Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies

Figures & data

Table 1. Modifications that form acidic species

Acidic species	References
Sialic acid	14,17,20,25,26
Deamidation	1,2,14,17,27-39
Non-classical disulfide linkage	40,41
Trisulfide bonds	42
High mannose	23
Thiosulfide modification	43
Glycation	14,44-46
Modification by maleuric acid	21
Cysteinylation	48
Reduced disulfide bonds	14
Non-reduced species	14
Fragments	23,25,36

Table 2. Major deamidation sites of recombinant monoclonal antibodies

Locations	Products	References
Light chain CDR1, Asn30	Asp	2
Light chain CDR2, Asn55	IsoAsp	2
Light chain CDR1, Asn33	Asp and isoAsp	27
Heavy chain CDR2, Asn55	Asp and isoAsp	28
Heavy chain CDR2, Asn55	Not determined	17
Heavy chain CDR2, Asn55	Not determined	29
Heavy chain CDR2, Asn55	Asp and isoAsp	30
Heavy chain, CH3 domain (First and second N in SNGPENNYK)		14,31-36
Heavy chain, CH2 domain (QDWLNGK)	Asp and isoAsp	32,34,37,38
Heavy chain, CH3 domain (LHNHYTQK)	Not determined	14
Light chain, CL domain (First N in LLNNFYPR)	Asp and isoAsp	32

Table 3. Impact of deamidation

Locations	Effects	Reference
Light chain, CDR1	Asp30/Asn30 only 70% as potent as Asn/Asn30	2
Light chain CDR1	Asp30/Asn30 has 39% and isoAsp30/Asn30 has 60% of binding affinity compared with Asn30/Asn30 of 96% binding affinity. Slight difference in the secondary structure of Fab with isoAsp compared with Fab with Asn30. Higher Tm for Fab with Asp30 and lower Tm for Fab with isoAsp30 compared with Fab with Asn30	27
Heavy chain CDR2	14 fold reduction in binding affinity compared with the antibody with Asn	28
Heavy chain CDR2	IsoAsp55 or Asp55 in one heavy chain only 20% potent as antibody with two Asn. Fab with either isoAsp55 or Asp55 has only 50% binding affinity compared with Fab with Asn.	23
Heavy chain CDR2	No impact on potency for fraction with lower level of Asn55 deamidation	17

Table 4. Modifications that form basic species

Basic species	References
C-terminal Lys	1,3,14,17-20,22,24,47,50
N-terminal Glu	3,17,26,51
Isomerization of Asp	2
Succinimide	2,23,56,57
Met oxidation	25,58,59
Amidation	60,61
Incomplete disulfide bonds	51
Incomplete removal of leader sequence	14,17,61
Mutation from Ser to Arg	26,62
Aglycosylation	63
Fragments	25
Aggregates	14,36

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