Continued interest in protein therapeutics has motivated the development of improved bioanalytical tools to support development programs. LC-MS offers specificity, sensitivity, and multiplexing ...capabilities without the need for target-specific reagents, making it a valuable alternative to ligand binding assays. Immunoaffinity purification (IP) and enzymatic digestion are critical, yet extensive and time-consuming components of the "gold standard" bottom-up approach to LC-MS-based protein quantitation. In the present work, commercially available technology, based on membrane-immobilized reagents in spin column and plate format, is applied to reduce IP and digestion times from hours to minutes. For a standard monoclonal antibody, the lower limit of quantitation was 0.1 ng μL
−1
compared to 0.05 ng μL
−1
for the standard method. A pharmacokinetics (PK) study dosing Herceptin in rat was analyzed by both the membrane and the standard method with a total sample processing time of 4 h and 20 h, respectively. The calculated concentrations at each time point agreed within 8% between both methods, and PK values including area under the curve (AUC), half-life (T1/2), mean residence time (MRT), clearance (CL), and volume of distribution (Vdss) agreed within 6% underscoring the utility of the membrane methodology for quantitative bioanalysis workflows.
Rapid spin membrane technology decreases the time for IP and digestion of therapeutic proteins.
Effective monoclonal antibody (mAb) therapies require a threshold mAb concentration in patient serum. Moreover, the serum concentration of the mAb Bevacizumab should reside in a specific range to ...avoid side effects. Methods for conveniently determining the levels of mAbs in patient sera could allow for personalized dosage schedules that lead to more successful treatments. This work utilizes microporous nylon membranes functionalized with antibody-binding peptides to capture Bevacizumab, Rituximab, or Panitumumab from diluted (25%) serum. Modification of the capture-peptide terminus is often crucial to creating the affinity necessary for effective binding. The high purity of eluted mAbs allows for their quantitation using native fluorescence, and membranes are effective in spin devices that can be used in any laboratory. The technique is effective over the therapeutic range of Bevacizumab concentrations. Future work aims at further modifications to develop rapid point-of-care devices and decrease detection limits.
This study develops procedures to rapidly screen conditions for purification of membrane proteins (MPs) using 96‐well plates containing nickel‐functionalized membranes. In addition to their ...application in the pharmaceutical industry, MPs are important components of new sensors, synthetic membranes, and bioelectronic devices. However, purification of MPs is challenging due to their hydrophobic exterior, which requires stabilization in amphipathic detergent micelles. We examined the extent of extraction of the light‐driven sodium transporter, Krokinobacter eikastus rhodopsin 2 (KR2) heterologously expressed in Escherichia coli using different salts and maltoside‐based detergents. The extraction was followed by subsequent affinity purification in membranes functionalized with Ni2+‐nitrilotriacetate complexes that bind the His‐tagged KR2. We also employed a hydrophobic chelator to separate detergent micelles from the aqueous phase as an initial isolation step prior to affinity purification. Unlike conventional resin‐based capture, which can take a full day or more, the membrane‐based screening of purification conditions takes only a few hours, and its scale‐up involves changing from a 96‐well format to a larger membrane module. The novelty of the method lies in utilizing membrane‐based ultracentrifugation‐free purification of MPs from cell membrane fragments; the optimized purification conditions from the screening method can potentially be applied to large‐scale/conventional resin‐based purification of MPs.
Proteolytic digestion is an important step in characterizing protein sequences and post-translational modifications (PTMs) using mass spectrometry (MS). This study uses pepsin- or trypsin-containing ...spin membranes for rapid digestion of single proteins or simple protein mixtures prior to ultrahigh-resolution Orbitrap MS analysis. Centrifugation of 100 μL of pretreated protein solutions through the functionalized membranes requires less than 1 min and conveniently digests proteins into large peptides that aid in confirming specific protein sequence variations and PTMs. Peptic and tryptic peptides from spin digestion of apomyoglobin and four commercial monoclonal antibodies (mAbs) typically cover 100% of the protein sequences in direct infusion MS analysis. Increasing the spin rate leads to a higher fraction of large peptic peptides for apomyoglobin, and MS analysis of peptic and tryptic peptides reveals mAb PTMs such as N-terminal pyroglutamate formation, C-terminal lysine clipping and glycosylation. Relative to overnight in-solution digestion of mAbs, spin digestion yields higher sequence coverages. Spin-membrane digestion followed by infusion MS readily differentiates a mAb to the Ebola virus from a related antibody that differs by addition of a single amino acid.
Centrifugation of antibodies through enzyme-containing spin membranes yields large proteolytic peptides that enable confirmation of protein sequences and post-translational modifications.
Proteolytic digestion is an important step in characterizing protein sequences and post-translational modifications (PTMs) using mass spectrometry (MS). This study uses pepsin- or trypsin-containing ...spin membranes for rapid digestion of single proteins or simple protein mixtures prior to ultrahigh-resolution Orbitrap MS analysis. Centrifugation of 100 μL of pretreated protein solutions through the functionalized membranes requires less than 1 min and conveniently digests proteins into large peptides that aid in confirming specific protein sequence variations and PTMs. Peptic and tryptic peptides from spin digestion of apomyoglobin and four commercial monoclonal antibodies (mAbs) typically cover 100% of the protein sequences in direct infusion MS analysis. Increasing the spin rate leads to a higher fraction of large peptic peptides for apomyoglobin, and MS analysis of peptic and tryptic peptides reveals mAb PTMs such as N-terminal pyroglutamate formation, C-terminal lysine clipping and glycosylation. Relative to overnight in-solution digestion of mAbs, spin digestion yields higher sequence coverages. Spin-membrane digestion followed by infusion MS readily differentiates a mAb to the Ebola virus from a related antibody that differs by addition of a single amino acid.
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