Page 12 - CuttingEdge April 2023

Key Changes in USP <621>

6. Adjustment of Chromatographic Conditions
Key Changes in USP <621>
- Future Expectations
Chromatography adjustments are outlined in Table 3.

Parameter Official from 01 Aug 2017 to 30 Nov 2022 Current USP : official as of 01 Dec 2022
Elution Type Isocratic Gradient Isocratic Gradient
Column parameters and flow rate
Condition 1: No change of the substituent (e.g., no replacement of
C18 by C8) and physicochemical characteristics of the stationary
phase.
Stationary Phase No changes allowed
Condition 2: A change from totally porous particles (TPP) columns to
Superficially porous particles (SPP) columns allowed if condition 1
requirements meet.
L and dP may be modified if the L and dP may be modified if the ratio
Column dimensions ratio (L/dP) remains constant or Changes not (L/dP) remains constant or in the Changes allowed. Same as
(particle size, length) in the range between -25% and allowed range between -25% and 50% of Isocratic
50% of prescribed ratio. prescribed ratio. 
Key Changes in USP <621>
Adjustments from Other combination of L and dP
- Future Expectations
totally porous to allowed, provided plate number Changes not Allowed, provided plate number (N) Changes allowed. Same as

superficially porous (N) is within -25% to +50% allowed is within -25% to +50% Isocratic
Official from 01 Aug 2017 to 30 Nov 2022
particles Parameter relative to prescribed column.  Current USP : official as of 01 Dec 2022 ♦ Where L= column length;
Changes not Adjustment allowed if no change in Changes allowed. Same as dP= particle size’ F=flow; dc=
Internal diameter Not mentioned
allowed dP and/or L  Isocratic internal diameter of the column.
**Based on particle size (formula
*Based on particle size and Changes in F, *The flow rate of ±50% is permitted Page 17 of 17 *When a change is made in
Flow Rate additionally ±50% (formula for dc and dP not is adjusted for both the change in for new gradient time calculation
from original gradient time, flow particle size from ≥3-µm to
flow rate change) allowed column diameter and particle size
rate and the column dimensions) <3-µm an additional increase
Column Temperature ± 10°C ± 10°C ± 5°C in flow rate may be justified
Mobile phase provided column efficiency
Minor component comprises ≤
Minor component (specified as ≤50%) Not mentioned should not drop by >20%.
100/n %) Similarly, when a change is
Adjustment allowed for minor
Adjustment allowed for minor components made in particle size from
components composition: ± 30 %
Composition Key Changes in USP <621> Adjustment acceptable if principal
composition: ± 30 % (any component change (any component change should not peak elute within ± 15% of RT <3-µm to ≥3-µm an additional
- Future Expectations
should not exceed ±10 % absolute i.e., in reduction in flow rate may be
relation to the total mobile phase) exceed ±10 % absolute i.e., in obtained with original condition
Current USP : officia
Parameter Official from 01 Aug 2017 to 30 Nov 2022 relation to the total mobile phase) l as of 01 Dec 2022 justified to avoid reduction in
Specified ratios 50:50, the mobile column efficiency should not
Specified ratios 50:50, the mobile phase ratio phase ratio may be adjusted only
may be adjusted only within the range of 40:60– drop by >20%.
within the range of 40:60–60:40.
Binary Mixture ratio 60:40. NA
And for 2:98, the maximum allowed 2 2
And for 2:98, the maximum allowed adjustment adjustment is within the range of Page 17 of 17 *F2 = F1 × [(dc2 × dp1)/ (dc1 × dp2)]
is within the range of 1.4:98.6–2.6: 97.4.
1.4:98.6–2.6: 97.4.
Specified ratio 70:25:5 , the mixture **When particle size changes
Specified ratio 60:35:5, the mixture ranges of flow rate requires changes
Ternary Mixture ratio 50:45:5–70:25:5 or 58.5:35:6.5–61.5:35:3.5 ranges of 62.5: 32.5: 5 to 77.5:17.5: NA because smaller -particle
5 or 68.5: 25: 6.5 to 71.5: 25: 3.5
would meet the requirement.
would meet the requirement columns will require higher
pH of aqueous ±0.2 units, unless otherwise prescribed for both ±0.2 units, unless otherwise prescribed for both isocratic and gradient velocities for the same
component isocratic and gradient Key Changes in USP <621> performance (as measured by
- Future Expectations
Concentration of salts in Within ±10 % if permitted pH variation met, Within ±10 % if permitted pH variation met, applies to both isocratic reduced plate height).
the buffer component applies to both isocratic and gradient and gradient.
of MP Parameter Official from 01 Aug 2017 to 30 Nov 2022 Current USP : official as of 01 Dec 2022
2
**tG2 = tG1 × (F1/F2) [L2 × dc ) / (L1 × dc 2 )]
Deviations not permitted. (For ±3nm at most 2 1
Detector wavelength error verification should be done using detector No adjustment permitted No adjustment permitted ♦♦ Where n= number of
manufacturer procedure)
Adjustment allowed for both isocratic and ***Formula added for adjustment components
gradient, as far as it is consistent with accepted allowed upon column dimension Page 17 of 17
Injection volume same as Isocratic *** Vinj2 = Vinj1 (L2 dc ) / (L dc )
2
2
precision, linearity, and detection limits. no changed (formula NA for changes 2 1 1
formula mentioned. from TPP columns to SPP column )
NA: Not applicable
Table 3 : Chromatography adjustments
Table 3 Chromatography adjustments
12 Spinco Biotech April 2023
 Where L= column length; dP= particle size’ F=flow; dc= internal diameter of the column.
*When a change is made in particle size from ≥3-µm to <3-µm an additional increase in flow rate may be justified provided column
efficiency should not drop by >20%. Similarly, when a change is made in particle size from <3-µm to ≥3-µm an additional reduction in
flow rate may be justified to avoid reduction in column efficiency should not drop by >20%.
2
2
*F2 = F1 × [(dc2 × dp1)/ (dc1 × dp2)]
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