ABSTRACT
The programmed synthesis of sequence-defined biomaterials whose monomer backbones diverge from those of canonical α-amino acids represents the next frontier in protein and biomaterial evolution. Such next-generation molecules provide otherwise nonexistent opportunities to develop improved biologic therapies, bioremediation tools, and biodegradable plastic-like materials. One monomer family of particular interest for biomaterials includes ß-hydroxy acids. Many natural products contain isolated ß-hydroxy acid monomers, and polymers of ß-hydroxy acids (ß-esters) are found in polyhydroxyalkanoate (PHA) polyesters under development as bioplastics and drug encapsulation/delivery systems. Here we report that ß2-hydroxy acids possessing both (R) and (S) absolute configuration are substrates for pyrrolysyl-tRNA synthetase (PylRS) enzymes in vitro and that (S)-ß2-hydroxy acids are substrates in cellulo. Using the orthogonal MaPylRS/MatRNAPyl synthetase/tRNA pair, in conjunction with wild-type E. coli ribosomes and EF-Tu, we report the cellular synthesis of model proteins containing two (S)-ß2-hydroxy acid residues at internal positions. Metadynamics simulations provide a rationale for the observed preference for the (S)-ß2-hydroxy acid and provide mechanistic insights that inform future engineering efforts. As far as we know, this finding represents the first example of an orthogonal synthetase that acylates tRNA with a ß2-hydroxy acid substrate and the first example of a protein hetero-oligomer containing multiple expanded-backbone monomers produced in cellulo.
ABSTRACT
BACKGROUND AND OBJECTIVE: ABP 654 is a proposed biosimilar to ustekinumab reference product (RP), a human immunoglobulin isotype class G subclass 1 kappa monoclonal antibody that acts as an antagonist of interleukin (IL)-23 and IL-12. Ustekinumab RP is indicated for the treatment of some forms of plaque psoriasis, active psoriatic arthritis, Crohn's disease, and ulcerative colitis. ABP 654 and ustekinumab RP utilize different expression systems, and the purpose of this study was to assess analytical similarity between ABP 654 and ustekinumab RP sourced from the United States (US) and the European Union (EU). METHODS: The analytical testing plan included general properties, primary structure, higher-order structure, product-related substances and impurities, particles and aggregates, biological activity, and thermal stability and degradation studies. RESULTS: ABP 654 was found to be analytically similar to ustekinumab RP with respect to physicochemical and biological properties, including structure, function, purity, and potency. CONCLUSIONS: Based on a comprehensive similarity assessment, ABP 654 was found to be similar to ustekinumab RP, notwithstanding minor physicochemical differences that are not expected to have a clinically meaningful effect on safety or efficacy.
Subject(s)
Arthritis, Psoriatic , Biosimilar Pharmaceuticals , Humans , United States , Ustekinumab/pharmacology , Ustekinumab/therapeutic use , Biosimilar Pharmaceuticals/pharmacology , Biosimilar Pharmaceuticals/chemistryABSTRACT
LC-MS based multi-attribute methods (MAM) have drawn substantial attention due to their capability of simultaneously monitoring a large number of quality attributes of a biopharmaceutical product. For successful implementation of MAM, it is usually considered a requirement that the method is capable of detecting any new or missing peaks in the sample when compared to a control. Comparing a sample to a control for rare differences is also commonly practiced in many fields for investigational purpose. Because MS signal variability differs greatly between signals of different intensities, this type of comparison is often challenging, especially when the comparison is made without enough replicates. In this report we describe a statistical method for detecting rare differences between two very similar samples without replicate analyses. The method assumes that an overwhelming majority of components have equivalent abundance between the two samples, and signals with similar intensities have similar relative variability. By analyzing several monoclonal antibody peptide mapping datasets, we demonstrated that the method is suitable for new-peak detection for MAM as well as for other applications when rare differences between two samples need to be detected. The method greatly reduced false positive rate without a significant increase of false negative rate.
Subject(s)
Tandem Mass Spectrometry , Chromatography, Liquid/methods , Peptide Mapping/methodsABSTRACT
The absence of orthogonal aminoacyl-transfer RNA (tRNA) synthetases that accept non-L-α-amino acids is a primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers and biomaterials. Here we report that pyrrolysyl-tRNA synthetase (PylRS) and certain PylRS variants accept α-hydroxy, α-thio and N-formyl-L-α-amino acids, as well as α-carboxy acid monomers that are precursors to polyketide natural products. These monomers are accommodated and accepted by the translation apparatus in vitro; those with reactive nucleophiles are incorporated into proteins in vivo. High-resolution structural analysis of the complex formed between one PylRS enzyme and a m-substituted 2-benzylmalonic acid derivative revealed an active site that discriminates prochiral carboxylates and accommodates the large size and distinct electrostatics of an α-carboxy substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose α-substituent diverges substantially from the α-amine of proteinogenic amino acids. These enzymes or derivatives thereof could synergize with natural or evolved ribosomes and/or translation factors to generate diverse sequence-defined non-protein heteropolymers.
Subject(s)
Amino Acyl-tRNA Synthetases , Amino Acyl-tRNA Synthetases/genetics , Lysine/chemistry , Amino Acids , RNA, Transfer/geneticsABSTRACT
Therapeutic IgG mAbs have shown presence of three variations of their heavy chain C-termini, including the unprocessed C-terminal lysine, the processed C-terminal lysine, and C-terminal amidation. These variants are also present in endogenous human IgGs, although the level of unprocessed C-terminal lysine is very low. Here we report a new heavy-chain C-terminal variant, i.e., the des-GK truncation, which exists in both recombinant and endogenous human IgG4. The des-GK truncation was found in negligible amount in IgG1, IgG2 and IgG3 subclasses. Observation of a significant level of heavy-chain C-terminal des-GK truncation in endogenous human IgG4 suggests that low level of this variant present in therapeutic IgG4 is unlikely to be a safety concern.
Subject(s)
Immunoglobulin G , Lysine , Humans , Immunoglobulin G/genetics , Antibodies, MonoclonalABSTRACT
One of the outcomes from the global COVID-19 pandemic caused by SARS-CoV-2 has been an acceleration of development timelines to provide treatments in a timely manner. For example, it has recently been demonstrated that the development of monoclonal antibody therapeutics from vector construction to IND submission can be achieved in five to six months rather than the traditional ten-to-twelve-month timeline using CHO cells [1], [2]. This timeline is predicated on leveraging existing, robust platforms for upstream and downstream processes, analytical methods, and formulation. These platforms also reduce; the requirement for ancillary studies such as cell line stability, or long-term product stability studies. Timeline duration was further reduced by employing a transient cell line for early material supply and using a stable cell pool to manufacture toxicology study materials. The development of non-antibody biologics utilizing traditional biomanufacturing processes in CHO cells within a similar timeline presents additional challenges, such as the lack of platform processes and additional analytical assay development. In this manuscript, we describe the rapid development of a robust and reproducible process for a two-component self-assembling protein nanoparticle vaccine for SARS-CoV-2. Our work has demonstrated a successful academia-industry partnership model that responded to the COVID-19 global pandemic quickly and efficiently and could improve our preparedness for future pandemic threats.
ABSTRACT
Since 2015 more than 34 biosimilars have been approved by the FDA. This new era of biosimilar competition has stimulated renewed technology development focused on therapeutic protein or biologic manufacturing. One challenge in biosimilar development is the genetic differences in the host cell lines used to manufacture the biologics. For example, many biologics approved between 1994 and 2011 were expressed in murine NS0 and SP2/0 cell lines. Chinese Hamster ovary (CHO) cells, however, have since become the preferred hosts for production due to their increased productivity, ease of use, and stability. Differences between murine and hamster glycosylation have been identified in biologics produced using murine and CHO cells. In the case of monoclonal antibodies (mAbs), glycan structure can significantly affect critical antibody effector function, binding activity, stability, efficacy, and in vivo half-life. In an attempt to leverage the intrinsic advantages of the CHO expression system and match the reference biologic murine glycosylation, we engineered a CHO cell expressing an antibody that was originally produced in a murine cell line to produce murine-like glycans. Specifically, we overexpressed cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) and N-acetyllactosaminide alpha-1,3-galactosyltransferase (GGTA) to obtain glycans with N-glycolylneuraminic acid (Neu5Gc) and galactose-α-1,3-galactose (alpha gal). The resulting CHO cells were shown to produce mAbs with murine glycans, and they were then analyzed by the spectrum of analytical methods typically used to demonstrate analytical similarity as a part of demonstrating biosimilarity. This included high-resolution mass spectrometry, biochemical, as well as cell-based assays. Through selection and optimization in fed-batch cultures, two CHO cell clones were identified with similar growth and productivity criteria to the original cell line. They maintained stable production for 65 population doubling times while matching the glycosylation profile and function of the reference product expressed in murine cells. This study demonstrates the feasibility of engineering CHO cells to express mAbs with murine glycans to facilitate the development of biosimilars that are highly similar to marketed reference products expressed in murine cells. Furthermore, this technology can potentially reduce the residual uncertainty regarding biosimilarity, resulting in a higher probability of regulatory approval and potentially reduced costs and time in development.
ABSTRACT
Therapeutic IgG mAbs expressed from Chinese hamster ovary (CHO) cells are known to contain three C-terminal variants in their heavy chains, namely, the unprocessed C-terminal lysine, the processed C-terminal lysine, and C-terminal amidation. Although the presence of C-terminal amidation in CHO-expressed IgGs is well studied, the biological impact of the variant on the safety and efficacy of biotherapeutics has not been well understood. To further our biological understanding of C-terminal amidation, we analyzed a series of IgG samples, including both endogenous human IgGs as well as recombinant IgGs of different subclasses expressed from both CHO and murine cell lines, for their heavy-chain C-terminal variants by LC-MS/MS based peptide mapping. The results demonstrate that heavy-chain C-terminal amidation is a common variant occurring in IgG of all four subclasses (IgG1, IgG2, IgG3 and IgG4). The variant is generally present in recombinant IgG mAbs expressed from CHO cell lines but not in IgG mAbs expressed from murine cell lines, whereas the IgGs expressed from murine cell lines contain a much larger amount of unprocessed C-terminal lysine. Additionally, a significant amount of heavy-chain C-terminal amidation is observed in endogenous human IgGs, indicating that small amount of the variant present in therapeutic IgGs does not pose a safety concern.
Subject(s)
Lysine , Tandem Mass Spectrometry , Animals , Antibodies, Monoclonal , CHO Cells , Chromatography, Liquid/methods , Cricetinae , Cricetulus , Humans , Immunoglobulin G/metabolism , MiceABSTRACT
During the development of a therapeutic protein, its quality attributes that pertain to the primary structure must be appropriately characterized, commonly by LC-MS/MS peptide mapping experiments. Extracting attribute information from LC-MS/MS data requires knowledge of the attribute of interest. Therefore, it is important to understand all potential modifications on the therapeutic proteins. In this work, we performed UV and visible light irradiation experiments on several therapeutic proteins, with or without the presence of a photosensitizer. Light-induced modifications were detected and characterized by tryptic digestion followed by LC-MS/MS analysis. A list of potential light-induced modifications, with their respective mass changes, was obtained. These modifications are primarily on methionine, tryptophan, histidine, cysteine, tyrosine and phenylalanine residues. Many of these modifications have not been previously reported on therapeutic proteins. Our findings therefore provide a database of potential light-induced modifications that would enable the routine characterization of light-induced modifications on therapeutic proteins.
Subject(s)
Methionine , Tandem Mass Spectrometry , Chromatography, Liquid/methods , Histidine , Methionine/chemistry , Peptide Mapping/methodsABSTRACT
A stable molecular structure is important in the development of a protein candidate into a therapeutic product. A therapeutic protein often contains many different variants; some of them may have an impact on the conformational stability of the protein. Conventionally, to evaluate the impact of a variant on stability, the variant must be enriched to a reasonable purity, and then its stability characterized by chromatographic or biophysical techniques. However, it is often impractical to purify and characterize each variant in a therapeutic protein. A workflow, based on limited proteolysis followed by MS detection, was established to simultaneously assess the impact of a large number of variants on conformational stability without enrichment. Because a less stable domain is more susceptible to proteolytic degradation, conformational stability of the domain can be reported from the release rate of a proteolytic peptide. A kinetic model is established to quantitatively determine the extent of domain stabilization/destabilization of different variants. The methodology is demonstrated by examining variants known to affect the stability of immunoglobulin domains, such as different N-glycoforms, methionine oxidations, and sequence variants. With this methodology, near 100 variants may be evaluated within 2 days in a single experiment. Insights into the sequence-stability relationship will be obtained by monitoring the large number of low-level sequence variants, facilitating engineering of more stable molecules.
Subject(s)
Proteolysis , Kinetics , Mass Spectrometry , Molecular Conformation , Protein StabilityABSTRACT
We report that the recently introduced commercial strain of Vibrio natriegens (Vmax X2) supports robust unnatural amino acid mutagenesis, generating exceptional yields of soluble protein containing up to 5 noncanonical α-amino acids (ncAA). The isolated yields of ncAA-containing superfolder green fluorescent protein (sfGFP) expressed in Vmax X2 are up to 25-fold higher than those achieved using commercial expression strains (Top10 and BL21) and more than 10-fold higher than those achieved using two different genomically recodedEscherichia colistrains that lack endogenous UAG stop codons and release factor 1 and have been optimized for improved fitness and preferred growth temperature (C321.ΔA.opt and C321.ΔA.exp). In addition to higher yields of soluble protein, Vmax X2 cells also generate proteins with significantly lower levels of misincorporated natural α-amino acids at the UAG-programmed position, especially in cases where the ncAA is a moderate substrate for the chosen orthogonal aminoacyl tRNA synthetase (aaRS). This increase in fidelity implies that the use of Vmax X2 cells as the expression host can obviate the need for time-consuming directed evolution experiments to improve the selectivity of an aaRS toward highly desired but suboptimal ncAA substrates.
ABSTRACT
Battery-powered drug delivery devices are widely used as primary containers for storing and delivering therapeutic protein products to improve patient compliance and quality of life. Compared to conventional delivery approaches such as pre-filled syringes, battery-powered devices are more complex in design requiring new materials/components for proper functionality, which could cause potential product safety and quality concerns from the extractable and leachables (E&L) of the new materials/components. In this study, E&L assessments were performed on a battery-powered delivery device during the development and qualification of the device, where novel compound 2hydroxy-2-methylpropiophenone (HMPP) and related compounds were observed in both E&L. The source of the HMPP and related compounds was identified to be the nonproduct contact device batteries, in which HMPP photo-initiator was used as a curing agent in the battery sealant to prevent leakage of the battery electrolytes. Toxicology assessment was performed, which showed the levels of HMPP observed in the device lots were acceptable relative to the permitted daily exposure. A drug product HMPP spike study was also performed, where no product impact was observed. Based on these assessments, an action threshold and specification limits could be established as a control strategy, if needed, to mitigate the potential risks associate with the observed leachables. As a full resolution, seven battery candidates from different suppliers were screened and one new battery was successfully qualified for the delivery devices. Overall, the holistic E&L approach was fully successful in the development and qualification of the battery-powered devices for biotherapeutic products delivery ensuring product quality and patient safety. Non-product contact materials are commonly rated as low or no risk and typically considered as out of scope of E&L activities for delivery systems following industry benchmark and regulatory agency guidance. This case study is novel as it brings into attention the materials that might not normally be in consideration during the development process. It is highly recommended to understand materials in the context of intended use on a case-by-case basis and not to generalize to ensure successful development and qualification.
Subject(s)
Pharmaceutical Preparations , Quality of Life , Biotechnology , Drug Contamination , Drug Packaging , HumansABSTRACT
Multi-attribute methods (MAM), based on proteolytic digestion followed by liquid chromatography-mass spectrometry analysis of proteolytic peptides, have gained substantial attention in the biopharmaceutical industry for quantifying a variety of quality attributes for therapeutic proteins. Most MAM developed so far have been based on high-resolution mass spectrometers, due to their superb resolving power to distinguish analyte signals from interferences. Lower-resolution instruments, if demonstrated suitable, may further promote the adoption of the technology due to their low cost, small footprint, and ease of use. In this work, we compared the performance of a high-resolution instrument with a few low-resolution quadrupole-type instruments in quantifying a diverse set of quality attributes in a monoclonal antibody product. Different modes of operation for the quadrupole instruments, including scan mode, selected-ion monitoring and multiple-reaction monitoring, were evaluated. The high-resolution instrument has superb performance, with a quantitation limit of 0.002%. Single-quadrupole instruments in scan mode, on the other hand, provide a quantitation limit of about 1%, which may be fit-for-purpose for many routine MAM applications.
Subject(s)
Antibodies, Monoclonal/analysis , Mass Spectrometry , Peptides/analysis , Chromatography, LiquidABSTRACT
Human IgG antibodies containing terminal alpha 2,6-linked sialic acid on their Fc N-glycans have been shown to reduce antibody-dependent cell-mediated cytotoxicity and possess anti-inflammatory properties. Although terminal sialylation on complex N-glycans can happen via either an alpha 2,3-linkage or an alpha 2,6-linkage, sialic acids on human serum IgG Fc are almost exclusively alpha 2,6-linked. Recombinant IgGs expressed in Chinese hamster ovary (CHO) cells, however, have sialic acids through alpha 2,3-linkages because of the lack of the alpha 2,6-sialyltransferase gene. The impact of different sialylation linkages to the structure of IgG has not been determined. In this work, we investigated the impact of different types of sialylation to the conformational stability of IgG through hydrogen/deuterium exchange (HDX) and limited proteolysis experiments. When human-derived and CHO-expressed IgG1 were analyzed by HDX, sialic acid-containing glycans were found to destabilize the CH2 domain in CHO-expressed IgG, but not human-derived IgG. When structural isomers of sialylated glycans were chromatographically resolved and identified in the limited proteolysis experiment, we found that only alpha 2,3-linked sialic acid on the 6-arm (the major sialylated glycans in CHO-expressed IgG1) destabilizes the CH2 domain, presumably because of the steric effect that decreases the glycan-CH2 domain interaction. The alpha 2,6-linked sialic acid on the 3-arm (the major sialylated glycan in human-derived IgG), and the alpha 2,3-linked sialic acid on the 3-arm, do not have this destabilizing effect.
Subject(s)
Immunoglobulin Fc Fragments/chemistry , Immunoglobulin G/chemistry , Polysaccharides/chemistry , Animals , CHO Cells , Cricetulus , Glycosylation , Humans , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/metabolism , Polysaccharides/metabolism , Protein DomainsABSTRACT
A major challenge of a mass-spectrometry-based quantitative multiattribute method (MAM) for biotherapeutics is its high variability between instruments. For reproducible attribute measurements, not only is a similar instrument model required, but the instruments must also be tuned to the same condition. This poses great long-term challenges, considering the rapid development of new instrumentations. In addition, differences in digestion efficiency, peptide recovery, and artificial modifications during sample preparation also contribute to variability between laboratories. To overcome these challenges, new mathematical methods are developed to calculate the attribute abundance in the sample, using the reference standard (RS) material as calibrant. Most quality attributes in the RS remain constant throughout the life of the standard, and therefore, the RS can serve as a calibrant to correct for the difference between instruments or sample preparation procedures. Because RS data are usually collected in a MAM assay, no additional work is required from the analyst. Data from a large number of attributes demonstrated that these methodologies greatly reduced instrument-to-instrument and sample preparation variabilities. With these methodologies, a consistent instrument model and sample preparation procedure is no longer a requirement. As a result, changes in digestion procedure and advances in instrumentations will not significantly affect the assay result.
Subject(s)
Biological Therapy , Chromatography, Liquid , Mass Spectrometry , Biological Therapy/standards , Calibration , Chromatography, Liquid/standards , Mass Spectrometry/standards , Peptides , Reference Standards , Time FactorsABSTRACT
High mannose (HM) glycan levels on secreted monoclonal antibodies can be influenced by external factors, including osmolality and copper deficiency, and by intrinsic factors determined by different cell lines. In order to identify the metabolic markers associated with HM glycan levels, metabolomics analysis was performed to assess the changes in the extracellular metabolites of recombinant cell lines at different time points during fed-batch production process. Ornithine was identified as the common metabolic marker influenced by both external and intrinsic factors when eight different medium conditions and eight different cell lines exhibiting different levels of HM were compared. A strong correlation was also observed between HM and mRNA expression levels of arginase 1, an enzyme that catalyzes the conversion of arginine to ornithine. The results from functional validation study showed that the supplementation of ornithine to the culture medium leads to an increased level of HM, while reduced concentration of spermine, a downstream product of ornithine metabolism, leads to a decreased level of HM. Additional metabolic markers correlating with HM glycan levels were identified from eight-cell line comparison analysis. A common feature shared by these identified markers is their previously described roles as contributors of cellular redox regulation.
Subject(s)
Antibodies, Monoclonal/metabolism , Mannose/metabolism , Polysaccharides/metabolism , Animals , Arginase/genetics , CHO Cells , Copper/metabolism , Cricetinae , Cricetulus , Culture Media , Metabolomics , Ornithine/metabolism , RNA, Messenger/metabolism , Recombinant Proteins/metabolism , Spermine/metabolismABSTRACT
Soy hydrolysates are widely used as a nutrient supplement in mammalian cell culture for the production of recombinant proteins. The batch-to-batch variability of a soy hydrolysate often leads to productivity differences. This report describes our metabolomics platform, which includes a battery of LC-MS/MS modes of operation, and advanced data analysis software for automated data processing. The platform was successfully used for screening productivity markers in soy hydrolysates during the production of two therapeutic antibodies in two Chinese hamster ovary cell lines. A total of 123 soy hydrolysate batches were analyzed, from which 62 batches were used in the production runs of cell line #1 and 12 batches were used in the production runs of cell line #2. For cell line #1, out of 19 amino acids, 106 other metabolites and 4,131 peptides identified in the soy hydrolysate batches being used, several nucleosides and short hydrophobic peptides showed negative correlation with antibody titer, while ornithine, citrulline and several amino acids and organic acids correlated positively with titer. For cell line #2, only ornithine and citrulline showed strong positive correlation. When ornithine was spiked into the culture media, both cell lines demonstrated accelerated cell growth, indicating ornithine as a root cause of the performance difference. It is proposed that better soy hydrolysate performance resulted from better bacterial fermentation during the hydrolysate production. A few selected markers were used to predict the performance of other soy hydrolysate batches for cell line #1. The predicted titers agreed with the experimental values with good accuracy.
Subject(s)
Biomarkers/analysis , Bioreactors , Metabolome/physiology , Metabolomics/methods , Protein Hydrolysates/analysis , Soybean Proteins/analysis , Animals , Biomarkers/metabolism , CHO Cells , Cell Survival , Cricetinae , Cricetulus , Mass Spectrometry , Principal Component Analysis , Protein Hydrolysates/metabolism , Recombinant Proteins , Soybean Proteins/metabolismABSTRACT
Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.
Subject(s)
Antibodies, Monoclonal/chemistry , Chromatography, Liquid/methods , Glycopeptides/chemistry , Polysaccharides/analysis , Tandem Mass Spectrometry/methods , Animals , CHO Cells , Cricetinae , Cricetulus , Peptide Fragments/chemistry , Peptide Mapping/methods , Polysaccharides/chemistry , Reproducibility of ResultsABSTRACT
A mass spectrometry-based method was developed to measure amino acid substitutions directly in proteins down to a level of 0.001%. When applied to recombinant proteins expressed in Escherichia coli, monoclonal antibodies expressed in mammalian cells, and human serum albumin purified from three human subjects, the method revealed a large number of amino acid misincorporations at levels of 0.001-0.1%. The detected misincorporations were not random but involved a single-base difference between the codons of the corresponding amino acids. The most frequent base differences included a change from G to A, corresponding to a G(mRNA)/U(tRNA) base pair mismatch during translation. We concluded that under balanced nutrients, G(mRNA)/U(tRNA) mismatches at any of the three codon positions and certain additional wobble position mismatches (C/U and/or U/U) are the main causes of amino acid misincorporations. The hypothesis was tested experimentally by monitoring the levels of misincorporation at several amino acid sites encoded by different codons, when a protein with the same amino acid sequence was expressed in E. coli using 13 different DNA sequences. The observed levels of misincorporation were different for different codons and agreed with the predicted levels. Other less frequent misincorporations may occur due to G(DNA)/U(mRNA) mismatch during transcription, mRNA editing, U(mRNA)/G(tRNA) mismatch during translation, and tRNA mischarging.
Subject(s)
Amino Acids/chemistry , RNA, Transfer/chemistry , Anticodon/chemistry , Codon/chemistry , Escherichia coli/geneticsABSTRACT
High levels of translational errors, both truncation and misincorporation in an Fc-fusion protein were observed. Here, we demonstrate the impact of several commercially available codon optimization services, and compare to a targeted strategy. Using the targeted strategy, only codons known to have translational errors are modified. For an Fc-fusion protein expressed in Escherichia coli, the targeted strategy, in combination with appropriate fermentation conditions, virtually eliminated misincorporation (proteins produced with a wrong amino acid sequence), and reduced the level of truncation. The use of full optimization using commercially available strategies reduced the initial errors, but introduced different misincorporations. However, truncation was higher using the targeted strategy than for most of the full optimization strategies. This targeted approach, along with monitoring of translation fidelity and careful attention to fermentation conditions is key to minimizing translational error and ensuring high-quality expression. These findings should be useful for other biopharmaceutical products, as well as any other transgenic constructs where protein quality is important.