Integrated Bottom-Up and Top-Down Liquid Chromatography–Mass Spectrometry for Characterization of Recombinant Human Growth Hormone Degradation Products

Abstract

With the advent of biosimilars to the U.S. market, it is important to have better analytical tools to ensure product quality from batch to batch. In addition, the recent popularity of using a continuous process for production of biopharmaceuticals, the traditional bottom-up method, alone for product characterization and quality analysis is no longer sufficient. Bottom-up method requires large amounts of material for analysis and is labor-intensive and time-consuming. Additionally, in this analysis, digestion of the protein with enzymes such as trypsin could induce artifacts and modifications which would increase the complexity of the analysis. On the other hand, a top-down method requires a minimum amount of sample and allows for analysis of the intact protein mass and sequence generated from fragmentation within the instrument. However, fragmentation usually occurs at the N-terminal and C-terminal ends of the protein with less internal fragmentation. Herein, we combine the use of the complementary techniques, a top-down and bottom-up method, for the characterization of human growth hormone degradation products. Notably, our approach required small amounts of sample, which is a requirement due to the sample constraints of small scale manufacturing. Using this approach, we were able to characterize various protein variants, including post-translational modifications such as oxidation and deamidation, residual leader sequence, and proteolytic cleavage. Thus, we were able to highlight the complementarity of top-down and bottom-up approaches, which achieved the characterization of a wide range of product variants in samples of human growth hormone secreted from Pichia pastoris.

Recent advances in structural mass spectrometry methods in the context of biosimilarity assessment: from sequence heterogeneities to higher order structures

“…More recently, Wang et al demonstrated the benefits of combining a bottom-up and TD-MS method for the characterization of a recombinant human growth hormone (hGH). This study can be considered as proof of concept in the context of biosimilar development, highlighting the high similarity between biosimilar products and those of innovators [68].…”

Evaluating protein cross-linking as a therapeutic strategy to stabilize SOD1 variants in a mouse model of familial ALS

“…Samples of SOD1 A4V with or without 10-fold molar excess of S -XL6 in 10 mM Tris HCl (pH 7.4) were incubated for 4 h at 37°C [ 77 , 78 ]. After incubation, samples were alkylated with iodoacetamide (100 mM for 30 min), heated to 75°C for 20 min, and then treated with 2 volumes of Poroszyme immobilized trypsin (Applied Biosystems, Life Technologies Corporation, Carlsbad, California, USA) at 37°C for 15 min, mixing every few minutes to keep beads suspended.…”

On-demand manufacturing of clinical-quality biopharmaceuticals

“…LCMS equipment was used as described previously 33 , except a microspray ion source was used. Mobile phase A and B were as described previously 33 and the flow rate was 200 μL/min.…”

“…LCMS equipment was used as described previously 33 , except a microspray ion source was used. Mobile phase A and B were as described previously 33 and the flow rate was 200 μL/min. The gradient was as follows: 0–2 min 2% B with curve level 5, 2–30 min to 40% B, 30–39 min to 60% B, 39–42 min to 85% B until 47 min, 48–52 min to 2% B again.…”