| 1. |
- Källsten, Malin, et al.
(författare)
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Investigating the Impact of Sample Preparation on Mass Spectrometry-Based Drug-To-Antibody Ratio Determination for Cysteine- and Lysine-Linked Antibody-Drug Conjugates
- 2020
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Ingår i: antibodies. - : MDPI AG. - 2073-4468. ; 9:3
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Antibody-drug conjugates (ADCs) are heterogeneous biotherapeutics and differ vastly in their physicochemical properties depending on their design. The number of small drug molecules covalently attached to each antibody molecule is commonly referred to as the drug-to-antibody ratio (DAR). Established analytical protocols for mass spectrometry (MS)-investigation of antibodies and ADCs often require sample treatment such as desalting or interchain disulfide bond reduction prior to analysis. Herein, the impact of the desalting and reduction steps-as well as the sample concentration and elapsed time between synthesis and analysis of DAR-values (as acquired by reversed phase liquid chromatography MS (RPLC-MS))-was investigated. It was found that the apparent DAR-values could fluctuate by up to 0.6 DAR units due to changes in the sample preparation workflow. For methods involving disulfide reduction by means of dithiothreitol (DTT), an acidic quench is recommended in order to increase DAR reliability. Furthermore, the addition of a desalting step was shown to benefit the ionization efficiencies in RPLC-MS. Finally, in the case of delayed analyses, samples can be stored at four degrees Celsius for up to one week but are better stored at -20 degrees C for longer periods of time. In conclusion, the results demonstrate that commonly used sample preparation procedures and storage conditions themselves may impact MS-derived DAR-values, which should be taken into account when evaluating analytical procedures.
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| 2. |
- Ramadhin, Jessica, et al.
(författare)
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Monoclonal Antibodies Generated against Glycoconjugates Recognize Chemical Linkers
- 2020
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Ingår i: ANTIBODIES. - : MDPI. - 2073-4468. ; 9:3
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Tidskriftsartikel (refereegranskat)abstract
- Monoclonal antibodies (mAbs) that recognize glycans are useful tools to assess carbohydrates' structure and function. We sought to produce IgG mAbs to the human milk oligosaccharide (HMO), lacto-N-fucopentaose III (LNFPIII). LNFPIII contains the Lewis(x)antigen, which is found on the surface of schistosome parasites. mAbs binding the Lewis(x)antigen are well-reported in the literature, but mAbs recognizing HMO structures are rare. To generate mAbs, mice were immunized with LNFPIII-DEX (P3DEX) plus CpGs in VacSIM(R), a novel vaccine/drug delivery platform. Mice were boosted with LNFPIII-HSA (P3HSA) plus CpGs in Incomplete Freund's Adjuvant (IFA). Splenocytes from immunized mice were used to generate hybridomas and were screened against LNFPIII conjugates via enzyme-linked immunosorbent assay (ELISA). Three positive hybridomas were expanded, and one hybridoma, producing IgG and IgM antibodies, was cloned via flow cytometry. Clone F1P2H4D8D5 was selected because it produced IgG1 mAbs, but rescreening unexpectedly showed binding to both LNFPIII and lacto-N-neotetraose (LNnT) conjugates. To further assess the specificity of the mAb, we screened it on two glycan microarrays and found no significant binding. This finding suggests that the mAb binds to the acetylphenylenediamine (APD) linker-spacer structure of the conjugate. We present the results herein, suggesting that our new mAb could be a useful probe for conjugates using similar linker spacer structures.
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| 3. |
- Rodrigo, Gustav, et al.
(författare)
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Antibody Fragments and Their Purification by Protein L Affinity Chromatography
- 2015
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Ingår i: ANTIBODIES. - : MDPI AG. - 2073-4468. ; 4:3, s. 259-277
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Forskningsöversikt (refereegranskat)abstract
- Antibodies and related proteins comprise one of the largest and fastest-growing classes of protein pharmaceuticals. A majority of such molecules are monoclonal antibodies; however, many new entities are antibody fragments. Due to their structural, physiological, and pharmacological properties, antibody fragments offer new biopharmaceutical opportunities. In the case of recombinant full-length antibodies with suitable Fc regions, two or three column purification processes centered around Protein A affinity chromatography have proven to be fast, efficient, robust, cost-effective, and scalable. Most antibody fragments lack Fc and suitable affinity for Protein A. Adapting proven antibody purification processes to antibody fragments demands different affinity chromatography. Such technology must offer the unit operation advantages noted above, and be suitable for most of the many different types of antibody fragments. Protein L affinity chromatography appears to fulfill these criteriasuggesting its consideration as a key unit operation in antibody fragment processing.
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| 4. |
- Sellberg, Anton, et al.
(författare)
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Model-Based Comparison of Antibody Dimerization in Continuous and Batch-Wise Downstream Processing
- 2015
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Ingår i: Antibodies. - : MDPI AG. - 2073-4468. ; 4:3, s. 157-169
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Tidskriftsartikel (refereegranskat)abstract
- Monoclonal antibodies are generally produced using a generic platform approach in which several chromatographic separations assure high purity of the product. Dimerization can occur during the fermentation stage and may occur also during the downstream processing. We present here simulations in which a traditional platform approach that consist of protein A capture, followed by cation-exchange and anion-exchange chromatography for polishing is compared to a continuous platform in which dimer removal and virus inactivation are carried out on a size-exclusion column. A dimerization model that takes pH, salt concentration and the concentration of antibodies into account is combined with chromatographic models, to be able to predicted both the separation and the degree to which dimers are formed. Purification of a feed composition that contained 1% by weight of dimer and a total antibody concentration of 1 g/L was modeled using both approaches, and the amount of antibodies in the continuous platform was at least 4 times lower than in the traditional platform. The total processing time was also lower, as the cation-exchange polish could be omitted.
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| 5. |
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| 6. |
- Eivazi, Alireza, et al.
(författare)
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On the development of all-cellulose capsules by vesicle-templated layer-by-layer assembly
- 2021
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Ingår i: Polymers. - : MDPI AG. - 2073-4360. ; 13:4
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Tidskriftsartikel (refereegranskat)abstract
- Polymeric multilayer capsules formed by the layer-by-layer (LbL) technique are interesting candidates for the purposes of storage, encapsulation, and release of drugs and biomolecules for pharmaceutical and biomedical applications. In the current study, cellulose-based core-shell particles were developed via the LbL technique alternating two cellulose derivatives, anionic carboxymethyl-cellulose (CMC), and cationic quaternized hydroxyethylcellulose ethoxylate (QHECE), onto a cationic vesicular template made of didodecyldimethylammonium bromide (DDAB). The obtained capsules were characterized by dynamic light scattering (DLS), ζ potential measurements, and high-resolution scanning electron microscopy (HR-SEM). DLS measurements reveal that the size of the particles can be tuned from a hundred nanometers with a low polydispersity index (deposition of 2 layers) up to micrometer scale (deposition of 6 layers). Upon the deposition of each cellulose derivative, the particle charge is reversed, and pH is observed to considerably affect the process thus demonstrating the electrostatic driving force for LbL deposition. The HR-SEM characterization suggests that the shape of the core-shell particles formed is reminiscent of the spherical vesicle template. The development of biobased nano-and micro-containers by the alternating deposition of oppositely charged cellulose derivatives onto a vesicle template offers several advantages, such as simplicity, reproducibility, biocompatibility, low-cost, mild reaction conditions, and high controllability over particle size and composition of the shell.
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