Method development for quantitative monitoring of monoclonal antibodies in upstream cell‐culture process samples with limited sample preparation – An evaluation of various capillary coatings

Abstract

Monoclonal antibodies (mAbs) have become an important class of biopharmaceuticals used for the treatment of various diseases. Their quantification during the manufacturing process is important. In this work, a capillary zone electrophoresis (CZE) method was developed for the monitoring of the mAb concentration during cell‐culture processes. CZE method development rules are outlined, particularly discussing various capillary coatings, such as a neutral covalent polyvinyl alcohol coating, a dynamic successive multiple ionic‐polymer coating, and dynamic coatings using background electrolyte additives such as triethanolamine (T‐EthA) and triethylamine. The dynamic T‐EthA coating resulted in most stable electro‐osmotic flows and most efficient peak shapes. The method is validated over the range 0.1–10 mg/ml, with a linear range of 0.08–1.3 mg/ml and an extended range of 1–10 mg/ml by diluting samples in the latter concentration range 10‐fold in water. The intraday precision and accuracy were 2%–12% and 88%–107%, respectively, and inter‐day precision and accuracy were 4%–9% and 93%–104%, respectively. The precision and accuracy of the lowest concentration level (0.08 mg/ml) were slightly worse and still well in scope for monitoring purposes. The presented method proved applicable for analysing in‐process cell‐culture samples from different cell‐culture processes and is possibly well suited as platform method.

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition

“…There are a number of variants of both methods [170,191,193,195]. Those may be optimized for a specific mAb (see Section 9.1.1) [196].…”

“…(I) T-EthA method for the concentration determination of mAb (in example process samples) [193] Polyvinyl alcohol (PVA)-coated capillaries with 50-µm id, a total length of 33 cm with an effective length of 24.5 or 8.5 cm (e.g., Agilent Technologies) Separation voltage: 16 kV, ramped over 0.5 min (approx. 45 µA) Sample injection at 10 mbar for 5 s, followed by the injection of a BGE plug using the same conditions T = 20 The intra-day precision and accuracy were 2%-12% and 88%-107%, respectively, and inter-day precision and accuracy were 4%-9% and 93%-104%, respectively t ana = 6-11 min (II) eACA method for the charge heterogeneity determination in drug substance and drug product [170] The BGE comprising 400 mM eACA and 2.0 mM.…”

“…They are useful both as rinsing substances for some samples and essential for CGE methods, especially of course CE-SDS. Polysorbate 20, Poloxamer, or other neutral surfactants as BGE additive can prevent adsorption of matrix components [187,193,198,323,330]. As protein analysis often involves working under reduced conditions, reducing agents such as β-mercaptoethanol are also must-haves for the laboratory bench.…”

“…In the meantime, two methods have become particularly established for the analysis of mAbs. One uses triethanolamine (T‐EthA) and coated capillaries [193] and focuses on the mAb concentration determination, including complex matrices such as upstream process samples (see Figure 2). The other, often called the He–CZE‐method [194] or eACA–CZE method, uses ε‐aminocaproic acid (eACA) [170] and determines charge heterogeneity (see Figure 3).…”

An interlaboratory capillary zone electrophoresis‐UV study of various monoclonal antibodies, instruments, and ε‐aminocaproic acid lots

“…The use of a bare fused silica capillary can also be responsible for the loss in electrophoretic resolution based on adsorption effects on the capillary wall [11]. This can be solved by using dynamic coatings with, for example, triethanolamine [12] or TETA [13] as BGE additive or by using neutral coated capillaries [12]. These types of amines could dynamically coat the capillary wall and/or act as cationic ion pairing reagents with acidic functional groups on the protein [13].…”