1.5 Batch-to-Batch
Consistency
If different batches of a protein are used or if production, storage,
or assay conditions are altered, it is necessary to demonstrate batchto-batch consistency; i.e., the different lots must exhibit the same
purity, homogeneity, oligomeric state, and conformational/structural integrity. While the first three criteria can be assessed via the
methods described (Subheading 1.2), consistency in conformation
can be checked by recording a “spectral signature” or “spectral
fingerprint,” with a CD or NMR spectrum being the most common ones (Subheading 1.3). The spectral signature spectrum of a
target protein is recorded when it is successfully produced and
purified for the first time, when it fulfills all QC criteria, and when
it is fully characterized (“batch 0”). This spectrum is then used as a
reference for all future batches. If a newer batch shows differences
to the reference signature, further quality tests are required or
conditions must be altered before proceeding to downstream
applications.
2 Materials and Methods
2.1 UV Spectrum
and Concentration
Determination
The most common causes of inaccuracy in concentration determination are miscalculation of dilutions or pipetting errors during
sample preparation. Special care must therefore be taken during
the preparation of the sample for concentration measurement and
in subsequent downstream uses.
Prior to the sample measurement, the upper absorbance limit
of the UV/Vis spectrometer in the wavelength range used must be
determined. It is critical that the total absorbance of cuvette, buffer,
and protein must not exceed this limit, as above this value the
instrument will not produce a measurement in proportion to the
actual sample absorbance and consequently an incorrect concentration would be obtained [42]. Consult the instrument manual or
manufacturer to find this limit—if a specific value for the linear
range of the instrument cannot be found, then ensure that absorbance measurements fall between 0.1 and 1 AU. It is highly recommended to record the spectrum using a 1-cm quartz cuvette to
minimize errors in the measurement (see Note 5).
Although most spectrometer software will have options for
determining concentration from absorbance based on average proteins or other standards such as BSA, these are meant for working
with undefined protein mixtures, not pure recombinant proteins.
Each pure protein will have a distinct molar absorption coefficient
that depends on its amino acid composition. In order to determine
concentration from the UV spectrum using Eq. 1, an estimate of
the protein’s molar absorption coefficient must be made based on
its primary sequence, e.g., using the ProtParam program for ε at
280 nm (available on the web at https://web.expasy.org/pro
tparam/) or the Protein Calculator for ε at 205 nm (https://spin.
Protein Quality Control
21
Consistency
If different batches of a protein are used or if production, storage,
or assay conditions are altered, it is necessary to demonstrate batchto-batch consistency; i.e., the different lots must exhibit the same
purity, homogeneity, oligomeric state, and conformational/structural integrity. While the first three criteria can be assessed via the
methods described (Subheading 1.2), consistency in conformation
can be checked by recording a “spectral signature” or “spectral
fingerprint,” with a CD or NMR spectrum being the most common ones (Subheading 1.3). The spectral signature spectrum of a
target protein is recorded when it is successfully produced and
purified for the first time, when it fulfills all QC criteria, and when
it is fully characterized (“batch 0”). This spectrum is then used as a
reference for all future batches. If a newer batch shows differences
to the reference signature, further quality tests are required or
conditions must be altered before proceeding to downstream
applications.
2 Materials and Methods
2.1 UV Spectrum
and Concentration
Determination
The most common causes of inaccuracy in concentration determination are miscalculation of dilutions or pipetting errors during
sample preparation. Special care must therefore be taken during
the preparation of the sample for concentration measurement and
in subsequent downstream uses.
Prior to the sample measurement, the upper absorbance limit
of the UV/Vis spectrometer in the wavelength range used must be
determined. It is critical that the total absorbance of cuvette, buffer,
and protein must not exceed this limit, as above this value the
instrument will not produce a measurement in proportion to the
actual sample absorbance and consequently an incorrect concentration would be obtained [42]. Consult the instrument manual or
manufacturer to find this limit—if a specific value for the linear
range of the instrument cannot be found, then ensure that absorbance measurements fall between 0.1 and 1 AU. It is highly recommended to record the spectrum using a 1-cm quartz cuvette to
minimize errors in the measurement (see Note 5).
Although most spectrometer software will have options for
determining concentration from absorbance based on average proteins or other standards such as BSA, these are meant for working
with undefined protein mixtures, not pure recombinant proteins.
Each pure protein will have a distinct molar absorption coefficient
that depends on its amino acid composition. In order to determine
concentration from the UV spectrum using Eq. 1, an estimate of
the protein’s molar absorption coefficient must be made based on
its primary sequence, e.g., using the ProtParam program for ε at
280 nm (available on the web at https://web.expasy.org/pro
tparam/) or the Protein Calculator for ε at 205 nm (https://spin.
Protein Quality Control
21