a practical example.