Michael W. Laird

Tethering of CpxP to the inner membrane prevents spheroplast induction of the Cpx envelope stress response

The Cpx envelope stress response of Escherichia coli is controlled by a two‐component regulatory system that senses misfolded proteins in extracytoplasmic compartments and responds by inducing the expression of envelope protein folding and degrading factors. We have proposed that in the absence of envelope stress the pathway is maintained in a downregulated state, in part through interactions between the periplasmic inhibitor molecule CpxP and the sensing domain of the histidine kinase CpxA. In this study, we show that depletion of the periplasmic contents of the cell by spheroplast formation does indeed lead to induction of the Cpx envelope stress response. Further, removal of CpxP is an important component of this induction because tethering an MBP–CpxP fusion protein to the spheroplast inner membranes prevents full activation by this treatment. Spheroplast formation has previously been demonstrated to induce the expression of a periplasmic protein of unknown function, Spy. Analysis of spy expression in response to spheroplast formation by Western blot analysis and by lacZ operon fusion in various cpx mutant backgrounds demonstrated that spy is a member of the Cpx regulon. Interestingly, although the only known spy homologue is cpxP, Spy does not appear to perform the same function as CpxP as it is not involved in inhibiting the Cpx envelope stress response. Rather, deletion of spy leads to activation of the σE stress response. Because the σE response is specifically affected by alterations in outer membrane protein biogenesis, we think it possible that Spy may be involved in this process.

Identification and prevention of antibody disulfide bond reduction during cell culture manufacturing

In the biopharmaceutical industry, therapeutic monoclonal antibodies are primarily produced in mammalian cell culture systems. During the scale‐up of a monoclonal antibody production process, we observed excessive mechanical cell shear as well as significant reduction of the antibodys interchain disulfide bonds during harvest operations. This antibody reduction event was catastrophic as the product failed to meet the drug substance specifications and the bulk product was lost. Subsequent laboratory studies have demonstrated that cells subjected to mechanical shear release cellular enzymes that contribute to this antibody reduction phenomenon (manuscript submitted; Kao et al., 2009). Several methods to prevent this antibody reduction event were developed using a lab‐scale model to reproduce the lysis and reduction events. These methods included modifications to the cell culture media with chemicals (e.g., cupric sulfate (CuSO4)), pre‐ and post‐harvest chemical additions to the cell culture fluid (CCF) (e.g., CuSO4, EDTA, L‐cystine), as well as lowering the pH and air sparging of the harvested CCF (HCCF). These methods were evaluated for their effectiveness in preventing disulfide bond reduction and their impact to product quality. Effective prevention methods, which yielded acceptable product quality were evaluated for their potential to be implemented at manufacturing‐scale. The work described here identifies numerous effective reduction prevention measures from lab‐scale studies; several of these methods were then successfully translated into manufacturing processes. Biotechnol. Bioeng. 2010; 106: 452–461.

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.

Characterization of a Monoclonal Antibody Cell Culture Production Process Using a Quality by Design Approach

The goal of quality by design (QbD) in cell culture manufacturing is to develop manufacturing processes which deliver products with consistent critical quality attributes (CQAs). QbD approaches can lead to better process understanding through the use of process parameter risk ranking and statistical design of experiments (DOE). The QbD process starts with an analysis of process parameter risk with respect to CQAs and key performance indicators (KPIs). Initial DOE study designs and their factor test ranges are based on the outcomes of the process parameter risk ranking exercises. Initial DOE studies screen factors for significant influences on CQAs as well as characterize responses for process KPIs. In the case study provided here, multifactor process characterization studies using a scale-down model resulted in significant variation in charge heterogeneity of a monoclonal antibody (MAb) as measured by ion-exchange chromatography (IEC). Iterative DOE studies, using both screening and response surface designs, were used to narrow the operating parameter ranges so that charge heterogeneity could be controlled to an acceptable level. The data from the DOE studies were used to predict worst-case conditions, which were then verified by testing at those conditions. Using the approach described here, multivariate process parameter ranges were identified that yield acceptable CQA levels and that still provide operational flexibility for manufacturing.

Mechanism of antibody reduction in cell culture production processes

We recently observed a significant disulfide reduction problem during the scale‐up of a manufacturing process for a therapeutic antibody using a CHO expression system. Under certain conditions, extensive reduction of inter‐chain disulfide bonds of an antibody produced by CHO cell culture may occur during the harvest operations and/or the protein A chromatography step, resulting in the observation of antibody fragments (light chain, heavy chain, and various combination of both) in the protein A pools. Although all conditions leading to disulfide reduction have not been completely identified, an excessive amount of mechanical cell lysis generated at the harvest step appears to be an important requirement for antibody reduction (Trexler‐Schmidt et al., 2010 ). We have been able to determine the mechanism by which the antibody is reduced despite the fact that not all requirements for antibody reduction were identified. Here we present data strongly suggesting that the antibody reduction was caused by a thioredoxin system or other reducing enzymes with thioredoxin‐like activity. The intracellular reducing enzymes and their substrates/cofactors apparently were released into the harvest cell culture fluid (HCCF) when cells were exposed to mechanical cell shear during harvest operations. Surprisingly, the reducing activity in the HCCF can last for a long period of time, causing the reduction of inter‐chain disulfide bonds in an antibody. Our findings provide a basis for designing methods to prevent the antibody reduction during the manufacturing process. Biotechnol. Bioeng. 2010;107:622–632.

Effect of Individual Fc Methionine Oxidation on FcRn Binding: Met252 Oxidation Impairs FcRn Binding More Profoundly than Met428 Oxidation

The long serum half-lives of mAbs are conferred by pH-dependent binding of IgG-Fc to the neonatal Fc receptor (FcRn). The Fc region of human IgG1 has three conserved methionine residues, Met252, Met358, and Met428. Recent studies showed oxidation of these Met residues impairs FcRn binding and consequently affects pharmacokinetics of therapeutic antibodies. However, the quantitative effect of individual Met oxidation on Fc-FcRn binding has not been addressed. This information is valuable for defining critical quality attributes. In the present study, two sets of homodimeric site-directed IgG1 mutations were generated to understand how individual Fc Met oxidation affects FcRn binding. The first approach used Met to Leu mutants to block site-specific Met oxidation. In the other approach, Met to Gln mutants were designed to mimic site-specific Met oxidation. Both mutagenesis approaches show that either Met252 or Met428 oxidation alone significantly impairs Fc-FcRn binding. Met252 oxidation has a more deleterious effect on FcRn binding than M428 oxidation, whereas Met428 oxidation has a bigger destabilization effect on the thermal stability. Our results also show that Met358 oxidation does not affect FcRn binding. In addition, our study suggests that Met to Gln mutation may serve as an important tool to understand Met oxidation.

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.

Thioredoxin 1 is responsible for antibody disulfide reduction in CHO cell culture.

During large-scale manufacturing of an IgG1 monoclonal antibody in Chinese hamster ovary (CHO) cells, reduction of the antibodys disulfide bonds was observed. We present evidence that mammalian thioredoxin 1 (TXN1) is the terminal enzyme responsible for this reduction event. We demonstrate a marked prevention of IgG1 disulfide bond reduction in a cell-density dependent manner by knocking down expression of TXN1 via lentivirus transduction of short hairpin RNA.

High-yield expression of human vascular endothelial growth factor VEGF165 in Escherichia coli and purification for therapeutic applications

Vascular endothelial growth factor (VEGF(165)) is a potent mitogen that induces angiogenesis and vascular permeability in vivo and has demonstrated potential in therapeutic applications for accelerating wound healing. An industrial production method that provides high yield as well as high purity, quality, and potency is needed. The process described in this report involves a bacterial expression system capable of producing approximately 9g of rhVEGF per liter of broth and a downstream purification process consisting of protein refolding and three chromatography steps prior to formulation of the drug substance. A high cell density (HCD) fed-batch fermentation process was used to produce rhVEGF in periplasmic inclusion bodies. The inclusion bodies are harvested from the cell lysate and subjected to a single-step protein solubilization and refolding operation to extract the rhVEGF for purification. Overall recovery yields observed during development, including refolding and chromatography, were 30+/-6%. Host cell impurities are consistently cleared below target levels at both laboratory and large-scale demonstrating process robustness. The structure of the refolded and purified rhVEGF was confirmed by mass spectrometry, N-terminal sequencing, and tryptic peptide mapping while product variants were analyzed by multiple HPLC assays. Biological activity was verified by the proliferation of human umbilical vein derived endothelial cells.

Air sparging for prevention of antibody disulfide bond reduction in harvested CHO cell culture fluid.

During the scale‐up of several Chinese Hamster Ovary (CHO) cell monoclonal antibody production processes, significant reduction of the antibody interchain disulfide bonds was observed. The reduction was correlated with excessive mechanical cell shear during the harvest operations. These antibody reduction events resulted in failed product specifications and the subsequent loss of the drug substance batches. Several methods were recently developed to prevent antibody reduction, including modifying the cell culture media, using pre‐ and post‐harvest chemical additions to the cell culture fluid (CCF), lowering the pH, and air sparging of the harvested CCF (HCCF). The work described in this paper further explores the option of HCCF air sparging for preventing antibody reduction. Here, a small‐scale model was developed using a 3‐L bioreactor to mimic the conditions of a manufacturing‐scale harvest vessel and was subsequently employed to evaluate several air sparging strategies. In addition, these studies enabled further understanding of the relationships between cell lysis levels, oxygen consumption, and antibody reduction. Finally, the effectiveness of air sparging for several CHO cell lines and the potential impact on product quality were assessed to demonstrate that air sparging is an effective method in preventing antibody reduction. Biotechnol. Bioeng. 2015;112: 734–742.