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13.4 GOOD LABORATORY PRACTICES
13.4.1 oveRvieW
Quality and Compliance Systems
Good Laboratory Practice (GLP) constitutes a quality management system for research laboratories and organizations involved in non- clinical experimental research. It ensures uniformity, consistency, reliability, reproducibility, quality, and integrity of products in development for human
or animal health (including pharmaceuticals) through non- clinical safety tests. Scientic measures
(whether they are used to detect pollutants in pharmaceutical products, make clinical blood glucose
determinations, characterize forensic evidence, or test materials for space missions) are widely
acknowledged to inuence life and death decisions. As a personal acknowledgment of their responsibility, scientists have historically employed sound laboratory procedures to ensure the accuracy
of their ndings. However, until recently, these policies faced inconsistent acceptance, application, and auditing. Because of well- known historical instances of incorrect data leading to disastrous outcomes, national and international agencies have established GLP guidelines for various
industries (food, agricultural, pharmaceutical, medicinal, environmental, etc.) outlined in 22 CFR
Part 58.
Federal agencies in the United States, such as the FDA and the EPA, have published documents
outlining laboratory standards that must be met for technical results from laboratory studies to be
acceptable for legal or contractual purposes. Laboratories engaging with these entities must adhere
to GLP regulations. Ensuring compliance has become paramount, with many companies investing
considerable effort, ranging from 10% to 50% of their overall resources, in internal quality assurance.
On average, companies allocate about 25% of their efforts towards this.
Given the critical role of GLP in modern laboratory operations and its signicance for competent scientists, it is highly recommended that all bioprocess engineers thoroughly review these
guidelines. The guidelines are accessible on the websites mentioned in the reference section of this
chapter.
13.4.2 eleMents of good laboRatoRy PRactice
13.4.2.1 Quality Assurance: Establishing Confidence in Reported Data
The laboratory’s analytical data, the primary outcome of chemical analysis, undergoes meticulous
quality assurance (QA) procedures. These tasks encompass ensuring accurate chemical and physical measurements, interpreting and recording data with sufcient error estimates and condence
levels. QA activities also involve maintaining comprehensive records of specimen/ sample sources,
background information (sample tracking), procedures, raw data, and ndings associated with each
specimen/ sample. Although each of these QA components might warrant extensive volumes, I’ll
briey touch upon each here. Standard Operating Procedures (SOPs) are validated and certied
procedures crucial for specic determinations. Regulatory bodies like the EPA or FDA typically
approve and publish these procedures, disallowing the use of alternative procedures for collecting
analytical data on specic analytes. It is imperative for any commercial laboratory to have SOPs
aligned with appropriate standards. This ensures that analytical data obtained and recorded can be
traced back to a documented procedure, allowing for replication of determinations using the SOP
for similar specimens.
13.4.2.2 Instrumentation Validation
Instrument validation stands as a critical procedure in every analytical laboratory. Although default
instrument data may seem reliable, modern computer- controlled systems, isolating analysts from
data collection and instrument control functions, make it challenging to detect inaccuracies. Thus,
it’s crucial to establish objective protocols for regularly assessing instrumental data validity.
These protocols guarantee the continued safe operation of laboratory instruments within dened
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parameters. Control charts, as time- related graphical records, depict outcomes of instrument validation procedures. Typically, the “control limits,” which are assigned as upper and lower ranges
around the projected instrumental output, are linked to some agreed- upon estimation of the expected
random error for the entire process. Typically, the control limits are set at two standard deviations.
When an instrument’s performance exceeds these limits, QA procedures mandate its non- use for
analytical reports until the issue is identied, rectied, and certied to operate within control limits
again before being returned to service for determinations.
13.4.2.3 Reagent and Materials Certification
Reagents and product testing are integral to QA, following GLP guidelines that stress adherence
to established protocols and proper documentation. Guidelines necessitate labeling each laboratory reagent/ material container with certication details, date, and expiration. This protocol aims to
ensure that SOP- specied reagents are used.
QA also requires an Analytical Qualication (COA), demanding evidence of analyst training and
experience with suitable laboratory procedures. As the American Chemical Society lacks a ‘certication’ policy for chemists or analysts, certication standards are usually set by the lab, meeting
FDA or EMA requirements.
13.4.2.4 Certification of Laboratory Facilities
Certication of laboratory facilities is usually performed by a third- party entity. Representatives
from a government agency with which they have a contract, for example, may be audited at an
analytical laboratory. An independent laboratory should submit paperwork to the appropriate state
or federal agency. The evaluation considers space (quantity, efciency, and relevance), ventilation,
facilities, storage, hygiene, and other factors.
13.4.2.5 Specimen and Sample Tracking
The emergence of computer- based Laboratory Information Management Systems (LIMSs)
highlighted the signicance of tracking specimens and samples in quality control. Sample
monitoring, whether manual using paper les or modern bar- coding techniques, remains an essential part of quality assurance. Although the terms “specimen” and “sample” are often interchangeable, “specimen” typically refers to a chemically determined substance, while “sample” generally
denotes a nite portion of the specimen taken for analysis. In homogenous specimens, the sample
mirrors the overall composition, but in heterogeneous ones (e.g., metal alloys, rock, soil, foods,
etc.), a sample might not accurately represent the overall composition (e.g., metal alloys, rock, soil,
textiles, foods, polymer composites, and vitamin capsules). Maintaining analytical result reports’
context is crucial for data interpretation.
Different laboratories may employ varied procedures for proper specimen/ sample monitoring,
but they all must preserve the unmistakable link between analytical data and the specimens/ samples
they were derived from. Additionally, the source of the specimen/ sample(s) must be documented
and unequivocally linked to the analytical data collection. In specic situations, establishing and
validating a “chain of custody” becomes necessary. This is particularly crucial for forensic samples
in criminal cases but could be relevant in various other circumstances. For instance, a pharmaceutical company might need to demonstrate the authenticity of specimens used in clinical trials to
eliminate doubts about data validity. These safeguards might involve ensuring that trial specimens
were not tampered with and providing a complete chain of custody to eliminate any doubts about
the validity of specimens submitted for chemical analysis.
13.4.2.6 Documentation and Maintenance of Records
Protection of specimen/ sample origin records, chain of custody, raw analytical data, processed data,
SOPs, instrument validation and reagent certication reports, and analyst certication papers aligns
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with GLP guidelines. These records allow for post- evaluation of performance, often after several
years have passed. In the event of legal disputes, maintaining all specied records offers evidence
that may be needed because of the effect of decisions concerning original analytical ndings.
GLP’s record- keeping function is critical, with many features now incorporated into modern
instrument service computer packages. For instance, modern computer- based instruments allow
unrestricted storage of raw analytical data for specic samples in a secure, tamper- proof environment. They also ensure the preservation of historical control chart data, crucial for determining an
instrument’s operational quality during data acquisition.
The duration for retaining laboratory records varies. In supervised laboratories, the general rule
is to maintain records for at least ve years, with the possibility of extending this period thanks
to higher- density storage devices for digitized data. This kind of record- keeping is increasingly
crucial, especially with the rise in chemistry- related commercial goods lawsuits. For businesses
dealing with potential lawsuits, safeguarding stored data integrity becomes a signicant security
concern.
All of the record- keeping ingredients described above are captured in the traditional laboratory notebooks serve as repositories for the record- keeping elements described earlier, providing
scientists with a detailed guide for laboratory maintenance. Detailed instructions on maintaining a
lab notebook will be provided in a subsequent section.
Data collection in laboratories involves meticulous recording in notebooks using indelible
ink, strictly avoiding erasable materials. Corrections are not made by overwriting; instead, incorrect entries are minimally crossed out for legibility. A new entry, accompanied by the operator’s
initials, follows. This practice, mandated by GLP, applies universally, even in simple or preparatory
experiments. In instances where data is initially noted on loose paper (such as napkins) and later
transcribed, the original writing must remain intact, a requirement extending to the nal document.
The value of data hinges on several crucial questions: What is its intrinsic value? How condent are the results? How precise is the measurement, and what’s the reproducibility of recorded
numbers? Contrary to popular belief, the number of signicant gures does not necessarily equate
to accuracy. An excessive number of gures often hampers data presentation and might misrepresent
its accuracy.
A data value represents a material property with an element of uncertainty. This data may be
accurate, inaccurate, or a combination of both. The level of uncertainty associated with a value is
reected in how it is written, typically by the number of gures included, indicating potential uncertainty. Sometimes, there’s an excessive number of gures after the decimal point, but even the gures before the decimal point can be tailored to represent the data accurately. A simple way to gauge
this necessity is by counting the signicant numbers. For instance, the numbers 19,490 and 10.098
both contain ve signicant gures each. The required number of signicant gures depends on two
factors. First, the value’s signicance, i.e., how relevant it is. For instance, if an instrument can only
detect a 1% difference, signicant gures should not exceed two after the decimal point. Second,
the numerical multiplicative value matters. Consider the number π (22/ 7); while it can be extended
to an innite number of signicant gures, a standard calculator rounds it to 3.14285714285714 due
to the digits’ remainder.
13.4.3 electRonic data handling
13.4.3.1 Overview
Entering data directly into electronic devices like computers, tablets, or through handwriting conversion is now common. However, electronic data handling poses new challenges and requirements
that must be thoroughly understood by all handling such data. In the past ve years, the FDA has
issued more citations for non- compliance with electronic data handling than for any other reason,
including adherence to GMP. These citations have been given to companies of various sizes, from
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small regional businesses to large multinational corporations. Consequently, many facilities generating data for regulatory submission continue using manual recording systems until they feel comfortable transitioning to electronic systems.
Electronic data management requires establishing an elaborate validation and security system,
often inaccessible to smaller institutions. Stringent FDA requirements mandate validated hardware,
secure operator identication, multi- level passwords, and active data monitoring. Although nonregulatory research institutions might bypass these requirements, compliance becomes imperative
when planning a regulatory submission.
These regulatory requirements are detailed in CFR 21 Part 11 of the Federal Register. Students
should familiarize themselves with at least the basic aspects of this law to grasp the system’s
requirements.
13.4.3.2 Code of Federal Regulations 21 Part 11: Electronic Records
Part 11 of Title 21 of the Code of Federal Regulations acts as a reference for those keeping or
uploading records electronically to comply with FDA regulations. It covers all electronic records
produced, updated, maintained, archived, retrieved, or distributed in line with FDA Regulations.
Part 11 also encompasses electronic documents submitted to the FDA under the Federal Food, Drug,
and Cosmetic Act (the Act) and the Public Health Service Act (the PHS Act), even if these records
aren’t expressly dened in FDA regulations (11.1). Predicate laws are the foundational criteria
outlined in the Act, the PHS Act, and the FDA, other than Part 11.
The FDA enforces the provisions of part 11 including, but not limited to, certain controls for
closed systems in § 11.10, for example, the following controls and requirements:
• Limiting device access to only those who are allowed.
• The application of operating framework tests.
• Tests for authority.
• Unit checks are used.
• The determination that people who design, operate, or use electronic systems have the necessary qualications, training, and experience to do their jobs.
• Individuals are held responsible for acts taken with their electronic signatures if written policies are developed and followed.
• Additionally, there are requirements for system documentation controls, acceptable controls
over systems documentation for open systems (11.30), and requirements related to electronic
signatures (e.g., 11.50, 11.70, 11.100, 11.200, and 11.300).
For full compliance, individuals must adhere to relevant predicate rules, ensuring the preserva-
tion and accuracy of the documents intended for preservation or submission.
Part 11 applies when people opt for electronic documents over paper format. When computers
generate paper printouts of electronic records that meet all relevant predicate rule criteria and these
printouts are relied upon for controlled activities, the FDA typically doesn’t classify this as ‘using
electronic records in place of paper records’ under 11.2(a) and 11.2(b). In such cases, the use of
computer systems in creating paper records doesn’t trigger part 11.
Part 11 of the FDA’s regulations pertains to specic electronic records or signatures (part 11
records or signatures)
• Records must be held following predicate rule provisions and kept in electronic format rather
than paper format. Part 11 records, on the other hand, are records (and any related signatures)
that are not supposed to be kept under predicate rules but are kept in electronic format.
• Records that are supposed to be kept under predicate rules are kept in both electronic and
paper formats and are used to carry out supervised activities.
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Real business practices may determine whether electronic records or paper records are used under
11.2(a) in certain cases. For example, if a predicate rule requires record maintenance and a machine
generates a paper printout of electronic records, the dependency for supervised operations is on
the electronic record. In such cases, the FDA might opt for an electronic record over a paper one.
Essentially, when determining the applicability of part 11, the FDA considers your business practices.
Records submitted to the FDA in electronic format under predicate rules (even if not explicitly
listed in FDA regulations) (assuming identication in docket number 92S- 0251 as acceptable electronic submissions). However, a record used to generate a request is not considered part 11 compliant unless allowed under a predicate rule and maintained in electronic format.
Predicate laws mandate handwritten signatures, initials, and other signings, with electronic
signatures intended as substitutes. Part 11 signatures include electronic signatures used to document
specic events or activities per the predicate rule (e.g., accepted, checked, veried).
13.4.4 validation
FDA requires validation of computerized systems (11.10(a)) and corresponding criteria (11.30).
Assess the scope of computerized system validation based on their impact on meeting predicate
rule requirements and the accuracy, reliability, credibility, availability, and validity of necessary
records and signatures. Even without predicate rule requirements for validation, cases may necessitate validation.
Clients should focus their strategy on a documented risk assessment to evaluate system impacts
on product quality, protection, and record integrity. Validation may not be necessary for a word processor generating SOPs exclusively.
Computer systems encompass hardware and software; hardware validation protocols are welldened, but software validation poses signicant challenges. Most off- the- shelf software programs
do not meet FDA validation requirements.
13.4.5 audit tRails
Unique part 11 standards govern computer- generated, time- stamped audit trails, requiring compliance with relevant predicate rule provisions for recording date (e.g., 58.130(e)), time, or event
sequencing. Any modications to records must not obscure prior entries.
Even if no predicate rule requirements are available to track, for example, the date, time, or
sequence of events in a specic case, audit trails or other physical, logical, or procedural security
measures may be necessary to ensure the records’ trustworthiness and reliability. We should base
our decision on whether to use audit trails or other necessary steps on the need to comply with predicate rule requirements, a justied and recorded risk assessment, and a determination of the possible
impact on product quality, protection, and record integrity. Based on such an assessment, the FDA
recommends that we implement effective controls. When users are required to build, alter, or remove
controlled records during regular operations, audit trails can be particularly useful.
The copies of electronic records are provided to the FDA as follows:
• When records are held in common portable formats, making copies of them is a simple task.
• Where possible, using existing automated conversion or export methods to create copies in a more
popular format (examples of such formats include, but are not limited to, PDF, XML, or SGML).
13.4.6 RecoRd Retention
Predicate rule criteria, a justied and recorded risk evaluation, and a calculation of the records’
worth over time should all be considered when deciding how to keep records.
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The FDA permits archiving necessary records on non- electronic media like microlm, microche,
paper, or standard electronic le formats (e.g., PDF, XML, or SGML). Predicate rule provisions
must be followed, and both content and purpose of the required documents and their copies must
be preserved. The electronic version of records may be discarded if all predicate rule conditions are
met, and content and context are maintained and archived. A hybrid situation where paper and electronic recording and signature components coexist is acceptable as long as predicate rule criteria are
met, and the records’ accuracy and meaning are preserved.
13.4.7 data eRRoRs
13.4.7.1 Absolute and Relative Errors
The signicant digits are also decided frequently on the output of the instrument recording the data;
for example, if a balance is capable of giving ±1 g, then there is no sense reporting weight even
to a single decimal place. The last number should be rounded off, for example, a weight machine
recording 138.7 g (yes the output may be provided to any signicant value) then the number should
be reported as 139 g. In the example given above, we have introduced another concept of absolute
error, meaning that within a range of 1 g on each side, the values are not accurate. When the absolute
error is compared with the total value, we obtain a relative error. In the example above, 139 g weight
recorded on a machine with ± 1 g uncertainty represents a relative error of (1/ 139) × 100 = 0.72%
relative error.
As absolute error is an estimation rather than an actual measurement, reporting it to more than two
signicant gures is unnecessary. In the example calculation, reporting a relative error of 0.7269%
wouldn’t provide meaningful information.
Absolute errors and relative errors, as discussed earlier, pertain to individual data sets. When
multiple datasets are incorporated into a mathematical formula, the error can become substantial. As a rule of thumb, relative errors are additive when performing multiplication or division.
In the previously mentioned example, the relative error for weight measurement was 0.72%. If
the volume measurement (for density determination) has a relative error of 2%, then the overall
error for density will amount to 2.72%. For addition or subtraction, absolute errors, not relative
errors, are combined. However, when subtracting large numbers, the absolute errors may result in
smaller values, transforming into considerably large relative errors. For instance, if a value of 2890
is subtracted from 2900, both with an absolute error of 10, the nal answer of 10 will possess an
absolute error of 10 or a 100% relative error.
13.4.8 systeMatic and RandoM eRRoRs
The preceding paragraphs detailed signicant gures in numbers and addressed handling data reliability. Errors in measurements can stem from a xed factor, such as lack of calibration, or unpredictable human errors. The former is termed a systematic error; for instance, if the user overlooks
the 42g weight of the sample container, all measurements should be adjusted downward by 42
g. Additionally, calibration errors, resulting in all readings being 10% higher, can be rectied after
data collection. Systematic errors produce highly reproducible results, hence precise although not
accurate. The latter type, random or accidental error, arises from unknown causes such as human
or machine errors leading to varying measurements or a scattered distribution. For accuracy, results
should exhibit minimal systematic and random errors.
Finally, some errors fall in the category of blunders— needless to say; it takes a man to err, a
computer to blunder.
The goal of obtaining data remains to obtain accurate data recorded to reasonable signicant gures, thus obviating systematic and random errors.
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FIGURE 13.2 Precision- accuracy relationship chart.
Source: Copyright Millipore Sigma
FIGURE 13.3 Darts thrown representing how precision and accuracy are dened: (a) imprecise and inaccurate
(b) imprecise and accurate (note: the decision to call this observation accurate depends on the range of precision
required; for example, in most circumstances, a 5% lack of precision will be readily accepted, in others a much
lower range is desired); (c) precise and inaccurate; and (d) precise and accurate.
Source: Copyright Millipore Sigma
In cases where absolute and correct gures are reported, there’s no limit to signicant gures, as
seen with the value of π or an exact dollar amount. However, in collected data, reporting more than
three signicant gures often surpasses the sensitivity of the methods used, giving a false impression
of higher accuracy and reliability.
To represent the above discussion in a graphical form, Figure 13.2 shows the relative errors.
On a more graphical base, Figure 13.3 shows the description of precision and accuracy on a
dartboard.
Interestingly, the next time you look at the advertisement of a high- end manual Swiss watch,
notice that they tout their “precision movement”, not “accurate movement” because none of the
watches, particularly the manual ones, are accurate.
()
()
−+
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13.4.9 statistical analysis
335
Randomness in data is associated with unpredictable errors; however, another aspect of randomness
involves the data measured. While an electronic instrument might produce an electrical signal with
a random range of responses to an impulse, universal values encountering randomness are common
occurrence.
The age distribution of the American population reects a bell- shaped curve, termed a
Gaussian distribution phenomenon, when plotted against age groups and corresponding population numbers. This bell curve might be ideally symmetrical (representing a truly randomized
sample) or skewed due to an overall bias. For instance, the Japanese curve skews rightward, signifying longer life expectancy and slower population growth. Conversely, in many developing
countries, the curve skews leftward (lower age), symbolizing a high birth rate and elevated mortality at older ages.
When a coin is ipped numerous times, the outcomes would be represented as a at bar indicating the two outcomes rather than a bell- shaped curve, owing to the discrete nature of the results.
It’s crucial not to confuse the randomness in these examples with the randomness observed in the
data output that constitutes our dataset. These random measurement errors can be analyzed through
statistical procedures to determine the best estimate of the measured variable and assess how random
error inuences the data.
It is pertinent to note that, similar to the age distribution example, random errors also adhere to
a Gaussian (or normal) distribution. Theoretically, an innite number of readings would yield an
error- free arithmetic mean. However, a reasonable number of readings sufce to understand the
actual value.
The process begins with calculating the arithmetic mean by adding all values and dividing the
sum by the number of readings.
This represents an initial attempt to dene the real value. However, this value doesn’t account for
measurement precision, which is indicated by how each reading deviates from the arithmetic mean—
the residual value. This residual provides a statistical parameter known as standard deviation (σ).
σ
=
2
xx xx xx
()()
−+−+
1
n
xx
−
2
2
2
∑−
2
()
xx
n
(13.1)
2
3
Hence, in reporting the results of repeated measurements, the mean is quoted as the best estimate of the variable, while the standard deviation gauges the condence in the result. Both mean
and standard deviation share units and dimensions with the variable (x), and the variable is reported
as mean ± standard deviation. As the number of observations (n) increases, the standard deviation
decreases while simultaneously bringing the arithmetic mean closer to the real value. In cases where
a high standard deviation is observed, the focus should be on system improvement rather than reducing the deviation through repeated measurements. Typically, a few dozen readings sufce to grasp
the extent of deviation. Notably, systematic errors stemming from poor calibration or validation are
constant modulators linked to accuracy and aren’t subject to statistical analysis.
13.4.10 conclusions
Data analysis encompasses techniques for describing evidence, identifying patterns, forming theories, and testing hypotheses. Numerical results of data analysis tend to be straightforward, often
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revealing the most representative number and comparing them. It unveils averages (e.g., average pH
or temperature) and variations (e.g., differences in optical density during fermentation). However,
data analysis is not solely about numbers; it utilizes them. The challenge arises not in contemplating
“How does it work?” but in the realm where data analysis operates. For instance, in an 8- hour bacterial culture, the optical density increased from an initial 0.1 to 12. Mathematically, the culture
averaged an optical density increase of 1.49 h−1. However, without further data collection and analysis, the question “How does it work?” remains unanswered. Additional data analysis in this scenario might reveal that the culture doubled in density every 20 minutes until reaching around 12 and
then stabilized, which occurred approximately 4.5 hours in. Consequently, data analysis assists in
testing a particular model’s applicability to a process, estimating the signicance of coefcients in
process models, and visualizing a variable’s general pattern inuencing another.
Experimental data can be categorized into independent variables and dependent variables, with
the latter representing the uncontrolled response. Examples of independent variables include time,
pH, or temperature, while the dependent variable, such as optical density, is expressed as a function
of the independent variable.
Flow diagrams serve as visual representations of processes, effectively summarizing a vast
amount of data. They can be complex, outlining pertinent process information and data. Figure 13.2
illustrates a ow diagram depicting the manufacture of a recombinant protein. Experiment conditions
and recorded outputs can be integrated into ow diagrams. Some ow diagrams transform into decision trees, determining a specic path based on true or false conditions. Engineering ow diagrams
contain extensive details and are routinely used to describe large complex systems.
Data are typically represented through tables, graphs, or equations. Tables offer a way to display data of varying lengths and levels of detail, but longer tables can become difcult to interpret. Graphs, on the other hand, provide a quick visualization of results and trends. Understanding
these aspects of data analysis is crucial for grasping the overall process and identifying outliers. It
also facilitates the design of additional experiments based on different phases of the experiment.
Conventionally, independent variables are plotted on the abscissa (X- axis), while one or more
dependent variables are plotted on the ordinate (Y- axis). One simple method for plotting data is
by using Microsoft Excel, which provides extensive mathematical and statistical tools. Prociency
in Microsoft Excel is highly recommended for anyone working in a laboratory setting. However,
it’s important to note that these data manipulations might not comply with CFR 21 Part 11, which
may or may not be an issue depending on whether the data are submitted for regulatory approval
of products.
The relationship between independent and dependent variables can often be presented in equation
form, establishing a mathematical relationship. A regression t might yield a linear relationship,
such as y = Ax + B, where B represents the intercept of the straight line on the ordinate and A is the
slope; A and B are also known as coefcients or adjustable parameters.
Non- linear regression involves incorporating another mathematical function, such as an exponential growth model like X = X0e−kt, where k is the rate constant and t is time. This relationship
implies a natural log- linear relationship, where plotting the natural log of X against time produces
a straight line with a slope equal to −k. Fitting data to these equations allows for understanding and
predicting outcomes based on a certain equation.
Considering inherent errors in each data point and those that can be analyzed through statistical methods is crucial when drawing conclusions from data analysis. This emphasizes the use of
regression analysis to ensure proper understanding of a dataset. While Microsoft Excel simplies
the creation of various regression ts or models, it’s essential for students to manually calculate the
goodness of t to appreciate the role of data variability. Understanding statistical principles behind
calculations is fundamental, as human errors often contribute signicantly to experimental errors.
Recognizing the signicance of random errors, systematic errors, and blunders is essential for those
working in laboratories. Collecting data inaccurately severely limits their meaningfulness, adhering
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to the principle of “garbage in, garbage out.” Therefore, a thorough understanding of basic statistical
methods and their limitations is crucial in laboratory work.
When data are plotted, either manually or by computer, and a best- t line is drawn through them,
the question arises regarding the signicance of certain points over others. This concern becomes
more pronounced when employing manual data smoothing techniques. Computers, however, mitigate this bias by drawing lines that minimize variance between predicted points and actual values
at different intervals. Nevertheless, this method tends to give more weight to data points with larger
values. To address this, models are available that assign specic weights to data points, thereby
preventing such biases in automated computations. The least- squares analysis, a prevalent technique for determining the line or curve that minimizes residuals, involves minimizing the sum of
squares of the residuals in this statistical method. Different approaches exist for this operation. For
instance, Legendre’s approach decreases the number of squares of the residuals for the dependent
variable, while Gauss’s and Laplace’s methods reduce the sum of squares of weighted residuals,
with weights determined by the scatter of replicate data points. It’s crucial to note that each approach
yields distinct results; determining the “correct” tting curve is essentially subjective. In the leastsquares analysis, the generated curve may not closely align with specic data points known to be
more accurate, as it minimizes the number of squares of the residuals. Alternatively, characterizing
the best t by raising the number of residuals to the fourth power minimizes the absolute values of
the residuals. The selection of the sum of squares is somewhat arbitrary, as several other mathematical methods are equally valid. Outliers, points with large residuals, strongly inuence regression. In some cases, these outliers can be excluded, but only after analyzing the data both with and
without them. Statistical models specically designed to handle outliers exist, requiring a deeper
understanding of statistical modeling. It is important to note that Good Laboratory Practice (GLP)
standards prohibit the exclusion of any outliers.
To summarize, note that the least- squares analysis applies solely to data containing random errors,
necessitating independent variables. In essence, y cannot be a function of x and be determined by
the process’s nature. For instance, if x represents time and y is optical density, regardless of the time
readings taken, y should not depend on the clock hour. If readings at 2 pm consistently show a 10%
increase, there exists a dependence between x and y. Such instances require more sophisticated
models for analysis. Moreover, it assumes uniform data output regardless of the experiment. Consider
whether equipment heating up over time might introduce more random error measurements.
That will require necessitating additional corrections and potentially requiring weighted leastsquare analysis.
In the past, graph paper plotting was commonplace. However, in today’s electronic age, it’s
advisable to refrain from this practice. Not only is it time- consuming, but it also tends to result in
signicant errors and hinders electronic data storage. Instead, students are encouraged to develop
prociency in tools like Microsoft Excel. Computer plotting offers added benets such as error bars,
standard deviation bars, regression coefcients, and measures of goodness of t.
13.5 QUALITY CONTROL
13.5.1 oveRvieW
For biopharmaceuticals and chemically derived drugs, in- process testing, release testing based on
DS and DP specications, and product characterization testing follow similar protocols. Monitoring
various parameters and responses is part of a robust quality management program. This encompasses
essential parameter descriptions, in- process control of intermediate substances, and testing of both
drug substance and product. Quality control standard denitions include process management, substance/ product control, and a summary of analytical methods employed to classify intermediate and
nal products. In- process measurements like pH, conductivity, total protein, and redox potential
are common. Testing of drug substances/ products adheres to ICH standards, focusing on identity,
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