Table 1.

Ambiguous regions of sequence and strategy for resolving these.

Sequence region	Mass (Da)	†Possible Sequence(s)	Fix or randomize sequence?	Rationale
3H3-VL: 2, 10, 33, 40, 46, 48, 58, 75, 78 3H3-VH: 4, 45, 51, 69, 80 LOB12.3-VL: 2, 15, 18, 21, 46, 48, 54, 73, 75, 83, 85, 106 LOB12.3-VH: 4, 20, 45, 78, 80, 82c	113	Leu or Ile	Fix	Fix to Leu or Ile according to germline homology
3H3-VL: 11, 21, 47, 54, 73, 104, 106 3H3-VH: 11, 20, 78, 82c LOB12.3-VL: 11, 47, 78, 104 LOB12.3-VH: 11, 18	113	Leu or Ile	Fix	Fix as Leu based on germline homology & chymotryptic cleavage at these positions
LOB12.3-VH: 98-99	114	GlyGly or Asn	Fix	Fix as GlyGly as “YGGYSE” motif is exact match to +1 translation of IGHD1-6 D-segment germline
LOB12.3-VL: 29, 33, 89 LOB12.3-VH: 51	113	Leu or Ile	Randomize	Although germline homology could have been used to fix as Leu or Ile, these site were randomized given the CDR location and low overall complexity of LOB12.3 library
3H3-VH: 29, 94 LOB12.3-VH: 28, 56, 100c	113	Leu or Ile	Randomize	Localized in or near CDR. Germline homology non-guiding, or not applicable. Randomize to Leu/Ile in library (in case of 3H3-VH29, Leu and Ile were encoded in two separate libraries)
3H3-VH: 55-56	158	AlaSer, SerAla, GlyThr, ThrGly	Randomize	Localized in CDRs. Germline homology non-guiding, therefore randomize in library
3H3-VH: 61-62	216	GluSer, AspThr	Randomize
3H3-VH: 98-99	114	GlyGly or Asn	Randomize
3H3-VH: 100a-100b	144	SerGly or GlySer	Randomize

^†Based on mass constraints and germline homology considerations.