Purpose: Characterization of therapeutic proteins through in vitro assessment of their critical quality attributes is an important part of biopharmaceutical product development. Generally, in vivo modifications or changes in quality attributes of protein therapeutics are not monitored due to technical challenges and sample availability, especially in clinical studies. Advances in high resolution mass spectrometry, coupled with powerful immunoaffinity sample purification procedures, have created an opportunity to study in vivo critical quality attributes. Additionally, use of multiplexed proteomic approaches to assess early and late immune based signatures can help elucidate the biological relevance of such attributes. Here we present a general strategy on how to identify and quantitate in vivo predicted and unexpected modifications of therapeutic proteins.
Methods: Figure 1 illustrates the work flow. Cynomologus monkeys were dosed with a therapeutic IgG1 monoclonal antibody (mAb1) at 200 mg/kg via IV infusion. Serum samples were collected at pre-dose, 15 minutes, 2, 4 and 7 days and kept frozen under -70°C. The therapeutic antibody was spiked into pooled cynomologus monkey serum at 0.5 mg/ml and incubated at 37°C for comparison. Total mAb1 was then purified from 30 µL serum samples by a biotinylated anti-idiotype antibody and streptavidin coupled magnetic beads. The antibody was eluted, reduced, alkylated, and digested with trypsin. The resulting digests were analyzed on a Q-Exactive HF Orbitrap mass spectrometer coupled with nanoAcquity UPLC. Separation was achieved using an Acquity HSST3 C18 column at 40 µL/min with a 90-min linear acetonitrile gradient. The multiplexed cytokine assessments were performed using the luminex platform.
Results: To demonstrate proof of concept, we first performed characterization of a therapeutic IgG1 mAb1 using peptide mapping. A list of potential quality attributes was built by comparing peptide maps between reference material, stressed and stability samples in formulation buffer. Approximately 50 potential quality attributes were identified. We then developed an affinity purification procedure to pull down mAb1 from cynomolgus monkey serum. The capture reagent was a biotinylated anti-idiotype antibody developed for capturing total mAb1. Pooled cynomologus monkey serum spiked with different concentrations of reference material and stressed mAb1 were used to optimize the affinity purification and digestion procedures. The goal was to achieve reproducible recovery of the therapeutic protein and protein variants, and minimize sample processing artifacts.
Using the optimized sample preparation method, pharmacokinetic profiles of multiple quality attributes were obtained from mAb1 dosed cynomologus monkeys. In addition, the same procedure was performed to analyze those ex vivo samples where mAb1 was spiked into pooled serum and incubated at 37°C. All 50 potential quality attributes were detected in both in vivo and ex vivo samples. Several quality attributes like high-mannose glycan and C terminal lysine processing on the heavy chain showed minimal changes ex vivo but significant changes in vivo (Figure 2&3), highlighting the potential importance of monitoring quality attributes in vivo. Another critical aspect of the strategy was to identify unexpected modifications in drug product in vivo using several commercially available New Peak Detection algorithms. The ability to normalize differences in overall peak intensity, set up appropriate thresholds to remove false positives, align retention times, and perform peak picking was compared. Gaps in these tools were identified.
Conclusion: Taken together, our results demonstrate that it is possible to recover and quantify in vivo modified protein variants from serum. This approach will have great utility in understanding the potential impact of product quality attributes on biological activity and safety risk of the protein therapeutics.
Yi Wang– Merck & Co., Inc.
Richard Rogers– Just BioTherapeutics
Daniela Tomazela– Merck & Co., Inc.
Douglas Richardson– Merck & Co., Inc.
Michael Swanson– Merck & Co., Inc.
Mohammad Tabrizifard– Merck & Co., Inc.
Lucinda Hittle– Merck & Co., Inc.
David Mclaren– Merck & Co., Inc.
Vihba Jawa– Merck & Co., Inc.