Table 1.

Summary table of used strains, titers in fed-batch fermentation and the % Δ−16 Da variant

Strain	Genotype	Source	Nanobody A		Nanobody B		Nanobody C
			Titers (g l−1)	% Δ−16 Da variant	Titers (g l−1)	% Δ−16 Da variant	Titers (g l−1)	% Δ−16 Da variant
CBS7435 (NRRL Y-11430)	AOX1	ARSa	5.2	0	7	0
CBS7435MutS	aox1	[7]	4.5	4	10.5 (2.5)a	12 (0)a
X-33	AOX1	Invitrogen	6.4	0	9.1	0	0.9	0
KM71H (MutS)	aox1	Invitrogen					1.2	8

Nanobody productions were performed at 2 l scale, pH 6, 30 °C in complex medium with a methanol feed rate of 4 or 3 ml l⁻¹ h⁻¹ for wild type or Mut^S strains respectively. Except for ^a where the methanol feed rate was reduced to 0.5 ml l⁻¹ h⁻¹ in a co-feeding with sorbitol. Expression levels of Nanobodies were analyzed via a proteinA cleanup step followed by OD₂₈₀ measurement. Relative abundance of the Δ−16 Da variant of the different Nanobodies was analysed via RP-HPLC followed by total mass measurement by ESI-Q-TOF-MS. The Δ−16 Da Nanobody variant was only observed in fed-batch fermentations with the Mut^S strains. A strong reduction of the Δ−16 Da was observed when the methanol feed rate was reduced to 0.5 ml l⁻¹ h⁻¹ and using co-feeding with sorbitol