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This unit describes the solid-phase synthesis and downstream processing for RNA oligonucleotides with a length of up to 40 to 50 nucleotides on a 1- to 4-mmol scale with subsequent conjugation to PEG using the L-RNA spiegelmer NOX-E36 as an example. Following synthesis and two-step deprotection, the crude oligonucleotide is purified by preparative reversed-phase HPLC and desalted by tangential flow ultrafiltration. The resulting intermediate amino-modified oligonucleotide is reacted with NHS-ester-activated PEG, and the oligonucleotide-PEG conjugate is obtained after preparative AX-HPLC purification, followed by ultrafiltration and lyophilization. Critical process parameters are described, as well as time considerations and examples for analytical methods used as in-process and quality controls.

Analytical methodologies for the absolute quantitation of proteins typically include a digest step often using trypsin as the proteolytic enzyme. In the majority of cases, off-line and on-line digestion methods are implemented prior to an LC-MS analysis system, requiring a high sequence coverage for unambiguous protein identification. For proteins with a strong overlap in amino acid sequence, e.g., therapeutic proteins and their metabolites, it is essential to separate proteins prior to digestion and the subsequent electrospray mass spectrometry analysis of marker peptides. Here, we present an on-line postcolumn solution-phase digestion methodology that is based on the continuous infusion of the proteolytic enzyme pepsin downstream to the nano C18 reversed-phase column. Proteins are identified based on their retention time in combination with the detection of specific marker peptides formed in the postcolumn digest. The optimization of important parameters such as enzyme concentration, reaction time, and organic modifier concentration is described. We demonstrated that the continuous-flow solution-phase digest method can be coupled on-line to the reversed-phase gradient liquid chromatography separation of proteins. Detection limits obtained for five model proteins, detected as specific marker peptides with m/z values of 300-1000, range from 30 to 90 fmol, with a linear response up to 3 pmol.

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