Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins.

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify metabolic engineering targets limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant expression. In an expression comparison of 24 difficult to express proteins, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. Guided by a comprehensive transcriptomics comparison between cell lines, especially highlighting differences in secretory pathway utilization, a co-expression screening of 21 secretory pathway components validated ATF4, SRP9, JUN, PDIA3 and HSPA8 as productivity boosters in CHO. Moreover, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components for metabolic engineering of HEK293 and CHO.

Low Shear Stress Increases Recombinant Protein Production and High Shear Stress Increases Apoptosis in Human Cells.

Human embryonic kidney cells HEK293 can be used for the production of therapeutic glycoproteins requiring human post-translational modifications. High cell density perfusion processes are advantageous for such production but are challenging due to the shear sensitivity of HEK293 cells. To understand the impact of hollow filter cell separation devices, cells were cultured in bioreactors operated with tangential flow filtration (TFF) or alternating tangential flow filtration (ATF) at various flow rates. The average theoretical velocity profile in these devices showed a lower shear stress for ATF by a factor 0.637 compared to TFF. This was experimentally validated and, furthermore, transcriptomic evaluation provided insights into the underlying cellular processes. High shear caused cellular stress leading to apoptosis by three pathways, i.e. endoplasmic reticulum stress, cytoskeleton reorganization, and extrinsic signaling pathways. Positive effects of mild shear stress were observed, with increased recombinant erythropoietin production and increased gene expression associated with transcription and protein phosphorylation.

Evolution from adherent to suspension: systems biology of HEK293 cell line development.

The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.

Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin.

Process intensification in mammalian cell culture-based recombinant protein production has been achieved by high cell density perfusion exceeding 108 cells/mL in the recent years. As the majority of therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cells, intensified perfusion processes have been mainly developed for this type of host cell line. However, the use of CHO cells can result in non-human posttranslational modifications of the protein of interest, which may be disadvantageous compared with human cell lines. In this study, we developed a high cell density perfusion process of Human Embryonic Kidney (HEK293) cells producing recombinant human Erythropoietin (rhEPO). Firstly, a small-scale perfusion system from commercial bench-top screening bioreactors was developed for <250 mL working volume. Then, after the first trial runs with CHO cells, the system was modified for HEK293 cells (more sensitive than CHO cells) to achieve a higher oxygen transfer under mild aeration and agitation conditions. Steady states for medium (20 × 106 cells/mL) and high cell densities (80 × 106 cells/mL), normal process temperature (37 °C) and mild hypothermia (33 °C) as well as different cell specific perfusion rates (CSPR) from 10 to 60 pL/cell/day were applied to study the performance of the culture. The volumetric productivity was maximized for the high cell density steady state but decreased when an extremely low CSPR of 10 pL/cell/day was applied. The shift from high to low CSPR strongly reduced the nutrient uptake rates. The results from our study show that human cell lines, such as HEK293 can be used for intensified perfusion processes.

Controlling the bioactivity of a peptide hormone in vivo by reversible self-assembly.

The use of peptides as therapeutic agents is undergoing a renaissance with the expectation of new drugs with enhanced levels of efficacy and safety. Their clinical potential will be only fully realised once their physicochemical and pharmacokinetic properties have been precisely controlled. Here we demonstrate a reversible peptide self-assembly strategy to control and prolong the bioactivity of a native peptide hormone in vivo. We show that oxyntomodulin, a peptide with potential to treat obesity and diabetes, self-assembles into a stable nanofibril formulation which subsequently dissociates to release active peptide and produces a pharmacological effect in vivo. The subcutaneous administration of the nanofibrils in rats results in greatly prolonged exposure, with a constant oxyntomodulin bioactivity detectable in serum for at least 5 days as compared to free oxyntomodulin which is undetectable after only 4 h. Such an approach is simple, cost-efficient and generic in addressing the limitations of peptide therapeutics.

A pH-Induced Switch in Human Glucagon-like Peptide-1 Aggregation Kinetics.

Aggregation and amyloid fibril formation of peptides and proteins is a widespread phenomenon. It has serious implications in a range of areas from biotechnological and pharmaceutical applications to medical disorders. The aim of this study was to develop a better understanding of the mechanism of aggregation and amyloid fibrillation of an important pharmaceutical, human glucagon-like peptide-1 (GLP-1). GLP-1 is a 31-residue hormone peptide that plays an important role regulating blood glucose levels, analogues of which are used for treatment of type 2 diabetes. Amyloid fibril formation of GLP-1 was monitored using thioflavin T fluorescence as a function of peptide concentration between pH 7.5 and 8.2. Results from these studies establish that there is a highly unusual pH-induced switch in GLP-1 aggregation kinetics. At pH 8.2, the kinetics are consistent with a nucleation-polymerization mechanism for fibril formation. However, at pH 7.5, highly unusual kinetics are observed, where the lag time increases with increasing peptide concentration. We attribute this result to the formation of off-pathway species together with an initial slow, unimolecular step where monomer converts to a different monomeric form that forms on-pathway oligomers and ultimately fibrils. Estimation of the pKa values of all the ionizable groups in GLP-1 suggest it is the protonation/deprotonation of the N-terminus that is responsible for the switch with pH. In addition, a range of biophysical techniques were used to characterize (1) the start point of the aggregation reaction and (2) the structure and stability of the fibrils formed. These results show that the off-pathway species form under conditions where GLP-1 is most prone to form oligomers.

The effect of a point mutation on the stability of IgG4 as monitored by analytical ultracentrifugation.

There is presently considerable interest in the state of aggregation and biophysical integrity of antibody preparations, and recent advances in the analysis of data from the analytical ultracentrifuge renders it a powerful probe of these stability phenomena, under both storage and freeze-thaw conditions. Solutions of a wild-type IgG4 antibody and a single amino acid hinge mutant IgG4m (serine residue 241 converted to proline) were exposed to different accelerated stress conditions, namely (i) elevated temperature storage for various periods (up to 59 days at 37 degrees C) or (ii) a series of freeze-thaw cycles (storage at -80 degrees C then incubation at 20 degrees C for 1 h under different conditions). Analysis using the nondisruptive probe of sedimentation velocity in the analytical ultracentrifuge indicated that for both antibodies the monomer was always the most common species present whatever storage regime had been used. Sedimentation coefficient distribution analysis showed that other higher oligomer species and half-antibodies were present, and appeared to be not in chemical equilibrium with each other. Solution heterogeneity was found to increase considerably with treatment for both native and hinge-mutant antibodies although the latter appeared to be more resistant to freeze-thaw-induced aggregation.

Postoperative pain following coblation tonsillectomy: randomized clinical trial.

BACKGROUND: Tonsillectomy is one of the commonest surgical procedures, with postoperative pain being an important source of morbidity. Coblation (cold ablation) is a new technique for tonsillectomy, promoted by claims of reduced postoperative pain levels. This study was designed to compare postoperative pain after tonsillectomy using coblation and tonsillectomy using the standard dissection techniques. METHODS: Twenty adult patients underwent tonsillectomy, each having one randomly selected tonsil removed by dissection and the other removed by coblation. For each side, subjective pain levels were recorded on a daily basis for 10 postoperative days, using a visual analogue scale. RESULTS: Coblation tonsillectomy was significantly less painful than dissection tonsillectomy on day 1 (P < 0.001), day 2 (P = 0.003) and day 3 (P = 0.018). For all subsequent postoperative days, there was no significant difference in pain levels between the techniques. CONCLUSION: Coblation tonsillectomy causes significantly less pain during the first three postoperative days, when compared with dissection tonsillectomy. No demonstrable benefit was shown on days 4-10. The beneficial effects of coblation on early postoperative pain make it a potentially attractive technique for day-case tonsillectomy in adults with recurrent or chronic tonsillitis.

Screening for glycosylation changes on recombinant human IgG using lectin methods.

Two lectin-binding methods were investigated as possible ways of monitoring the glycosylation of human monoclonal antibodies during their development and production. Carbohydrate composition was assessed in various preparations that were produced in different host cell types, cell sublines or batches of the same cells. The lectin binding was measured with ELISA and surface plasmon resonance (SPR). For comparative purposes, the monosaccharide content of many of the preparations was also measured by high-pressure anion exchange chromatography (HPAEC)/pulsed amperometric detection (PAD). Both lectin methods detected modifications in glycosylation when antibodies were produced in different ways; SPR was more sensitive than ELISA for some lectins and vice versa. Generally, the lectin results agreed with those obtained by the monosaccharide analysis; however, the former were much better for assessing N -acetylneuraminic acid changes. The latter were impossible to assess by HPAEC/PAD because of their low levels. The lectin-based methods also had the advantages that they were quicker to perform and required less expertise and could quickly identify structures that monosaccharide analysis might miss. It is suggested that, in the development of therapeutic proteins, monosaccharide analysis and/or oligosaccharide profiling is initially performed but later routine batches of the glycoprotein are screened with a lectin method. Of the two lectin methods used, SPR is much quicker when performing a screen, whereas ELISA is particularly useful for comparing a particular carbohydrate feature on different samples of the same glycoprotein.

Selective cleavage of human IgG by the matrix metalloproteinases, matrilysin and stromelysin.

We have shown that two of the matrix metalloproteinases (MMPs), matrilysin and stromelysin-1, are capable of cleaving all of the human IgG subclasses. The cleavage occurs at a conserved site in the CH(2) domain of the heavy chain of IgG, releasing a single chain Fc-like fragment. We have not been able to demonstrate cleavage of IgA, IgD, IgM or IgE classes, which lack the cleavage site, nor could we show cleavage of IgG by collagenase, gelatinase, macrophage metalloelastase or membrane-type (MT)-MMP. This cleavage of IgG, by separating the antigen-binding (Fabprime prime or minute)(2) from the Fc portion, will remove much of the immunoglobulins' functionality, e.g. complement fixation, Fc receptor binding. In the context of a tumour producing matrilysin or stromelysin, this may represent a way in which the tumour protects itself from ADCC. In inflamed or damaged tissues where plasma protein leakage occurs, degradation by MMPs may be a mechanism for clearance of IgG.