X. Christopher Yu

Mechanisms of unintended amino acid sequence changes in recombinant monoclonal antibodies expressed in Chinese Hamster Ovary (CHO) cells.

An amino acid sequence variant is defined as an unintended amino acid sequence change and contributes to product heterogeneity. Recombinant monoclonal antibodies (MAbs) are primarily expressed from Chinese Hamster Ovary (CHO) cells using stably transfected production cell lines. Selections and amplifications with reagents such as methotrexate (MTX) are often required to achieve high producing stable cell lines. Since MTX is often used to generate high producing cell lines, we investigated the genomic mutation rates of the hypoxanthine–guanine phosphoribosyltransferase (HGPRT or HPRT) gene using a 6‐thioguanine (6‐TG) assay under various concentrations of MTX selection in CHO cells. Our results show that the 6‐TG resistance increased as the MTX concentration increased during stable cell line development. We also investigated low levels of sequence variants observed in two stable cell lines expressing different MAbs. Our data show that the replacement of serine at position 167 by arginine (S167R) in the light chain of antibody A (MAb‐A) was due to a genomic nucleotide sequence change whereas the replacement of serine at position 63 by asparagine (S63N) in the heavy chain of antibody B (MAb‐B) was likely due to translational misincorporation. This mistranslation is codon specific since S63N mistranslation is not detectable when the S63 AGC codon is changed to a TCC or TCT codon. Our results demonstrate that both a genomic nucleotide change and translational misincorporation can lead to low levels of sequence variants and mistranslation of serine to asparagine can be eliminated by substituting the TCC or TCT codon for the S63 AGC codon without impacting antibody productivity. Biotechnol. Bioeng. 2010;107: 163–171.

Identification of codon-specific serine to asparagine mistranslation in recombinant monoclonal antibodies by high-resolution mass spectrometry.

Translation errors in protein biosynthesis may result in low level amino acid misincorporation and contribute to product heterogeneity of recombinant protein therapeutics. We report the use of peptide map analysis by reversed-phase high-performance liquid chromatography and high-resolution mass spectrometry to detect and identify mistranslation events in recombinant monoclonal antibodies expressed in mammalian cell lines including Chinese hamster ovary (CHO) cells. Misincorporation of an asparagine residue at multiple serine positions was detected as earlier-eluting peptides with masses 27.01 Da higher than expected. The exact positions at which misincorporation occurred were identified by tandem mass spectrometry of the asparagine-containing variant peptides. The identified asparagine misincorporation sites correlated with the use of codon AGC but with none of the other five serine codons. The relative levels of misincorporation ranged from 0.01%-0.2% among multiple serine positions detected across three different antibodies by targeted analysis of expected and variant peptides. The low levels of misincorporation are consistent with published predictions for in vivo translation error rates. Our results demonstrate that state-of-the-art mass spectrometry with a combination of high sensitivity, accuracy, and dynamic range provides a new ability to discover and characterize low level protein variants that arise from mistranslation events.

Accurate Determination of Succinimide Degradation Products Using High Fidelity Trypsin Digestion Peptide Map Analysis

We report an efficient, high fidelity trypsin digestion method for peptide map analysis. This method minimizes artifacts caused by the sample preparation process, and we show its utility for the accurate determination of succinimide formation in a degraded monoclonal antibody product. A basic charge variant was detected by imaged capillary isoelectric focusing and was shown with reduced antigen binding and biological activity. Samples were reduced under denaturing conditions at pH 5.0, and digestion of the reduced protein with porcine trypsin was performed at pH 7.0 for 1 h. Following reversed phase high-performance liquid chromatography and online mass spectrometric analysis, succinimide formation was identified at Asp30 in the light chain. This result contrasts with the observation of only iso-Asp and Asp residues under conventional sample preparation conditions, which are therefore concluded to be artificially generated. The Asp30 residue is seen in the cocrystal structure model to participate in favorable charge interaction with an antigen molecule. Formation of succinimide and the resulting loss of negative charge are therefore hypothesized to be the degradation mechanism. After treatment of the degraded antibody sample to mildly alkaline pH conditions, we observed only Asp residue as the succinimide hydrolysis product and concurrent recovery of biological activity.

Eliminating tyrosine sequence variants in CHO cell lines producing recombinant monoclonal antibodies

Amino acid sequence variants are defined as unintended amino acid sequence changes that contribute to product variation with potential impact to product safety, immunogenicity, and efficacy. Therefore, it is important to understand the propensity for sequence variant (SV) formation during the production of recombinant proteins for therapeutic use. During the development of clinical therapeutic products, several monoclonal antibodies (mAbs) produced from Chinese Hamster Ovary (CHO) cells exhibited SVs at low levels (≤3%) in multiple locations throughout the mAbs. In these examples, the cell culture process depleted tyrosine, and the tyrosine residues in the recombinant mAbs were replaced with phenylalanine or histidine. In this work, it is demonstrated that tyrosine supplementation eliminated the tyrosine SVs, while early tyrosine starvation significantly increased the SV level in all mAbs tested. Additionally, it was determined that phenylalanine is the amino acid preferentially misincorporated in the absence of tyrosine over histidine, with no other amino acid misincorporated in the absence of tyrosine, phenylalanine, and histidine. The data support that the tyrosine SVs are due to mistranslation and not DNA mutation, most likely due to tRNATyr mischarging due to the structural similarities between tyrosine and phenylalanine. Biotechnol. Bioeng. 2013; 110: 1087–1097.

More similar than different: Host cell protein production using three null CHO cell lines

To understand the diversity in the cell culture harvest (i.e., feedstock) provided for downstream processing, we compared host cell protein (HCP) profiles using three Chinese Hamster Ovary (CHO) cell lines in null runs which did not generate any recombinant product. Despite differences in CHO lineage, upstream process, and culture performance, the cell lines yielded similar cell‐specific productivities for immunogenic HCPs. To compare the dynamics of HCP production, we searched for correlations between the time‐course profiles of HCP (as measured by multi‐analyte ELISA) and those of two intracellular HCP species, phospholipase B‐like 2 (PLBL2) and lactate dehydrogenase (LDH). Across the cell lines, proteins in the day 14 supernatants analyzed by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) showed different spot patterns. However, subsequent analysis by liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) indicated otherwise: the total number of peptides and proteins identified were comparable, and 80% of the top 1,000 proteins identified were common to all three lines. Finally, to assess the impact of culture viability on extracellular HCP profiles, we analyzed supernatants from a cell line whose viability dropped after day 10. The amounts of HCP and PLBL2 (quantified by their respective ELISAs) as well as the numbers and major populations of HCPs (identified by LC‐MS/MS) were similar across days 10, 14, and 17, during which viabilities declined from ∼80% to <20% and extracellular LDH levels increased several‐fold. Our findings indicate that the CHO‐derived HCPs in the feedstock for downstream processing may not be as diverse across cell lines and upstream processes, or change as dramatically upon viability decline as originally expected. In addition, our findings show that high density CHO cultures (>107 cells/mL)—operated in fed‐batch mode and exhibiting high viabilities (>70%) throughout the culture duration—can accumulate a considerable amount of immunogenic HCP (∼1–2 g/L) in the extracellular environment at the time of harvest (day 14). This work also demonstrates the potential of using LC‐MS/MS to overcome the limitations associated with ELISA and 2D‐PAGE for HCP analysis. Biotechnol. Bioeng. 2015;112: 2068–2083.

Absolute Quantitation of Intact Recombinant Antibody Product Variants Using Mass Spectrometry

Accurate and precise quantitative measurement of product-related variants of a therapeutic antibody is essential for product development and testing. Bispecific antibodies (bsAbs) are Abs composed of two different half antibody arms, each of which recognizes a distinct target, and recently they have attracted substantial therapeutic interest. Because of the increased complexity of its structure and its production process, as compared to a conventional monoclonal antibody, additional product-related variants, including covalent and noncovalent homodimers of half antibodies (hAbs), may be present in the bsAb product. Sufficient separation and reliable quantitation of these bsAb homodimers using liquid chromatography (LC) or capillary electrophoresis-based methods is challenging because these homodimer species and the bsAb often have similar physicochemical properties. Formation of noncovalent homodimers and heterodimers can also occur. In addition, since homodimers share common sequences with their corresponding halves and bsAb, it is not suitable to use peptides as surrogates for their quantitation. To tackle these analytical challenges, we developed a mass spectrometry-based quantitation method. Chip-based nanoflow LC-time-of-flight mass spectrometry coupled with a standard addition approach provided unbiased absolute quantitation of these drug-product-related variants. Two methods for the addition of known levels of standard (multi- or single-addition) were evaluated. Both methods demonstrated accurate and reproducible quantitation of homodimers at the 0.2% (w/w) level, with the single-addition method having the promise of higher analytical throughput.

Strain engineering to prevent norleucine incorporation during recombinant protein production in Escherichia coli

Incorporation of norleucine in place of methionine residues during recombinant protein production in Escherichia coli is well known. Continuous feeding of methionine is commonly used in E. coli recombinant protein production processes to prevent norleucine incorporation. Although this strategy is effective in preventing norleucine incorporation, there are several disadvantages associated with continuous feeding. Continuous feeding increases the operational complexity and the overall cost of the fermentation process. In addition, the continuous feed leads to undesirable dilution of the fermentation medium possibly resulting in lower cell densities and recombinant protein yields. In this work, the genomes of three E. coli hosts were engineered by introducing chromosomal mutations that result in methionine overproduction in the cell. The recombinant protein purified from the fermentations using the methionine overproducing hosts had no norleucine incorporation. Furthermore, these studies demonstrated that the fermentations using one of the methionine overproducing hosts exhibited comparable fermentation performance as the control host in three different recombinant protein production processes.

A modular and adaptive mass spectrometry-based platform for support of bioprocess development toward optimal host cell protein clearance

ABSTRACT A modular and adaptive mass spectrometry (MS)-based platform was developed to provide fast, robust and sensitive host cell protein (HCP) analytics to support process development. This platform relies on one-dimensional ultra-high performance liquid chromatography (1D UHPLC) combined with several different MS data acquisition strategies to meet the needs of purification process development. The workflow was designed to allow HCP composition and quantitation for up to 20 samples per day, a throughput considered essential for real time bioprocess development support. With data-dependent acquisition (DDA), the 1D UHPLC-MS/MS method had excellent speed and demonstrated robustness in detecting unknown HCPs at ≥ 50 ng/mg (ppm) level. Combining 1D UHPLC with sequential window acquisition of all theoretical spectra (SWATH) MS enabled simultaneous detection and quantitation of all HCPs in single-digit ng/mg range within 1 hour, demonstrating for the first time the benefit of SWATH MS as a technique for HCP analysis. As another alternative, a targeted MS approach can be used to track the clearance of specific known HCP under various process conditions. This study highlights the importance of designing a robust LC-MS/MS workflow that not only allows HCP discovery, but also affords greatly improved process knowledge and capability in HCP removal. As an orthogonal and complementary detection approach to traditional HCP analysis by enzyme-linked immunosorbent assay, the reported LC-MS/MS workflow supports the development of bioprocesses with optimal HCP clearance and the production of safe and high quality therapeutic biopharmaceuticals.

A 2D LC-MS/MS Strategy for Reliable Detection of 10-ppm Level Residual Host Cell Proteins in Therapeutic Antibodies

Methodologies employing LC-MS/MS have been increasingly used for characterization and identification of residual host cell proteins (HCPs) in biopharmaceutical products to ensure their consistent product quality and safety for patients. To improve the sensitivity and reliability for HCP detection, we developed a high pH-low pH two-dimensional reversed phase LC-MS/MS approach in conjunction with offline fraction concatenation. Proof-of -concept was established using a model in which seven proteins spanning a size range of 29-78 kDa are spiked into a purified antibody product to simulate the presence of low-level HCPs. By incorporating a tandem column configuration and a shallow gradient through the second-dimension, all seven proteins were consistently identified at 10 ppm with 100% success rate following LC-MS/MS analysis of six concatenated fractions across multiple analysts, column lots and injection loads. Using the more complex Universal Proteomic Standard 1 (UPS-1) as an HCP model, positive identification was consistently achieved for 19 of the 22 proteins in 8-12 ppm range (10 ppm ±20%). For the first time, we demonstrate an effective LC-MS/MS strategy that not only has high sensitivity but also high reliability for HCP detection. The method performance has high impact on pharmaceutical company practices in using advanced LC-MS/MS technology to ensure product quality and patient safety.