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.

Comprehensive analysis of host cell impurities in monoclonal antibodies with improved sensitivity by capillary zone electrophoresis mass spectrometry.

Four methods were compared for analysis of host-cell protein (HCP) impurities in a recombinant mAb. First, CZE-MS/MS was used to analyze the digest of an HCP sample following extraction of the mAb with proteins A and L affinity columns; 220 protein groups and 976 peptides were identified from the depleted HCP digest. Second, a nanoACQUITY UltraPerformance LCH system was also used to analyze the depleted HCP digest; 34 protein groups and 53 peptides from 50 ng of the depleted HCP digest and 290 protein groups and 1011 peptides were identified from 1 µg of the depleted HCP digest. Third, 185 protein groups and 709 peptides were identified by CZE-MS/MS from the HCP digest without depletion. Fourth, a strong cation exchange SPE was coupled to CZE-ESI-MS/MS using online pH gradient elution for analysis of the HCP digest without depletion. A series of five pH bumps were applied to elute peptides from the strong cation exchange monolith followed by analysis using CZE coupled to a Q Exactive HF mass spectrometer; 230 protein groups and 796 peptides were identified from the HCP digest without depletion.

Storage Conditions of Conjugated Reagents Can Impact Results of Immunogenicity Assays.

Consistent performance of anti-drug antibody (ADA) assays through all stages of clinical development is critical for the assessment of immunogenicity and interpretation of PK, PD, safety, and efficacy. The electrochemiluminescent assays commonly employed for ADA measurement use drug conjugated with ruthenium and biotin to bind ADA in samples. Here we report an association between high nonspecific ADA responses in certain drug-naïve individuals and the storage buffer of the conjugated reagents used in a monoclonal antibody ADA assay. Ruthenylated reagents stored in phosphate-buffered saline (PBS) buffer had increased levels of aggregate and produced variable and high baseline responses in some subjects. Reagents stored in a histidine-sucrose buffer (HSB) had lower aggregate levels and produced low sample responses. In contrast to PBS, conjugated reagents formulated in HSB remained low in aggregate content and in sample response variability after 5 freeze/thaw cycles. A reagent monitoring control (RMC) serum was prepared for the real-time evaluation of conjugated reagent quality. Using appropriate buffers for storage of conjugated reagents together with RMCs capable of monitoring of reagent aggregation status can help ensure consistent, long-term performance of ADA methods.

Capillary zone electrophoresis tandem mass spectrometry detects low concentration host cell impurities in monoclonal antibodies.

We have evaluated CZE-ESI-MS/MS for detection of trace amounts of host cell protein impurities in recombinant therapeutics. Compared to previously published procedures, we have optimized the buffer pH used in the formation of a pH junction to increase injection volume. We also prepared a 5-point calibration curve by spiking 12 standard proteins into a solution of a human mAb. A custom CZE-MS/MS system was used to analyze the tryptic digest of this mixture without depletion of the antibody. CZE generated a ∼70-min separation window (∼90-min total analysis duration) and ∼300-peak capacity. We also analyzed the sample using ultra-performance LC-MS/MS. CZE-MS/MS generated approximately five times higher base peak intensity and more peptide identifications for low-level spiked proteins. Both methods detected all proteins spiked at ∼100 ppm level with respect to the antibody.