Liposome Technology for Industrial Purposes

Liposomes, spherical vesicles consisting of one or more phospholipid bilayers, were first described in the mid 60s by Bangham and coworkers. Since then, liposomes have made their way to the market. Today, numerous lab scale but only a few large-scale techniques are available. However, a lot of these methods have serious limitations in terms of entrapment of sensitive molecules due to their exposure to mechanical and/or chemical stress. This paper summarizes exclusively scalable techniques and focuses on strengths, respectively, limitations in respect to industrial applicability. An additional point of view was taken to regulatory requirements concerning liposomal drug formulations based on FDA and EMEA documents

Antibody charge heterogeneity formation in a mammalian cell culture fed-batch process

The charge heterogeneity of a monoclonal antibody (mAb) is as a sum factor of several post translational modifications and most of them are of high importance regarding product quality and efficacy. For this reason monitoring and controlling of this sum factor can be beneficial. The work presented here builds the basis for on-line monitoring and will help to achieve Quality by Control (Sommeregger et al., 2017). The aim of this work was to develop a method that allows fast and accurate determination of the charge profile of monoclonal antibodies directly from cell culture supernatants. We were able to circumvent a pre-purification step by adapting a cation exchange method (CEX) using a highly linear pH gradient (Lingg et al, 2013). The established method was then used to gain information about the formation of charge variants during a fed-batch process of an industrial relevant mAb produced by a Chinese Hamster Ovary (CHO) cell line. Please click Additional Files below to see the full abstract

Conjugation of Native-Like HIV-1 Envelope Trimers onto Liposomes Using EDC/Sulfo-NHS Chemistry: Requirements and Limitations

The display of native-like human immunodeficiency virus type 1 envelope (HIV-1 Env) trimers on liposomes has gained wide attention over the last few years. Currently, available methods have enabled the preparation of Env-liposome conjugates of unprecedented quality. However, these protocols require the Env trimer to be tagged and/or to carry a specific functional group. For this reason, we have investigated N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide/N-Hydroxysulfosuccinimide (EDC/Sulfo-NHS) chemistry for its potential to covalently conjugate tag-free, non-functionalized native-like Env trimers onto the surface of carboxyl-functionalized liposomes. The preservation of the liposome’s physical integrity and the immunogen’s conformation required a fine-tuned two-step approach based on the controlled use of β-mercaptoethanol. The display of Env trimers was strictly limited to activated liposomes of positive charge, i.e., liposomes with a positive zeta potential that carry amine-reactive Sulfo-NHS esters on their surface. In agreement with that, conjugation was found to be highly ionic strength- and pH-dependent. Overall, we have identified electrostatic pre-concentration (i.e., close proximity between negatively charged Env trimers and positively charged liposomes established through electrostatic attraction) to be crucial for conjugation reactions to proceed. The present study highlights the requirements and limitations of potentially scalable EDC/Sulfo-NHS-based approaches and represents a solid basis for further research into the controlled conjugation of tag-free, non-functionalized native-like Env trimers on the surface of liposomes, and other nanoparticles

Conjugation of Native-Like HIV-1 Envelope Trimers onto Liposomes Using EDC/Sulfo-NHS Chemistry: Requirements and Limitations

The display of native-like human immunodeficiency virus type 1 envelope (HIV-1 Env) trimers on liposomes has gained wide attention over the last few years. Currently, available methods have enabled the preparation of Env-liposome conjugates of unprecedented quality. However, these protocols require the Env trimer to be tagged and/or to carry a specific functional group. For this reason, we have investigated N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide/N-Hydroxysulfosuccinimide (EDC/Sulfo-NHS) chemistry for its potential to covalently conjugate tag-free, non-functionalized native-like Env trimers onto the surface of carboxyl-functionalized liposomes. The preservation of the liposome’s physical integrity and the immunogen’s conformation required a fine-tuned two-step approach based on the controlled use of β-mercaptoethanol. The display of Env trimers was strictly limited to activated liposomes of positive charge, i.e., liposomes with a positive zeta potential that carry amine-reactive Sulfo-NHS esters on their surface. In agreement with that, conjugation was found to be highly ionic strength- and pH-dependent. Overall, we have identified electrostatic pre-concentration (i.e., close proximity between negatively charged Env trimers and positively charged liposomes established through electrostatic attraction) to be crucial for conjugation reactions to proceed. The present study highlights the requirements and limitations of potentially scalable EDC/Sulfo-NHS-based approaches and represents a solid basis for further research into the controlled conjugation of tag-free, non-functionalized native-like Env trimers on the surface of liposomes, and other nanoparticles

Generation of Biologically Active Multi-Sialylated Recombinant Human EPOFc in Plants

<div><p>Hyperglycosylated proteins are more stable, show increased serum half-life and less sensitivity to proteolysis compared to non-sialylated forms. This applies particularly to recombinant human erythropoietin (rhEPO). Recent progress in <em>N</em>-glycoengineering of non-mammalian expression hosts resulted in <em>in vivo</em> protein sialylation at great homogeneity. However the synthesis of multi-sialylated <em>N-</em>glycans is so far restricted to mammalian cells. Here we used a plant based expression system to accomplish multi-antennary protein sialylation. A human erythropoietin fusion protein (EPOFc) was transiently expressed in <em>Nicotiana benthamiana</em> ΔXTFT, a glycosylation mutant that lacks plant specific N-glycan residues. cDNA of the hormone was co-delivered into plants with the necessary genes for (i) branching (ii) β1,4-galactosylation as well as for the (iii) synthesis, transport and transfer of sialic acid. This resulted in the production of recombinant EPOFc carrying bi- tri- and tetra-sialylated complex <em>N-</em>glycans. The formation of this highly complex oligosaccharide structure required the coordinated expression of 11 human proteins acting in different subcellular compartments at different stages of the glycosylation pathway. <em>In vitro</em> receptor binding assays demonstrate the generation of biologically active molecules. We demonstrate the <em>in planta</em> synthesis of one of the most complex mammalian glycoforms pointing to an outstanding high degree of tolerance to changes in the glycosylation pathway in plants.</p> </div

Generation of GnGn structures in rhEPOFc.

<p>Mass spectra of trypsin and endoproteinase Glu-C double-digested rhEPOFc expressed in <i>N. benthamiana</i> ΔXTFT (rhEPOFc_ΔXTFT; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone-0054836-g002" target="_blank">Figure 2B</a>, lane 1). Glycosylation patterns of rhEPO glycopeptide 1 (Gp1): E/A²²E<u>NIT</u>TGCAE³¹; glycopeptide 2 (Gp2): E/H³²CSLNE<u>NIT</u>VPDTK⁴⁵ and glycopeptide 3 (Gp3): R/G⁷⁷QALLV<u>NSS</u>QPWEPLQHLVDK⁹⁷ are shown. The corresponding <i>N-</i>glycosylation profile of the Fc glycopeptide (R/EEQY<u>NST</u>YR) is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s001" target="_blank">Figure S1</a>. Peak labels were made according to the ProGlycAn system (<a href="http://www.proglycan.com" target="_blank">www.proglycan.com</a>). Illustrations display <i>N</i>-glycans on assigned peaks, for interpretation of other assigned glycoforms see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s005" target="_blank">Figure S5</a>.</p

Expression of rhEPOFc in <i>N. benthamiana</i>.

<p>A. Western blot analysis of total soluble proteins extracted from <i>N. benthamiana</i> expressing rhEPOFc. 55 kDa protein reacts to both anti-EPO (α-EPO) and anti-Fc (α-Fc) antibodies while the ∼30 kDa band reacts only with α-Fc antibodies. B. Protein A purified rhEPOFc fractionated by SDS PAGE and stained with Coomassie-brilliant blue R-250. lane 1: rhEPOFc expressed in <i>N. benthamiana</i> mutants lacking plant specific β1,2-xylose and α1,3-fucose (rhEPOFc_ΔXTFT); lane 2: rhEPOFc co-expressed with mammalian genes for protein sialylation (GNE, NANS, CMAS, CST, ^STGalT and ST) (rhEPOFc_Sia,); lane 3: rhEPOFc co-expressed with mammalian genes necessary for sialylation and synthesis of tri-antennary <i>N-</i>glycans GnTIV or GnTV, (rhEPO_TriSia,); lane 4: rhEPOFc co-expressed with mammalian genes for sialylation and synthesis of tetra-antennary <i>N-</i>glycans, GnTIV and GnTV (rhEPO_TetraSia). A and B represent distinct protein fractions from the 55 kDa band of rhEpoFc_TriSia and rhEPO_TetraSia, used for N-glycan analysis; the ∼30 kDa band represent free Fc. C. Western blot analysis of total soluble proteins (5 µg TSP) extracted from <i>N. benthamiana</i> ΔXTFT mutants (control; lane 1) and of purified rhEPOFc_ΔXTFT (lane 2) using antibodies against Lewis-A epitopes (JIM 84). Several proteins in TSP and the 55 kDa protein band corresponding to intact rhEPOFc reacted to JIM 84 revealing the presence of <i>N-</i>glycans with Lewis-a epitopes. (M) protein marker.</p

Generation of tri-sialylated structures in rhEPOFc.

<p>Mass spectra of trypsin and endoproteinase Glu-C double-digested rhEPOFc co-expressed in <i>N. benthamiana</i> ΔXTFT with mammalian genes for synthesis of tri-antennary sialylated <i>N-</i>glycans (rhEPO_TriSia). The analysis was performed on rhEPOFc_TriSia present on fraction A of the 55kDa band (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone-0054836-g002" target="_blank">Figure 2B</a>, lane 3). Glycosylation patterns of rhEPO Gp1: E/A²²E<u>NIT</u>TGCAE³¹; Gp2: E/H³²CSLNE<u>NIT</u>VPDTK⁴⁵ and Gp3: R/G⁷⁷QALLV<u>NSS</u>QPWEPLQHLVDK⁹⁷ are shown. <i>N-</i>glycosylation profile of the Fc glycopeptide is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s001" target="_blank">Figure S1</a>. Glycosylation profile of rhEPOFc present on fraction B of the 55 kDa band is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s002" target="_blank">Figure S2</a>. Peak labels were made according to the ProGlycAn system (<a href="http://www.proglycan.com" target="_blank">www.proglycan.com</a>). Illustrations display <i>N</i>-glycans on assigned peaks, for interpretation of other assigned glycoforms see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054836#pone.0054836.s005" target="_blank">Figure S5</a>.</p