Efficient Development of Nanobody-Based Affinity Chromatography for AAV8 Purification

Deamidation of Amino Acids on the Surface of Adeno-Associated Virus Capsids Leads to Charge Heterogeneity and Altered Vector Function.

A scalable, serotype-agnostic, and fully chromatographic downstream platform was developed for the purification of adeno-associated virus (AAV) based on affinity chromatography (AC), anion-exchange chromatography (AEX), and multi-modal polishing chromatography (MMC). Quantitative proteomic profiling across each purification stage was performed using sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) to characterize residual host cell protein (HCP) retention for four AAV serotypes (AAV2, -5, -8, and -9) produced with suspension HEK293 cells. The downstream process showed cumulative vector genome (VG) yields ranging from 44.6 to 69.2% with full capsid enrichments ranging from 2.62- to 5.93-fold across all AAV samples. A total of 880, 99, and 21 HCPs were identified in all AAV samples after AC, AEX, and MMC, respectively. Vector-mediated mechanisms of retention are proposed (i.e., capsid or genome association) based on enrichment in protein- and nucleic acid-binding properties of difficult-to-remove HCPs, little clearance across multiple modes of separation, and divergence of HCP profiles for AAV-containing samples vs. null lysate material. The characterization of HCP retention described here advances the understanding of process-related impurity clearance in scalable AAV downstream systems, with implications for both purification process design and AAV interactions with host cell factors.

Serotype-agnostic affinity purification of adeno-associated virus (AAV) via peptide-functionalized chromatographic resins

Cytochrome c maturation (Ccm) is a post-translational and post-export protein modification process that involves ten (CcmABCDEFGHI and CcdA or DsbD) components in most Gram-negative bacteria. The absence of any of these components abolishes the ability of cells to form cytochrome c, leading in the case of Rhodobacter capsulatus to the loss of photosynthetic proficiency and respiratory cytochrome oxidase activity. Based on earlier molecular genetic studies, we inferred that R. capsulatus CcmF, CcmH, and CcmI interact with each other to perform heme-apocytochrome c ligation. Here, using functional epitope-tagged derivatives of these components coproduced in appropriate mutant strains, we determined protein-protein interactions between them in detergent-dispersed membranes. Reciprocal affinity purification as well as tandem size exclusion and affinity chromatography analyses provided the first biochemical evidence that CcmF, CcmH, and CcmI associate stably with each other, indicating that these Ccm components form a membrane-integral complex. Under the conditions used, the CcmFHI complex does not contain CcmG, suggesting that the latter thio-reduction component is not always associated with the heme ligation components. The findings are discussed with respect to defining the obligatory components of a minimalistic heme-apocytochrome c ligation complex in R. capsulatus.

The photochemical degradation of 2,4,6-trichlorophenol (2,4,6-TCP) has been studied in the presence of a nonionic surfactant. A faster decay of 2,4,6-TCP could be achieved by the addition of surfactant depending on the pH levels. By examining the TCP decay at different solution pH and surfactant concentrations, the variations in photodecay rates were dominated by the existence of surfactant micelles. In water or when surfactant dosage was below the critical micellar concentration, the photodecay of 2,4,6-TCP was found to be faster at high pH levels, mainly due to the higher absorption of TCP and additional hydrogen source (from surfactant monomer). On the contrary, in the presence of micelles, faster decay of 2,4,6-TCP was obtained at lower pH levels. This was possibly due to the domination of both photochemical homolysis and electron transfer processes in acidic condition, but the latter was inhibited at alkaline condition. In addition, the cage effect of surfactant micelles may possibly hinder the OH - -catalyzed photohydrolysis in the solution, so the TCP decay in micellar solution was reduced at high pH levels.

We present here a gel-based method for rapid purification of apolipoprotein A-I (apoA-I) from small volumes of human plasma. After isolation of high density lipoprotein from plasma, the apoA-I protein was separated by electrophoresis and the apoA-I band excised from the gel. The apoA-I was then eluted from the gel strip, concentrated, and delipidated ready for use. The structure and function of the gel-purified apoA-I protein was compared against apoA-I purified by the traditional size-exclusion chromatography method. The α-helical content of the gel-purified apoA-I as determined by circular dichroism was similar to chromatography-purified apoA-I. The functional activity of gel-purified apoA-I, as determined by cholesterol efflux assays in primary human fibroblasts and RAW264.7 macrophages, was also comparable with chromatography-purified apoA-I. This method is a valid alternative for apoA-I purification with some advantages over traditional chromatography purification including a much reduced plasma volume requirement, less time and cost, and a higher percentage protein recovery. The method is particularly suitable for applications requiring the purification of apoA-I from multiple human or animal samples of interest.

Adeno-Associated Virus Vectors in Clinical Trials

Biogas is an environmentally friendly alternative biomass and combustible gas. It contains a mixture of methane (CH4), Carbon dioxide (CO2) and other gases, which is obtained from the fermentation of organic materials by anaerobic bacteria. Biogas production needs to be developed in order to address the environmental and energy concerns. The aim of the present study is to obtain the highest biogas volume production through acetic and base solution into substrate. The research was performed in 30 days to know a better understanding on the effect of substrate pH level on the produced biogas. Five different pH levels of substrate were obtained by adding lime water and acetic acid. The pH level of the substrate without any treatment is 7, and a higher pH level is gained by adding acetic acid while a lower pH level is gained by adding lime water. The results reveal that the substrate pH level increases during the fermentation followed by a reduction of biogas volume production. The drawback is overcome by reducing the pH level of the substrate, in which it shows better result in producing biogas volume compared to those with higher pH levels. It also verifies that the rate of biogas formation is better on lower substrate pH.

The effects of six target media pH levels (4.5,5.0,5.5,6.0,6.5) on cut‐flower (glasshouse) and tuber production (outdoor) in Sandersonia aurantiaca (Hook.) were compared in two experiments. The effects of these pH levels on tuber storage and subsequent cut‐flower production were assessed in a third experiment in a glasshouse. Actual media pH levels achieved varied from 4.1 to 6.1 in the glasshouse and from 4.4 to 6.5 outdoors. The pH levels in the glasshouse produced small differences in stem length but no significant differences in stem weight, flower number, or vase life. At the highest pH level, the number of stems harvested was less and the number of rejected flowers greater than at lower pH levels because of decreased stem length or physiological disorders, such as leaf chlorosis and leaf tip browning on the lower leaves. Leaf concentrations of S, Mn, and Zn declined with increasing pH level whereas Mg increased in the cut‐flower experiment. The number of tubers lifted outdoors was least at the highest pH level but there was no difference in mean tuber weight. Tuber nutrient concentrations of N, Ca, Mn, Zn, and Cu declined with increasing pH level whereas Mg increased. There were no production differences at forcing with the lifted tubers. For sandersonia cut‐flower production a media pH range of 4.4–5.3 appears to be suitable, whereas for tuber production pH levels of 4.4–6.1 were best. As sandersonia production usually involves both growth phases, a range pH of 4.4–5.3 would be suitable with a target pH of 5.0–5.3 recommended for growth in peat‐pumice media. This will ensure mid‐range tuber and leaf nutrient concentrations and optimised production.

Affinity ligands for immunoglobulins based on the multicomponent Ugi reaction

Affinity chromatography for vaccines manufacturing: Finally ready for prime time?

Protein splicing involves the excision of an intervening polypeptide, the intein, from flanking polypeptides, the exteins, concomitant with the specific ligation of the exteins. The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be overexpressed and purified as an unspliced precursor, which allows for a detailed in vitro kinetic analysis of the individual steps of protein splicing. The first order rate constant for splicing of this intein, which has a non-canonical Gln at its C terminus, is 9.3 x 10(-6) s(-1) at 60 degrees C. The rate constant for splicing increases 3-fold with substitution of Asn for the C-terminal Gln. The pseudo first order rate constant of dithiothreitol-dependent N-terminal cleavage is 1 x 10(-4) s(-1). The first order rate constant of C-terminal cleavage is 1.2 x 10(-5) s(-1) with Gln at the C-terminal position, 2.8 x 10(-4) s(-1) with Asn, and decreases significantly with mutation of the penultimate His of the intein to Ala. N-terminal cleavage is most efficient between pH 7 and 7.5 and decreases at both more acidic and alkaline pH values, whereas C-terminal cleavage and splicing are both efficient over a broader range of pH values.

Affinity chromatography plays a significant role in the separation and purification of biologically active macromolecules in laboratory and large-scale applications. There is a need for models which could be used to predict accurately the dynamic behavior of affinity chromatography separations, in order to permit the design, optimization, control, and process scale-up of affinity chromatography systems. Furthermore, the construction and use of such models will contribute to a better fundamental understanding of the physicochemical and biospecific mechanisms involved in affinity chromatography processes. The parameters of the models should be obtainable by using information from a small number of experiments. This work reviews the modeling of affinity chromatography, and presents general models that could be used to describe the dynamic behavior of the adsorption, wash, and elution stages of affinity chromatography systems. Certain model structures, modeling approaches and operational strategies f...

Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Regardless of producing high protein titers, various cellular and process level bottlenecks restrict the expression of recombinant proteins in P. pastoris. In the present review, we have summarized the recent developments in the expression of foreign proteins in P. pastoris. Further, we have discussed various cellular engineering strategies which include codon optimization, pathway engineering, signal peptide processing, development of protease deficient strain and glyco-engineered strains for the high yield protein secretion of recombinant protein. Bioprocess development of recombinant proteins in large-scale bioreactor including medium optimization, optimum feeding strategy and co-substrate feeding in fed-batch as well as continuous cultivation have been described. The recent advances in system and synthetic biology studies including metabolic flux analysis in understanding the phenotypic characteristics of recombinant Pichia and genome editing with CRISPR-CAS system have also been summarized.

Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.