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New Perspectives on Cleaning.Rizwan Sharnez and Angela To[

ABOUT THE AUTHORSRizwan Sharnez, Ph.D., is principal engineer at Amgen, Colorado. He has more than 15 years of experience in the pharmaceutical industry. He may be reached by e-mail at rsharnez@amgen.com. Angela To has a bachelors degree in chemical engineering from Rice University.

Multiproduct Cleaning Validation: Acceptance Limits for the Carryover of Inactivated APIPart I–The Comparable Quality Approach

“New Perspectives on Cleaning” is an ongoing series of articles dedicated to cleaning process development, validation, and monitoring. This column addresses scientific principles, strategies, and approaches associ-ated with cleaning that are faced in everyday work situations.

Reader questions, comments, and suggestions are requested for future discussion topics. These can be submitted to the column coordinator Rizwan Sharnez at rsharnez@amgen.com.

SUMMARYAn important consideration in multiproduct cleaning validation is to demonstrate that the carryover of the previously manufactured active pharmaceutical ingredi-ent (API) into a batch of the subsequently manufactured product is below an acceptable limit. If, however, the previously manufactured API becomes therapeutically inactive during cleaning, then there is limited value in verifying clearance of the API. Instead, it is more appro-priate to demonstrate clearance of inactivated product. This approach is gaining acceptance in the industry.

A methodology for evaluating the degree of inactiva-tion of a product during cleaning and setting accep-tance limits for the carryover of inactivated product in multiproduct equipment is described. A new approach for justifying acceptance limits for inactivated product, known as the comparable quality (CQ) approach, is described in Part I; the application of this approach to biopharmaceutical cleaning will be described in Part II. The general principles of the CQ approach are appli-cable to most active pharmaceutical ingredients (APIs).

INTRODUCTIONFor multiproduct cleaning validation, acceptable car-ryover of the previously manufactured API (Product A) into the subsequently manufactured API (Product B) is determined through a maximum allowable carryover (MAC) calculation (1-3). A limitation of the conventional MAC approach is that it does not account for the carry-over of the inactivated molecule between lots of different products (i.e., A → B, or B → A). This is an important fac-tor to consider when aggressive cleaning conditions are used. For example, biopharmaceutical cleaning cycles

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Rizwan Sharnez and Angela To.

are generally designed to expose product contact equip-ment to extremes of pH (i.e., 2-13) and temperature (i.e., 60-80°C) for several minutes. The equipment may also be steam sterilized or sanitized after cleaning. Under these conditions, monoclonal antibodies, therapeutic proteins, and other biological APIs are known to degrade and denature rapidly (4, 5) and are, therefore, likely to become therapeutically inactive (6).

Inactivation of the product during cleaning has impor-tant implications for cleaning validation of multiproduct equipment. If it can be demonstrated that the product becomes therapeutically inactive during cleaning, a MAC assessment for the API would not be required. It also obviates the need to develop product specific assays (PSA) for cleaning validation.

Inactivation of the product during cleaning can be assessed by exposing the process soil to simulated clean-ing conditions at bench scale (4, 5). The bench scale stud-ies are designed to simulate the conditions that are least conducive (worst-case) for inactivation. For example, for alkaline washes, the lowest applicable pH, temperature, duration, and ratio of cleaning solution to process soil is used to simulate the cleaning cycle at bench scale. The sample is then neutralized and cooled to minimize any further inactivation. The degree of inactivation is evalu-ated by subjecting the sample and an untreated control to the appropriate assays (e.g., Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis [SDS PAGE] and bioassay for biological products).

A literature search did not uncover any scientific approaches or regulatory guidances for setting accept-able limits for the carryover of inactivated product for cleaning validation.

LIMITATIONS OF THE MAC APPROACHThe MAC approach is often used to set cleaning valida-tion acceptance criteria for the carryover of the previously manufactured API (Product A) into the subsequently manufactured API (Product B) (1-3). A limitation of this approach is that it does not account for the carryover of the inactivated molecule between lots of different products (i.e., A → B, or B → A).

Another limitation of the MAC approach is that the acceptance limits for cleaning validation are often below process capability limits and/or below the limit of quan-titation (LOQ) of non-specific assays (e.g., total organic carbon [TOC]; the LOQ of TOC is typically between 0.05 and 0.2 ppm). This issue is further exacerbated by the low recovery of APIs from process soils. PSAs such as enzyme-linked immunosorbent assay (ELISA) and enzyme immunoassay (EIA) are sometimes used

to address this issue because they have very low LOQs (typically below 10 ppb). However, PSAs are difficult and laborious to qualify, and can give inaccurate results if the API degrades during cleaning (7). That is because PSAs detect activity indirectly, by recognizing specific epitopes (i.e., short amino acid sequences that PSAs are designed to detect). The epitopes can be destroyed by buffer and cleaning agent components. However, a biological API can be therapeutically active even if the epitopes are destroyed, and this can lead to false nega-tives. Similarly, it is possible for the API to be inactivated even though the epitopes are not completely destroyed, and this can lead to false positives. Another issue with PSAs is that it is difficult to get an accurate recovery fac-tor. That is because the experimental conditions of the recovery study (e.g., direct spotting of coupons with the API) do not represent the actual sample matrix (i.e., a small amount of API in the presence of degradants and cleaning agent residues). For the above reasons, PSAs should be used judiciously for verifying clearance of biological APIs after cleaning (7).

Another issue with the MAC approach is that every time a new product is introduced into a facility there is a risk that one or more of the new MAC limits for the previously validated products could be below the existing acceptance limits for cleaning validation.

PROTEIN DEGRADATION AND INACTIVATION APPROACH

Performing Degradation and Inactivation StudiesA bench-scale approach for evaluating the bioactivity of the residual API and the molecular weight distribution of the degradants after exposure to cleaning conditions is described in this section.

Full-scale cleaning conditions of shared product con-tact equipment are evaluated to determine the conditions that are least conducive for inactivation (e.g., lowest ratio of wash volume to protein, lowest cleaning agent con-centration, lowest temperature, and shortest duration of exposure). The product is exposed to these conditions at bench scale. The objective of the study is to ascertain whether the API in the process sample is inactivated when exposed to cleaning conditions.

Product is spiked into tubes containing alkaline cleaning solution, heated to the appropriate tempera-ture, and allowed to incubate for the duration of the alkaline wash. Samples may also be titrated with the acidic cleaning solution to the pH of the acidic wash and held for the duration of the acidic wash. Samples

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New Perspectives on Cleaning.

are then titrated to a neutral pH and cooled to 4°C to minimize further degradation and inactivation. The samples are then subjected to SDS-PAGE and bioassay to determine the degree of degradation and TOC analysis to determine whether the degradants can be adequately recovered by TOC.

AssaysSDS-PAGE and bioassays are used to evaluate protein degradation and inactivation, respectively. The product is exposed to cleaning conditions at small scale and then analyzed with the above assays to determine degree of degradation (i.e., molecular weight distribution) and bioactivity. Additionally, samples are analyzed for TOC to determine the recovery of the inactivated protein and the applicability of the TOC assay for demonstrating clearance of the inactivated product at full scale.

SDS-PAGE solubilizes aggregated and degraded pro-teins and separates them based on molecular weight (MW). The inclusion of MW standards allows for esti-mation of the MWs of the degradants and any aggre-gates, and the inclusion of control samples at a defined protein load allows for the estimated quantitation of protein concentration by densitometry. Staining of gels

is sensitive to 5-10 ng for Silver Staining and 100 ng for Coomassie Staining. SDS-PAGE has the advantage of providing a wide range of specificity for detecting proteins with unknown primary structures, size, charge, and hydrophobic states. This feature is particularly useful for protein degradation analysis because the level of protein degradation due to cleaning is highly unpredictable and can extend over a wide range of MW. SDS-PAGE also provides high sensitivity for detecting trace amounts of protein.

Bioassays measure the relative amount of biologically active product present in a sample. Thus, bioassays can be used to determine the effect of cleaning conditions on the inactivation of biologicals.

SETTING ACCEPTANCE LIMITS BASED ON PROTEIN INACTIVATION The methodology for setting acceptance limits is summa-rized in the Figure. Degradation and inactivation studies are first performed under simulated cleaning conditions. If the sample is shown to have therapeutically active product, the MAC approach is used to limit carryover of previous product to an acceptable level (1-3). If the MAC limit is higher than the LOQ of TOC, TOC is used

No Yes

Is API in process

sample inactivated after exposure to cleaning

conditions?1

Perform

inactivation study

Use CQ approach to

set acceptance limit (AL) for inactivated

API

Use MAC

approach to

set acceptance

limit (AL) for API

Inputs • Process samples and

reference standard

• Cleaning cycle and

equipment parameters

1 Based on

results of

bioassay

continued continued

Figure: Methodology for setting acceptance limits flowchart. Continued on page 35

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Rizwan Sharnez and Angela To.

Yes

Is AL LOQ

of TOC?

No

Inputs •Dose and batch sizes of

products

• Surface area and rinse

parameters of shared

equipment

•Recovery study for API

•Carbon content of API

Use TOC to

demonstrate clearance of API

Develop alternative

assay to demonstrate

clearance of API

Use MAC

approach to

set acceptance

limit (AL) for API

Continued from page 34

Yes

No Yes

Can

inactivated API be recovered

by TOC?

Develop alternative assay

to demonstrate clearance of inactivated API2

Use TOC to

demonstrate clearance of

inactivated API

Is AL LOQ

of TOC?

No 2 This is

done on a

case-by-

case basis

Inputs •Dose and batch sizes of products

• Surface area of dedicated and

shared equipment

•Recovery study for inactivated API

•Carbon content of inactivated API

Use CQ approach to set

acceptance limit (AL) for inactivated API

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New Perspectives on Cleaning.

to verify clearance of previous product at full scale. If the MAC limit is below the LOQ of TOC, an alternate assay may need to be developed to verify clearance of the API.

The MAC approach limits the amount of API of the previously manufactured product (A) in a dose of the subsequently manufactured product (B) to the accept-able daily exposure (ADE) of A (8), or to 1/1000th of the minimum dose of A (9).

COMPARABLE QUALITY APPROACHIf the protein becomes therapeutically inactive during cleaning, then the acceptance limit for the inactivated molecule of the previously manufactured product (Prod-uct A) in the subsequently manufactured product (Prod-uct B) can be set based on the CQ approach. With the CQ approach, the amount of inactivated Product A in a dose of Product B is limited to the amount of inactivated API of Product A in a dose of Product A. Appropriate adjust-ments are made to account for differences in process parameters of the two products. If inactivated Product A can be recovered and detected by TOC, and its acceptance limit is greater than the LOQ of TOC, then TOC is used to verify clearance of the inactivated API. Otherwise, an alternative assay is developed to demonstrate clearance of the inactivated API at full scale.

CONCLUSIONThe inactivation of a product during cleaning and steam-ing has important implications for cleaning validation of multiproduct equipment. Demonstrating that the product becomes inactivated during these operations obviates the need to perform arduos MAC assessments for the API. It also eliminates the need to develop PSAs for cleaning validation. PSAs are designed to detect specific epitopes; thus, if the API degrades, the result from the assay may not necessarily correlate to therapeutic activity.

The CQ approach can be used to set acceptance limits for the carryover of inactivated product for multiproduct cleaning validation. This approach is designed to ensure that the amount of inactivated Product A in a dose of Product B is less than the amount of inactivated Product A in a dose of Product A. Application of the CQ approach to biopharmaceutical cleaning will be described in Part II of this series.

REFERENCES1. Sharnez, R., “Strategies for Setting Rational MAC-based

Limits–Part I: Reassessing the Carryover Criterion,” Journal of Validation Technology, Vol 16, No. 1, p. 71-74, 2010.

2. Sharnez, R., To, A., Klewer, L., “Strategies for Setting Ra-tional MAC-based Limits–Part II: Application to Rinse

Samples,” Journal of Validation Technology, Vol 17, No. 2, p. 43-46, 2011.

3. Sharnez, R., To, A., “Strategies for Setting Rational MAC-based Limits–Part III: Leveraging Toxicology and Cleanability Data,” Journal of Validation Technology, Vol 17, No. 3, p. 24-28, 2011.

4. Kendrick, K., Canhoto, A., Kreuze, M., “Analysis of Degra-dation Properties of Biopharmaceutical Active Ingredients as Caused by Various Process Cleaning Agents and Tem-perature,” Journal of Validation Technology, Vol 15, No. 3, p. 69, 2009.

5. Rathore, N., Qi, W., Chen, C., Ji, W., “Bench-scale charac-terization of cleaning process design space for biopharma-ceuticals,” Biopharm Int, Vol 22, No. 3, 2009.

6. Martinez, J.E., “Immunogenic Potential of Therapeutic Protein Residues after Cleaning,” Bioprocess International, Vol. 9, P. 38-44, 2004.

7. Health Canada, Cleaning Validation Guidelines (GUIDE-0028); Section 8.3, Health Products and Food Branch In-spectorate, 2008. http://www.hc-sc.gc.ca/dhp-mps/compli-conform/gmp-bpf/validation/index-eng.php

8. ISPE, Risk-Based Manufacture of Pharmaceutical Products: A Guide to Managing Risks Associated with Cross-Contamination, 1st Edition, Vol. 7, ISPE, 2010.

9. Fourman, Gary L. and Michael V. Mullen, “Determining Cleaning Validation Acceptance Limits for Pharmaceutical Manufacturing Operations,” Pharmaceutical Technology, 17 (4), 54-60, 1993. JVT

ACKNOWLEDGEMENTSThe authors are grateful to Arun Tholudur and Joel Bercu for their helpful suggestions and support.

ARTICLE ACRONYM LISTINGA Product A–previously

manufactured productADE Acceptable Daily ExposureAL Acceptance LimitAPI Active Pharmaceutical IngredientB Product B–subsequently

manufactured productCQ Comparable QualityEIA Enzyme ImmunoassayELISA Enzyme-Linked Immunosorbent AssayLOQ Limit of QuantitationMAC Maximum Allowable CarryoverMW Molecular WeightPSA Product Specific AssaySDS PAGE Sodium Dodecyl Sulfate

Polyacrylamide Gel ElectrophoresisTOC Total Organic Carbon